WO2025038494A1

WO2025038494A1 - Compositions, systems, and methods for lymphoid cell differentiation using targeted gene activation

Info

Publication number: WO2025038494A1
Application number: PCT/US2024/041832
Authority: WO
Inventors: Lucas James Thompson; Charles Chapman; Cameron Lee; Oliver Kraemer
Original assignee: Tune Therapeutics Inc
Current assignee: Tune Therapeutics Inc
Priority date: 2023-08-11
Filing date: 2024-08-09
Publication date: 2025-02-20
Anticipated expiration: 2026-02-11

Abstract

Provided are DNA-targeting systems, such as CRISPR-Cas/guide RNA (gRNA) systems, that bind to or target a target site in a gene or regulatory element thereof in a stem cell. In some embodiments, the provided DNA-targeting systems promote transcriptional activation of genes that promote lymphoid cell differentiation. In some embodiments, the provided DNA-targeting systems promote differentiation of hematopoietic progenitor cells (HPCs) into a differentiated population of cells. In some embodiments, the differentiated population of cells are lymphoid progenitor cells. In some embodiments, the differentiated population of cells are lymphoid cells. Also provided herein are methods and uses related to the provided compositions, for example in facilitating lymphoid cell differentiation without the provision of extrinsic differentiation signals.

Description

COMPOSITIONS, SYSTEMS, AND METHODS FOR LYMPHOID CELL DIFFERENTIATION USING TARGETED GENE ACTIVATION

Cross-Reference to Related Applications

[0001] This application claims priority from U.S. provisional application No. 63/532,347 filed August 11, 2023, and U.S. provisional application No. 63/669,228 filed July 9, 2024, the contents of which are incorporated by reference in their entireties.

Incorporation by Reference of Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 22474-20025.40_SeqList.xml, created August 8, 2024, which is 350,981 bytes in size. The information in the electronic format of the Sequence Listing is herein incorporated by reference in its entirety.

Field

[0003] The present disclosure relates in some aspects to DNA-targeting systems, such as CRISPR-Cas/guide RNA (gRNA) systems, that bind to or target a target site in a gene or regulatory element thereof in a stem cell. In some aspects, the provided DNA-targeting systems of the present disclosure promote differentiation of hematopoietic progenitor cells (HPCs) into a differentiated population of cells. In some aspects, the differentiated population of cells are lymphoid progenitor cells. In other aspects, the differentiated population of cells are lymphoid cells. In particular, the present disclosure relates to the transcriptional activation of genes that promote lymphoid cell differentiation. In some aspects, the present disclosure is directed to methods and uses related to the provided compositions, for example in facilitating lymphoid cell differentiation without the provision of extrinsic differentiation signals.

Background

[0004] The ability to modulate the Notch signaling pathway is the foundation for producing highly functional cells for immunotherapy. However, current methods for differentiating stem cells into lymphoid cells (i.e., NK cells or T-cells) rely on the provision of external Notchsignaling ligands, which is laborious, costly, and difficult to scale. Therefore, there is a need for new and improved methods to overcome these challenges. The present disclosure addresses these and other needs.

Summary

[0005] Provided herein is a DNA-targeting system comprising one or more DNA-targeting modules, wherein the one or more DNA-targeting modules target one or more lymphoid cell differentiation (LCD) genes, and wherein each of the one or more DNA-targeting modules comprises a fusion protein comprising: (a) a DNA-binding domain that binds to a target site for one of the one or more lymphoid differentiation genes; and (b) at least one transcriptional activation domain that increases transcription of the one or more lymphoid differentiation genes.

[0006] Also provided herein is a DNA-targeting system comprising one or more DNA- targeting modules, wherein the one or more DNA-targeting modules increase transcription of one or more lymphoid cell differentiation (LCD) genes, and wherein each of the one or more DNA-targeting modules comprises a fusion protein comprising: (a) a DNA-binding domain that binds to a target site for one of the one or more lymphoid cell differentiation genes; and (b) at least one transcriptional activation domain that increases transcription of the one or more lymphoid cell differentiation genes.

[0007] In some of any of the provided embodiments, the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B.

[0008] Provided herein is a DNA-targeting system comprising one or more DNA-targeting modules for increasing transcription of one or more lymphoid cell differentiation (LCD) genes, wherein each of the one or more DNA-targeting modules comprises a fusion protein comprising: (a) a DNA-binding domain that binds to a target site for one of the one or more lymphoid cell differentiation genes, wherein the one or more lymphoid cell differentiation genes is selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B; and (b) at least one transcriptional activation domain that increases transcription of the one or more lymphoid cell differentiation genes.

[0009] In some of any of the provided embodiments, the one or more LCD genes is selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY 1 ; optionally where the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, the one or more DNA targeting modules is a plurality of DNA-targeting modules, and wherein each DNA-targeting module targets one of the one or more LCD genes. In some of any of the provided embodiments, the plurality of DNA-targeting modules is two, three, four, five, or six DNA-targeting modules, and wherein each DNA-targeting module targets one of the one or more LCD genes. In some of any of the provided embodiments, the plurality of DNA- targeting modules is two DNA-targeting modules, each targeting one of the one or more LCD genes. In some of any of the provided embodiments, the one or more LCD genes is two genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the two genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, the plurality of DNA-targeting modules is three DNA-targeting modules, each targeting one of the one or more LCD genes. In some of any of the provided embodiments, the one or more LCD genes is three genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the three genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, the plurality of DNA-targeting modules is four DNA-targeting modules, each targeting one or more LCD genes. In some of any of the provided embodiments, the one or more LCD genes is four genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the four genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, the plurality of DNA-targeting modules is five DNA-targeting modules, each targeting one of the one or more LCD genes. In some of any of the provided embodiments, the one or more LCD genes is five genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the five genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, the plurality of DNA-targeting modules is six DNA-targeting modules, each targeting one of the one or more LCD genes. In some of any of the provided embodiments, the one or more LCD genes is six genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the six genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. [0010] In some of any of the provided embodiments, at least one of the one or more LCD genes is RUNX3. In some of any of the provided embodiments, at least one of the one or more LCD genes is IL7Ra. In some of any of the provided embodiments, the one or more LCD genes include RUNX3 and IL7Ra. In some of any of the provided embodiments, at least one of the one or more LCD genes is TBX21. In some of any of the provided embodiments, at least one of the one or more LCD genes is CBFB. In some of any of the provided embodiments, at least one of the one or more LCD genes is LEFL In some of any of the provided embodiments, at least one of the one or more LCD genes is MYB. In some of any of the provided embodiments, at least one of the one or more LCD genes is RUNXL In some of any of the provided embodiments, at least one of the one or more LCD genes is SPIL In some of any of the provided embodiments, at least one of the one or more LCD genes is HEY 1.

[0011] In some of any of the provided embodiments, the one or more LCD genes are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; RUNX3, IL7Ra, and TBX21; RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB. In some of any of the provided embodiments, the one or more DNA-targeting modules comprise: (a) a first set of DNA-targeting modules, wherein the first set of DNA-targeting modules targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, and CBFB; and (b) a second set of DNA-targeting modules, wherein the second set of DNA-targeting modules targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, the one or more LCD genes targeted by the first set of DNA-targeting modules are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; or RUNX3, IL7Ra, and TBX21. In some of any of the provided embodiments, the one or more LCD genes targeted by the second set of DNA-targeting modules are: RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB.

[0012] In some of any of the provided embodiments, the one or more LCD genes are selected from the group consisting of TCF7, GATA3 and BCL1 IB.

[0013] In some of any of the provided embodiments, the one or more DNA-targeting modules comprise two DNA-targeting modules that target a combination of two lymphoid differentiation genes selected from the group consisting of TCF7, GATA3 and BCL11B. In some of any of the provided embodiments, the one or more DNA-targeting modules comprise three DNA-targeting modules that target TCF7, GATA3 and BCL1 IB.

[0014] In some of any of the provided embodiments, at least one of the one or more LCD genes is TCF7. In some of any of the provided embodiments, at least one of the one or more LCD genes is GATA3. In some of any of the provided embodiments, at least one of the one or more LCD genes is BCLB11. In some of any of the provided embodiments, the one or more LCD genes include TCF7, GATA3, and BCLB11.

[0015] In some of any of the provided embodiments, the regulatory DNA element is a promoter of the gene. In some of any of the provided embodiments, the target site for each of the one or more LCD genes is within 1000 base pairs of the transcription start site (TSS) of the gene. In some of any of the provided embodiments, the target for each of the one or more LCD genes is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS of the gene. In some of any of the provided embodiments, the target site for each of the one or more LCD genes is within 550 base pairs of the TSS of the gene.

[0016] In some of any of the provided embodiments, the target site for RUNX3 has the sequence forth in SEQ ID NO: 99 or SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for IL7Ra has the sequence set forth in SEQ ID NO: 103, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for TBX21 has the sequence set forth in SEQ ID NO: 104, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for CBFB has the sequence set forth in SEQ ID NO: 106, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for LEF1 has the sequence set forth in SEQ ID NO: 105, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for MYB has the sequence set forth in SEQ ID NO: 93 or SEQ ID NO: 94, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for RUNX1 has the sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 96, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for SPI1 has the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for HEY 1 has the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 98, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0017] In some of any of the provided embodiments, introduction of the DNA-targeting system to a stem cell promotes lymphoid differentiation.

[0018] In some of any of the provided embodiments, the stem cell is an induced pluripotent stem cell (iPSC). In some of any of the provided embodiments, the stem cell is a hematopoietic progenitor cell (HPC).

[0019] In some of any of the provided embodiments, lymphoid differentiation of the stem cell is characterized by decreased expression of CD34. In some of any of the provided embodiments, lymphoid differentiation of the stem cell is characterized by increased expression of CD56.

[0020] In some of any of the provided embodiments, the DNA-targeting system does not introduce a genetic disruption or a DNA break.

[0021] In some of any of the provided embodiments, the fusion protein of each DNA- targeting module comprises a DNA-binding domain selected from: a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or a variant thereof; a zinc finger protein (ZFP); a transcription activator- like effector (TALE); a meganuclease; a homing endonuclease; or an I-Scel enzyme or a variant thereof, optionally wherein the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing, wherein, when the DNA-binding domain of the fusion protein comprises a Cas protein, the DNA-targeting system further comprises one or more gRNAs, each capable of targeting the Cas protein to the target site for one of the one or more LCD genes.

[0022] In some of any of the provided embodiments, the target site for each of the one or more LCD genes is in the gene or a regulatory DNA element thereof. In some of any of the provided embodiments, the regulatory DNA element is an enhancer or a promoter of the gene.

[0023] In some of any of the provided embodiments, the target site is selected from: (a) a target site for TCF7 having the sequence set forth in any one of SEQ ID NOS: 1-6, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (b) a target site for GATA3 having the sequence set forth in any one of SEQ ID NOS:7-12, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; and (c) a target site for BCL1 IB having the sequence set forth in any one of SEQ ID NOS: 13-18, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0024] In some of any of the provided embodiments, the target site for TCF7 has the sequence set forth in SEQ ID NO: 1, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for GATA3 has the sequence set forth in SEQ ID NO: 11, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for BCLB11 has the sequence set forth in SEQ ID NO: 16, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing.

[0025] In some of any of the provided embodiments, the target site is selected from: (a) a target site for TCF7 having the sequence set forth in SEQ ID NO: 1, or a complementary sequence thereof; (b) a target site for GATA3 having the sequence set forth in SEQ ID NO: 11, or a complementary sequence thereof; and (c) a target site for BCL1 IB having the sequence set forth in SEQ ID NO: 16, or a complementary sequence thereof.

[0026] In some of any of the provided embodiments, the DNA-binding domain is a zinc finger protein. In some of any of the provided embodiments, the fusion protein of each of the plurality of DNA-targeting modules is different. In some of any of the provided embodiments, the DNA-targeting system comprises one fusion protein that is shared by each of the plurality of DNA-targeting modules and wherein each DNA-targeting module is characterized by comprising a different guide nucleic acid for targeting the DNA-binding domain to the target site. In some of any of the provided embodiments, the DNA-binding domain of the fusion protein is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof.

[0027] In some of any of the provided embodiments, the DNA-binding domain of each of the one or more DNA-targeting modules is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof, and each of the one or more DNA-targeting modules further comprises one or more gRNAs for targeting the DNA-binding domain to the target site of a LCD gene. In some of any of the provided embodiments, the DNA-binding domain of each of the one or more DNA-targeting modules is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof, and each of the one or more DNA-targeting modules further comprises one or more guide nucleic acids for targeting the DNA-binding domain to the target site of one of the one or more LCD gene.

[0028] In some of any of the provided embodiments, the one or more guide nucleic acids are one or more guide RNAs (gRNAs). In some of any of the provided embodiments, the Cas protein or variant thereof is a deactivated (dCas) protein. In some of any of the provided embodiments, the dCas protein lacks nuclease activity. In some of any of the provided embodiments, the dCas protein is a dCas9 protein. In some of any of the provided embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

[0029] In some of any of the provided embodiments, the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 83. In some of any of the provided embodiments, the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 84 or SEQ ID NO: 191, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSaCas9 protein is set forth in SEQ ID NO: 84 or SEQ ID NO: 191.

[0030] In some of any of the provided embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

[0031] Provided herein is a DNA-targeting system comprising one or more DNA-targeting modules, wherein each of the one or more DNA-targeting modules comprises : (a) a fusion protein comprising a DNA binding domain comprising a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and (b) one or more gRNAs that target a target site for one or more lymphoid cell differentiation (LCD) genes, wherein the DNA-targeting system increases transcription of the one or more LCD genes. In some of any of the provided embodiments, the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B.

[0032] Also provided herein is a DNA targeting system comprising: (a) a fusion protein comprising a DNA-binding domain comprising a Streptococcus pyogenes dCas9 (dSpCas9) protein and at least one transcriptional activation domain that increases transcription of one or more lymphoid cell differentiation (LCD) genes; and (b) one or more gRNAs that target a target site for one or more LCD genes selected from the group consisting of TCF7, GATA3, and BCL1 IB. Provided herein is a DNA-targeting system comprising one or more DNA-targeting modules, wherein each of the one or more DNA-targeting modules comprises: (a) a fusion protein comprising a DNA binding domain comprising a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and (b) one or more gRNAs that target a target site for one or more lymphoid cell differentiation (LCD) genes, wherein the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B.

[0033] In some of any of the provided embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 63. In some of any of the provided embodiments, the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 64 or SEQ ID NO: 196, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSpCas9 protein is set forth in SEQ ID NO: 64 or SEQ ID NO: 196.

[0034] In some of any of the provided embodiments, the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1 and HEY1. In some of any of the provided embodiments, the DNA targeting system comprises two, three, four, five, or six different guide RNAs, wherein each gRNA targets a target site of one of the one or more LCD genes. In some of any of the provided embodiments, the one or more LCD genes are two genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the two genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, the one or more LCD genes are three genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the three genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, the one or more LCD genes are four genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the four genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, the one or more LCD genes are five genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the five genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, the one or more LCD genes are six genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the six genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, at least one of the one or more LCD genes is RUNX3. In some of any of the provided embodiments, at least one of the one or more LCD genes is IL7Ra. In some of any of the provided embodiments, the one or more LCD genes include RUNX3 and IL7Ra. In some of any of the provided embodiments, at least one of the one or more LCD genes is TBX21. In some of any of the provided embodiments, at least one of the one or more LCD genes is CBFB. In some of any of the provided embodiments, at least one of the one or more LCD genes is LEF1. In some of any of the provided embodiments, at least one of the one or more LCD genes is MYB. In some of any of the provided embodiments, at least one of the one or more LCD genes is RUNX1. In some of any of the provided embodiments, at least one of the one or more LCD genes is SPI1. In some of any of the provided embodiments, at least one of the one or more LCD genes is HEY1. In some of any of the provided embodiments, the one or more LCD genes are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; RUNX3, IL7Ra, and TBX21; RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB.

[0035] In some of any of the provided embodiments, the one or more DNA-targeting modules comprise: (a) a first DNA-targeting module comprising a first fusion protein and a first set of gRNAs; and (b) a second DNA-targeting module comprising a second fusion protein and a second set of gRNAs, wherein the first and second fusion proteins comprise a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and wherein the first and second set of gRNAs target a target site for one or more lymphoid cell differentiation (LCD) genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1.

[0036] In some of any of the provided embodiments, the first set of gRNAs and the second set of gRNAs are the same. In some of any of the provided embodiments, the first set of gRNAs and the second set of gRNAs each target a combination of two or more of the LCD genes that are the same. In some of any of the provided embodiments, the first set of gRNAs and the second set of gRNAs are different. In some of any of the provided embodiments, the first set of gRNAs and the second set of gRNAs each target a combination of two or more of the LCD genes that are different.

[0037] Provided herein is a DNA-targeting system comprising one or more DNA-targeting modules comprising : (1) a first DNA-targeting module comprising (a) a first fusion protein comprising a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and (b) a first set of gRNAs comprising two or more gRNAs that target a target site for two or more lymphoid cell differentiation (LCD) genes; and (2) a second DNA-targeting module comprising (a) a second fusion protein comprising a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and (b) a second set of gRNAs comprising two or more gRNAs that target a target site for two or more lymphoid cell differentiation (LCD) genes, wherein the two or more LCD genes in the first and second set of gRNAs are independently selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1.

[0038] In some of any of the provided embodiments, the first set of gRNAs targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, and CBFB; and (b) a second set of gRNAs, wherein the second set of gRNAs targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, the one or more LCD genes targeted by the first set of gRNAs are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; or RUNX3, IL7Ra, and TBX21. In some of any of the provided embodiments, the one or more LCD genes targeted by the second set of gRNAs are: RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB. In some of any of the provided embodiments, the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1 and TBX21; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target RUNX3, CBFB, LEF1 and MYB; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1 and MYB; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1, MYB and TBX21; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target RUNX3, CBFB, LEF1, MYB and TBX21; or the first set of gRNAs target RUNX3 and IL7RA and the second set for gRNAs target RUNX3, CBFB, LEF1, MYB, TBX21 and IL7Ra. [0039] In some of any of the provided embodiments, the dCas and the transcriptional activation domain of the first and second fusion protein are the same. In some of any of the provided embodiments, the first DNA-targeting module is present in a first lipid nanoparticle and the second DNA-targeting module is present in a second lipid nanoparticle. In some of any of the provided embodiments, the one or more LCD genes are selected from the group consisting of TCF7, GATA3, and BCL1 IB.

[0040] In some of any of the provided embodiments, the dCas protein lacks nuclease activity. In some of any of the provided embodiments, the dCas protein is a dCas9 protein. In some of any of the provided embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 ( dSaCas9) protein. In some of any of the provided embodiments, the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 83. In some of any of the provided embodiments, the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 84, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSaCas9 protein is set forth in SEQ ID NO: 84. In some of any of the provided embodiments, the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 191 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSaCas9 protein is set forth in SEQ ID NO:191. In some of any of the provided embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 63. In some of any of the provided embodiments, In some of any of the provided embodiments, the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 64, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSpCas9 protein is set forth in SEQ ID NO: 64. In some of any of the provided embodiments, In some of any of the provided embodiments, the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 196, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSpCas9 protein is set forth in SEQ ID NO: 196.

[0041] In some of any of the provided embodiments, each of the one or more gRNAs comprise a gRNA spacer that is complementary to the target site of the gene. In some of any of the provided embodiments, the one or more gRNAs comprise a gRNA spacer that is complementary to the target site of the gene.

[0042] In some of any of the provided embodiments, the gRNA is selected from: (a)targeting a target site for RUNX3 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114. In some of any of the provided embodiments, the gRNA targeting a target site for IL7Ra comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 117, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO: 117. In some of any of the provided embodiments, the gRNA targeting a target site for TBX21 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 118, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO: 118. In some of any of the provided embodiments, the gRNA targeting a target site for CBFB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 120, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA targeting a target site for CBFB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 120. In some of any of the provided embodiments, the gRNA targeting a target site for LEF1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA targeting a target site for LEF1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119. In some of any of the provided embodiments, the gRNA targeting a target site for MYB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA targeting a target site for MYB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108. In some of any of the provided embodiments, the gRNA targeting a target site for RUNX1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA targeting a target site for RUNX1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110. In some of any of the provided embodiments, the gRNA targeting a target site for SPI1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, a target site for SPI1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116. In some of any of the provided embodiments, the gRNA targeting a target site for HEY 1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA targeting a target site for HEY 1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112.

[0043] In some of any of the provided embodiments, the gRNA is selected from: (a) a gRNA targeting a target site for TCF7 comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 19-24, or a contiguous portion thereof of at least 14 nt; (b) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 25-30, or a contiguous portion thereof of at least 14 nt; and (c) a gRNA targeting a target site for BCLB 11 comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 31-36, or a contiguous portion thereof of at least 14 nt.

[0044] In some of any of the provided embodiments, (a) the gRNA targeting a target site for TCF7 comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 19-24, or a contiguous portion thereof of at least 14 nt; (b) the gRNA targeting a target site for GATA3 comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 25-30, or a contiguous portion thereof of at least 14 nt; and (c) the gRNA targeting a target site for BCLB 11 comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 31-36, or a contiguous portion thereof of at least 14 nt.

[0045] In some of any of the provided embodiments, the gRNA is selected from: (a) a gRNA targeting a target site for TCF7 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19; (b) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 29; and (c) a gRNA targeting a target site for BCLB 11 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 34. [0046] In some of any of the provided embodiments, (a) a gRNA targeting a target site for TCF7 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19; (b) the gRNA targeting a target site for GATA3 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 29; and (c) the gRNA targeting a target site for BCLB 11 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 34.132

[0047] In some of any of the provided embodiments, the gRNA comprises a spacer sequence that is between 14 nt and 24 nt, or between 16 nt and 22 nt in length. In some of any of the provided embodiments, the gRNA comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

[0048] In some of any of the provided embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 56. In some of any of the provided embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 91. In some of any of the provided embodiments, the gRNA futher comprises a scaffold sequence set forth in SEQ ID NO: 122 .

[0049] In some of any of the provided embodiments, the gRNA futher comprises 2’ MeO modified bases and/or phosphorothiate backbone modifications.

[0050] In some of any of the provided embodiments, the at least one transcriptional activator effector domain is selected from the group consisting of: a VP64 domain, a p65 activation domain, a p300 domain, an Rta domain, a CBP domain, a VPR domain, a VPH domain, an HSF1 domain, a TET protein domain, optionally wherein the TET protein is TET1, a SunTag domain, or a domain, portion, variant, or truncation of any of the foregoing.

[0051] In some of any of the provided embodiments, the at least one transcriptional activator effector domain comprises at least one VP 16 domain, and/or a VP 16 tetramer (“VP64”) or a variant thereof.

[0052] In some of any of the provided embodiments, the at least one transcriptional activator effector domain comprises a VP64 domain or a variant or portion thereof that exhibits transcriptional activation activity. In some of any of the provided embodiments, the at least one transcriptional activator effector domain is VP64.

[0053] In some of any of the provided embodiments, the VP64 is positioned N-terminal and/or C-terminal to the DNA-binding domain.

[0054] In some of any of the provided embodiments, the at least one transcriptional activator effector domain comprises two copies of VP64. [0055] In some of any of the provided embodiments, the at least one transcriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 60, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing. In some of any of the provided embodiments, the at least one transcriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 62, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0056] In some of any of the provided embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO: 58, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0057] In some of any of the provided embodiments, introduction of the DNA-targeting system to a hematopoietic progenitor cell (HPC) promotes lymphoid differentiation. In some of any of the provided embodiments, the lymphoid differentiation is to a lymphoid progenitor cell (LPC) phenotype. In some of any of the provided embodiments, the LPC phenotype is an induced common lymphoid progenitor (iCLP) phenotype. In some of any of the provided embodiments, the lymphoid differentiation is characterized by decreased expression of CD34 relative to a HPC that was not introduced with the DNA-targeting system.

[0058] In some of any of the provided embodiments, the lymphoid differentiation is characterized by differentiation to a CD34- cell. In some of any of the provided embodiments, the lymphoid differentiation is characterized by increased expression of CD45 relative to a HPC that was not introduced with the DNA-targeting system. In some of any of the provided embodiments, the lymphoid differentiation is characterized by differentiation to a CD45+ cell. In some of any of the provided embodiments, the lymphoid differentiation is characterized by increased expression of CD7 relative to a HPC that was not introduced with the DNA-targeting system. In some of any of the provided embodiments, the lymphoid differentiation is characterized by differentiation to a CD7+ cell. In some of any of the provided embodiments, the lymphoid differentiation is characterized by increased expression of both CD5 and CD7 relative to a HPC that was not introduced with the DNA-targeting system. In some of any of the provided embodiments, the lymphoid differentiation is characterized by differentiation to a CD5+CD7+ cell. In some of any of the provided embodiments, the lymphoid differentiation is characterized by increased expression of CD56 relative to a HPC that was not introduced with the DNA-targeting system. In some of any of the provided embodiments, the lymphoid differentiation is characterized by differentiation to a CD56+ cell. In some of any of the provided embodiments, the lymphoid differentiation is characterized by decreased expression of c-KIT relative to a HPC that was not introduced with the DNA-targeting system. In some of any of the provided embodiments, the lymphoid differentiation is characterized by differentiation to a c-KIT- cell.

[0059] Also provided herein is a guide RNA (gRNA) that targets a target site for a lymphoid cell differentiation (LCD) gene selected from the group consisting of TCF7, GATA3 and BCL1 IB. In some of any of the provided embodiments, the target site for the LCD gene is in the gene or a regulatory DNA element thereof. In some of any of the provided embodiments, the target site for RUNX3 comprises the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0060] In some of any of the provided embodiments, the target site for RUNX3 comprises the sequence set forth in any one of SEQ ID NO:99 or SEQ ID NO: 100. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114.

[0061] Provided herein is a gRNA targeting a target site for IL7Ra, wherein the target site for IL7Ra comprises the sequence set forth in SEQ ID NO: 103, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for IL7Ra comprises the sequence set forth in SEQ ID NOS: 103. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 117, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 117.

[0062] Provided herein is a gRNA targeting a target site for TBX21, wherein the target site for TBX21 comprises the sequence set forth in SEQ ID NO: 104, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for TBX21 comprises the sequence set forth in SEQ ID NO: 104. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 118, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119.

[0063] Provided herein is a gRNA targeting a target site for CBFB wherein the target site for CBFB comprises the sequence set forth in SEQ ID NO: 106, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for CBFB comprises the sequence set forth in SEQ ID NO: 106. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 120, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 120.

[0064] Provided herein is a gRNA targeting a target site for LEF1 wherein the target site for LEF1 comprises the sequence set forth in SEQ ID NO: 105, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for LEF1 comprises the sequence set forth in SEQ ID NO: 105. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119.

[0065] Provided herein is a gRNA targeting a target site for MYB, wherein the target site for MYB comprises the sequence set forth in SEQ ID NO: 93 or SEQ ID NO: 94, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for MYB comprises the sequence set forth in SEQ ID NO: 93 or SEQ ID NO: 94. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108.

[0066] Provided herein is a gRNA targeting a target site for RUNX1 wherein the target site for RUNX1 comprises the sequence set forth in SEQ ID NO:95 or SEQ ID NO: 96, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for RUNX1 comprises the sequence set forth in SEQ ID NO:95 or SEQ ID NO: 96. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110.

[0067] Provided herein is a gRNA targeting a target site for SPI1, wherein the target site for SPI1 comprises the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for SPI1 comprises the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116.

[0068] Provided herein is a gRNA targeting a target site for HEY1, wherein the target site for HEY1 comprises the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 98, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for HEY1 comprises the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 98. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112.

[0069] In some of any of the provided embodiments, the regulatory DNA element is an enhancer or a promoter.

[0070] In some of any of the provided embodiments, the target site is selected from: (a) a target site for TCF7 having the sequence set forth in any one of SEQ ID NOS: 1-6, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (b) a target site for GATA3 having the sequence set forth in any one of SEQ ID NOS:7-12, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; and (c) a target site for BCL1 IB having the sequence set forth in any one of SEQ ID NOS: 13-18, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0071] Provided herein is a gRNA that targets a target site for TCF7, wherein the target site has the sequence set forth in any one of SEQ ID NOS: 1-6, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0072] In some of any of the provided embodiments, the target site is selected from: (a) a target site for TCF7 having the sequence set forth in SEQ ID NO: 1, or a complementary sequence thereof; (b) a target site for GATA3 having the sequence set forth in SEQ ID NO: 11, or a complementary sequence thereof; and (c) a target site for BCL1 IB having the sequence set forth in SEQ ID NO: 16, or a complementary sequence thereof. In some of any of the provided embodiments, the target site for TCF7 comprises the sequence set forth in SEQ ID NO: 1, or a complementary sequence thereof.

[0073] In some of any of the provided embodiments, the gRNA is selected from: (a) a gRNA targeting a target site for TCF7 comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 19-24, or a contiguous portion thereof of at least 14 nt; (b) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 25-30, or a contiguous portion thereof of at least 14 nt; and (c) a gRNA targeting a target site for BCLB 11 comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 31-36, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 19-24, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 19-24.

[0074] In some of any of the provided embodiments, the gRNA is selected from: (a) a gRNA targeting a target site for TCF7 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19; (b) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 29; and (c) a gRNA targeting a target site for BCLB 11 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 34. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19. [0075] Provided herein is a gRNA that targets a target site for GATA3 having, wherein the target site comprises the sequence set forth in any one of SEQ ID NOS:7-12, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing, In some of any of the provided embodiments, the target site for GATA3 comprises the sequence set forth in SEQ ID NO: 11, or a complementary sequence thereof. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 25-30, or a contiguous portion thereof of at least 14 nt.

[0076] In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 25-30. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 29.

[0077] Provided herein is a gRNA that targets a target site for BCLB11, wherein the target site for BCL11B comprises the sequence set forth in any one of SEQ ID NOS:13-18, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for BCL1 IB comprises the sequence set forth in SEQ ID NO: 16, or a complementary sequence thereof. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 31-36, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 31-36. In some of any of the provided embodiments, the gRNA targeting a target site for BCLB11 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 34.

[0078] In some of any of the provided embodiments, the gRNA comprises a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length. In some of any of the provided embodiments, the gRNA comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

[0079] In some of any of the provided embodiments, spacer sequence is between 14 nt and 24 nt, or between 16 nt and 22 nt in length. In some of any of the provided embodiments, the spacer sequence is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length. In some of any of the provided embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 56. [0080] In some of any of the provided embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 56. In some of any of the provided embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 91. In some of any of the provided embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 122

[0081] In some of any of the provided embodiments, the gRNA futher comprises 2’ MeO modified bases and/or phosphorothiate backbone modifications.

[0082] Also provided herein is a combination of gRNAs comprising two or more gRNAs, each selected from the gRNA of some of any embodiments. Provided herein is a combination comprising two or more of the provided gRNAs.

[0083] In some of any of the provided embodiments, the combination comprises two, three, four, five, or six different guide RNAs, wherein each gRNA targets a target site of a different LCD genes.

[0084] Provided herein is a combination of gRNAs comprising two or more gRNAs, wherein the combination of gRNAs targets a target site for one or more lymphoid cell differentiation (LCD) genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1.

[0085] In some of any of the provided embodiments, the target site for each of the one or more LCD genes is in the gene or a regulatory DNA element thereof. In some of any of the provided embodiments, the regulatory DNA element is an enhancer or a promoter. In some of any of the provided embodiments, the regulatory DNA element is a promoter of the gene. In some of any of the provided embodiments, the target site for each of the one or more LCD genes is independently within 1000 base pairs of the transcription start site (TSS). In some of any of the provided embodiments, the target site for each of the one or more LCD genes is independently within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS of the gene. In some of any of the provided embodiments, the target site for each of the one or more LCD genes is within 550 base pairs of the TSS of the gene. In some of any of the provided embodiments, the combination comprises two, three, four, five, or six different guide RNAs, wherein each gRNA targets a target site of one of the one or more LCD genes. In some of any of the provided embodiments, the one or more LCD genes are two genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the two genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. . In some of any of the provided embodiments, the one or more LCD genes are three genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the three genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, the one or more LCD genes are four genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the four genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, the one or more LCD genes are five genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPIl,and HEY1; optionally where the five genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, the one or more LCD genes are six genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the six genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

[0086] In some of any of the provided embodiments, at least one of the one or more LCD genes is RUNX3. In some of any of the provided embodiments, at least one of the one or more LCD genes is IL7Ra. In some of any of the provided embodiments, the one or more LCD genes include RUNX3 and IL7Ra. In some of any of the provided embodiments, at least one of the one or more LCD genes is TBX21. In some of any of the provided embodiments, at least one of the one or more LCD genes is CBFB. In some of any of the provided embodiments, at least one of the one or more LCD genes is LEF1. In some of any of the provided embodiments, at least one of the one or more LCD genes is MYB. In some of any of the provided embodiments, at least one of the one or more LCD genes is RUNX1. In some of any of the provided embodiments, at least one of the one or more LCD genes is SPI1. In some of any of the provided embodiments, at least one of the one or more LCD genes is HEY1. In some of any of the provided embodiments, the one or more LCD genes are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; RUNX3, IL7Ra, and TBX21; RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB. In some of any of the provided embodiments, the two or more gRNAs comprise: (a) a first set of gRNAs, wherein the first set of gRNAs target one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, and CBFB; and (b) a second set of gRNAs, wherein the second set gRNAs target one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, the one or more LCD genes targeted by the first set of gRNAs are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; or RUNX3, IL7Ra, and TBX21. In some of any of the provided embodiments, the one or more LCD genes targeted by the second set of gRNAs are: RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB. In some of any of the provided embodiments, the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1 and TBX21; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target RUNX3, CBFB, LEF1 and MYB; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1 and MYB; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1, MYB and TBX21; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target RUNX3, CBFB, LEF1, MYB and TBX21; or the first set of gRNAs target RUNX3 and IL7RA and the second set for gRNAs target RUNX3, CBFB, LEF1, MYB, TBX21 and IL7Ra.

[0087] In some of any of the provided embodiments, the target site for RUNX3 has the sequence set forth in NO: 99 or SEQ ID NO: 100 , a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the gRNA targeting a target site for RUNX3 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the target site for IL7Ra has the sequence set forth in SEQ ID NO: 103, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the gRNA targeting a target site for IL7Ra comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 117, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the target site for TBX21 has the sequence set forth in SEQ ID NO: 104, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the gRNA targeting a target site for TBX21 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 118, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the target site for CBFB has the sequence set forth in SEQ ID NO: 106, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the gRNA targeting a target site for CBFB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 120, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the target site for LEF1 has the sequence set forth in SEQ ID NO: 105, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the gRNA targeting a target site for LEF1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the target site for MYB has the sequence set forth in SEQ ID NO: 93 or SEQ ID NO: 94, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the gRNA targeting a target site for MYB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the target site for RUNX1 has the sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 96, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the gRNA targeting a target site for RUNX1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the target site for SPI1 has the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the gRNA targeting a target site for SPI1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the target site for HEY1 has the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 98, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the gRNA targeting a target site for HEY 1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112, or a contiguous portion thereof of at least 14 nt. [0088] In some of any of the provided embodiments, the two or more guide RNAs independently comprise a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length. In some of any of the provided embodiments, the two or more guide RNAs independently comprise a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length. In some of any of the provided embodiments, the two or more guide RNAs each further comprise a scaffold sequence set forth in SEQ ID NO: 56. In some of any of the provided embodiments, two or more guide RNAs each further comprise a scaffold sequence set forth in SEQ ID NO: 91. In some of any of the provided embodiments, the two or more guide RNAs each further comprise a scaffold sequence set forth in SEQ ID NO: 122. In some of any of the provided embodiments, the two or more gRNAs independently further comprise 2’ MeO modified bases and/or phosphorothiate backbone modifications.

[0089] Provided herein is a Cas-guide RNA (gRNA) combination comprising: (a) a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof fused to a transcriptional activation domain; and (b) the combination of any of the provided gRNAs.

[0090] Provided herein is a Cas-guide RNA (gRNA) combination comprising: (a) a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof fused to a transcriptional activation domain; and (b) one or more of the provided guide RNAs.

[0091] Also provided herein is a Cas-guide RNA (gRNA) combination comprising: (a) a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof fused to a transcriptional activation domain; and (b) one or more guide RNAs, each selected from the gRNA of some of any embodiments.

[0092] In some of any of the provided embodiments, the Cas protein or variant thereof is a deactivated (dCas) protein. In some of any of the provided embodiments, the dCas protein lacks nuclease activity. In some of any of the provided embodiments, the dCas protein is a dCas9 protein.

[0093] In some of any of the provided embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

[0094] In some of any of the provided embodiments, the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 83. In some of any of the provided embodiments, the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 84 or SEQ ID NO: 191, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSaCas9 protein is set forth in SEQ ID NO: 84.

[0095] In some of any of the provided embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

[0096] In some of any of the provided embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 63. In some of any of the provided embodiments, the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 64 or SEQ ID NO: 196, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSpCas9 protein is set forth in SEQ ID NO: 64 or SEQ ID NO: 196.

[0097] Also provided herein is a polynucleotide encoding the DNA-targeting system of some of any embodiments.

[0098] Also provided herein is a polynucleotide encoding at least one DNA-targeting module of the DNA-targeting system of some of any embodiments.

[0099] Also provided herein is a polynucleotide encoding the gRNA some of any embodiments.

[0100] Also provided herein is a polynucleotide encoding the combination of gRNAs of some embodiments.

[0101] Also provided herein is a polynucleotide encoding the Cas-gRNA combination of some of any embodiments.

[0102] Also provided herein is a vector comprising the polynucleotide of some of any embodiments. In some of any of the provided embodiments, the vector is a viral vector. In some of any of the provided embodiments, the vector is a lipid nanoparticle.

[0103] Provided herein is a pharmaceutical composition comprising any of the provided DNA-targeting systems, any of the provided Cas-gRNA combination, any of the provided polynucleotides, or any of the provided vectors. In some of any of the provided embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0104] Provided herein is a method of differentiating a population of hematopoietic progenitor cells (HPCs) to a differentiated population of cells, the method comprising 1 introducing any of the provided DNA-targeting systems, any of the provided Cas-gRNA combination, any of the provided polynucleotides, any of the provided vectors, or a combination thereof, into a population of HPCs, and culturing the HPCs under conditions for their differentiation.

[0105] Provided herein is a method of differentiating a population of hematopoietic progenitor cells (HPCs) to a differentiated population of cells, the method comprising introducing any of the provided pharmaceutical compositions into a population of HPCs, and culturing the HPCs under conditions for their differentiation.

[0106] Also provided herein is a method of differentiating a population of stem cells comprising introducing the DNA-targeting system of some of any embodiments, the gRNA of some of any embodiments, the combination of gRNAs of some embodiments, the Cas-gRNA combination of some of any embodiments, the polynucleotide of some of any embodiments, the vector of some embodiments, or a combination thereof, into a population of stem cells.

[0107] In some of any of the provided embodiments, the population of stem cells differentiate into lymphocytes. In some of any of the provided embodiments, the population of stem cells differentiate into T cells, B cells or natural killer (NK) cells.

[0108] In some of any of the provided embodiments, the population of stem cells are induced pluripotent stem cells (iPSCs). In some of any of the provided embodiments, the population of stem cells are derived from induced pluripotent stem cells (iPSCs). In some of any of the provided embodiments, the population of stem cells are hematopoietic progenitor cells (HPCs). In some of any of the provided embodiments, the population of HPCs are induced hematopoietic progenitor cells (iHPCs). In some of any of the provided embodiments, the population of stem cells are primary hematopoietic progenitor cells.

[0109] In some of any of the provided embodiments, the HPCs comprise cells engineered with a recombinant receptor, optionally a chimeric antigen receptor. In some of any of the provided embodiments, the differentiated cells comprise cells that express a recombinant receptor, optionally a chimeric antigen receptor. In some of any of the provided embodiments, cells of the differentiated population of cells are lymphoid progenitor cells. In some of any of the provided embodiments, the lymphoid progenitor cells are induced common lymphoid progenitor cells (iCLPs). In some of any of the provided embodiments, the introducing decreases expression of CD34 in the differentiated population of cells relative to the population of HPCs. In some of any of the provided embodiments, cells of the differentiated population of cells are CD34- cells.

[0110] In some of any of the provided embodiments, the introducing increases expression of CD45 in the differentiated population of cells relative to the population of HPCs. In some of any of the provided embodiments, cells of the differentiated population of cells are CD45+ cells. In some of any of the provided embodiments, the introducing increases expression of CD7 in the differentiated population of cells relative to the population of HPCs. In some of any of the provided embodiments, cells of the differentiated population of cells are CD7+ cells. In some of any of the provided embodiments, the introducing increases expression of both CD5 and CD7 in the differentiated population of cells relative to the population of HPCs. In some of any of the provided embodiments, cells of the differentiated population of cells are CD5+CD7+ cells. In some of any of the provided embodiments, the introducing increases expression of CD56 in the differentiated population of cells relative to the population of HPCs. In some of any of the provided embodiments, cells of the differentiated population of cell are CD56+ cells. In some of any of the provided embodiments, the introducing decreases expression of c-KIT in the differentiated population of cells relative in the population of HPCs. In some of any of the provided embodiments, cells of the differentiated population of cells are c-KIT" cells.

[0111] In some of any of the provided embodiments, the introducing is by transient delivery into the population of stem cells. In some of any of the provided embodiments, the introducing is by transient delivery into the population of stem HPCs. In some of any of the provided embodiments, the transient delivery comprises electroporation, transfection, or transduction. In some of any of the provided embodiments, the transient delivery comprises transfection using lipid nanoparticles (LNPs). In some of any of the provided embodiments, the transient delivery is repeated at least once.

[0112] In some of any of the provided embodiments, the DNA-targeting system of some of any embodiments, the gRNA of some of any embodiments, the combination of gRNAs of some embodiments, the Cas-gRNA combination of some of any embodiments, the polynucleotide of some of any embodiments, the vector of some embodiments, or a combination thereof, is transiently expressed and/or transiently present in the population of stem cells. In some of any of the provided embodiments, any of the provided the DNA-targeting systems, any of the provided the Cas-gRNAs, any of the provided combinations of gRNAs, any of the provided Cas-gRNA combinations, any of the provided polynucleotides, any of the provided vectors, or a combination thereof, is transiently expressed and/or transiently present in the population of HPCs.

[0113] In some of any of the provided embodiments, the introducing increases transcription of one or more lymphoid cell differentiation (LCD) genes, selected from the group consisting of TCF7, GATA3, and BCL1 IB, in the population of stem cells. In some of any of the provided embodiments, the introducing increases transcription of TCF7, GATA3, and BCL11B in the population of stem cells. In some of any of the provided embodiments, the introducing increases transcription of one or more lymphoid cell differentiation (LCD) genes, selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL1 IB, in the population of HPCs. In some of any of the provided embodiments, the introduced DNA-targeting system comprises at least two DNA-targeting modules and each of the at least two modules are introduced at different times. In some of any of the provided embodiments, at least two DNA-targeting modules is a first set and second set of DNA-targeting modules that are introduced at different times.

[0114] In some of any of the provided embodiments, a) the first set of DNA-targeting modules targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, and CBFB; and (b) a second set of DNA-targeting modules, wherein the second set of DNA-targeting modules targets one or more LCD genes selected from the group consisting ofRUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some of any of the provided embodiments, the one or more LCD genes targeted by the first set of DNA-targeting modules are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; or RUNX3, IL7Ra, and TBX21. In some of any of the provided embodiments, the one or more LCD genes targeted by the second set of DNA-targeting modules are: RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB. In some of any of the provided embodiments, the lymphoid progenitor cells express a recombinant receptor, optionally a chimeric antigen receptor.

[0115] In some of any of the provided embodiments, the introducing decreases expression of CD34 in the population of stem cells.

[0116] In some of any of the provided embodiments, the introducing increases expression of CD56 in the population of stem cells. [0117] Provided herein is a population of lymphoid progenitor cells produced by any of the provided methods. In some of any of the provided embodiments, cells of the differentiated population of cells are lymphoid cells (LCs).

[0118] Also provided herein is a population of differentiated stem cells produced by the method of some of any embodiments.

[0119] Provided herein is a method of generating lymphoid cells (LCs), the method comprising culturing the population of lymphoid progenitor cells produced by any of the provided methods or any of the provided populations of lymphoid progenitor cells under conditions to differentiate cells of the population to lymphoid cells (LCs) to produce a population comprising LCs.

[0120] Also provided herein is a method of treating a disease or condition in a subject, the method comprising administering to the subject the population of differentiated stem cells some embodiments.

[0121] In some of any of the provided embodiments, LCs are induced T (iT) cells, induced B (iB) cells or induced natural killer (iNK) cells. In some of any of the provided embodiments, the LCs are induced Natural Killer (iNK) cells. In some of any of the provided embodiments, the iNK cells are CD56+CD3- cells. In some of any of the provided embodiments, the iNK cells are further characterized by one or more of the following: DNAM1+, NKG2D+, NKP30+ and/or CD16+. In some of any of the provided embodiments, the LCs express a recombinant receptor, optionally a chimeric antigen receptor. In some of any of the provided embodiments, the method is carried out in vitro or ex vivo. In some of any of the provided embodiments, the HPCs are human HPCs.

[0122] Provided herein is a population of differentiated cells produced by any of the provided methods. Provided herein is a population of lymphoid cells (LCs) produced by any of the provided methods.

[0123] Provided herein is a method of treating a disease or condition in a subject, the method comprising administering to the subject any of the provided population of differentiated cells, any of the provided lymphoid progenitor cells, any of the provided populations of lymphoid cells.

Brief Description of the Drawings [0124] FIG. 1 shows results from RT-qPCR to assess expression of BCL11B, GATA3 and TCF7 mRNA in hematopoietic progenitor cells (HPCs) electroporated with dSpCas9-2xVP64 and gRNAs targeting each gene. Expression is shown as fold change relative to Day 1 postelectroporation and normalized to GADPH.

[0125] FIG. 2 shows results from a viability assay at various time points postelectroporation to assess % viability of hematopoietic progenitor cells (HPCs) electroporated with dSpCas9-2xVP64 and gRNAs targeting BCL11B, GATA3 and TCF7.

[0126] FIG. 3 shows results from RT-qPCR to assess expression of BCL11B, GATA3 and TCF7 mRNA in hematopoietic progenitor cells (HPCs) electroporated with dSpCas9-2xVP64 and a gRNA targeting each gene. Expression is shown as fold change relative to Day 1 postelectroporation and normalized to GADPH.

[0127] FIG. 4A shows results from RT-qPCR to assess expression of TCF7 mRNA in hematopoietic progenitor cells (HPCs) electroporated with dSpCas9-2xVP64 and multiplexed gRNAs targeting BCE11B, GATA3 and TCF7. FIG. 4B shows results from RT-qPCR to assess expression of GATA3 mRNA in hematopoietic progenitor cells (HPCs) electroporated with dSpCas9-2xVP64 and multiplexed gRNAs targeting BCE11B, GATA3 and TCF7. FIG. 4C shows results from RT-qPCR to assess expression of BCE1 IB mRNA in hematopoietic progenitor cells (HPCs) electroporated with dSpCas9-2xVP64 and multiplexed gRNAs targeting BCE1 IB, GATA3 and TCF7. Expression is shown as fold change relative to Day 0 postelectroporation and normalized to GADPH. Results are shown at Day 3, Day 6 and Day 9 postelectroporation for cells electroporated at Day 0 and at both Day 0 and Day 3.

[0128] FIG. 5 shows results from a flow cytometry assay to assess CD34 expression for hematopoietic progenitor cells (HPCs) electroporated with dSpCas9-2xVP64 and multiplexed gRNAs targeting BCL11B, GATA3 and TCF7. Results are shown as mean fluorescence intensity (MFI) at Day 6 post-electroporation for cells electroporated at Day 0 and at both Day 0 and Day 3.

[0129] FIG. 6A shows results from a flow cytometry assay to assess CD56 expression for hematopoietic progenitor cells (HPCs) electroporated with dSpCas9-2xVP64 and multiplexed gRNAs targeting BCL11B, GATA3 and TCF7. Results are shown as %CD56+ cells at Day 6 post-electroporation for cells electroporated at Day 0 and at both Day 0 and Day 3. FIG. 6B shows results from a flow cytometry assay to assess CD56 expression for HPCs electroporated with dSpCas9-2xVP64 and multiplexed gRNAs targeting BCL11B, GATA3 and TCF7. Results are shown as %CD56+ cells at Day 9 post-electroporation for cells electroporated at Day 0 and at both Day 0 and Day 3. FIG. 6C shows exemplary flow cytometry plots showing expression of CD56 in HPCs electroporated with dSpCas9-2xVP64 and multiplexed gRNAs targeting BCL1 IB, GATA3 and TCF7 (DNA-Targeting System EP) or a mock control (Mock EP) for cells electroporated at Day 0 and at both Day 0 and Day 3.

[0130] FIG. 7A shows an exemplary workflow for a transient CRISPR activation screen. Hematopoietic Progenitor Cells (HPCs) are transduced with a lentiviral gRNA library and transfected with lipid nanoparticles (LNPs) for delivery of an epi-editor, such as a dCas fused to a transcriptional activator. Cells are then screened for the desired cell surface marker phenotype using flow cytometry. FIG. 7B shows an exemplary flow cytometry plot showing expression of CD45 in HPCs delivered with the gRNA library and epi-editor, with a box indicating the CD45high populations to be sorted for further analysis. FIG. 7C shows an exemplary plot from the CRISPR activation screen for gRNAs and genes that modulate CD45 expression. Dots represent individual gRNAs. gRNAs on the right are those that target genes whose activation results in increased CD45 expression. X-axis represents log2 fold change of gRNA abundance in CD45high sorted cells versus unsorted cells. Y-axis represents significance (-loglO adjusted p- value).

[0131] FIG. 8A shows results from RT-qPCR to assess expression of MYB, RUNX1, HEY1, RUNX3, and SPI mRNA. Hematopoietic progenitor cells (HPCs) were transfected on Day 0 with LNPs for delivery of mRNA encoding dSpCas9-2xVP64 and a combination of three guide RNAs, each targeting a different gene. Expression is shown as fold change relative to a non-targeting (NT) gRNA control. Results are shown for Day 2 post-transfection. FIG. 8B shows results from RT-qPCR to assess expression of IL7Ra, TBX21, LEF1, or CBFB mRNA. Hematopoietic progenitor cells (HPCs) were transfected on Day 0 with LNPs for delivery of mRNA encoding dSpCas9-2xVP64 and a single guide RNA for each respective gene. Expression is shown as fold change relative to a non-targeting (NT) gRNA control. Results are shown for Day 2 post-transfection.

[0132] FIG. 9A shows flow cytometry plots for assessing expression of CD45RA and CD34. HPCs were transfected on Day 0 with LNPs for delivery of mRNA encoding dSpCas9- 2xVP64 with a non-targeting (NT) gRNA or a gRNA targeting HEY1, TBX21, SPI1, or RUNX1. Results are shown for Day 4 post-transfection with a box indicating the CD45RA+CD34- population. FIG. 9B shows flow cytometry plots for assessing expression of CD7 and CD 14. HPCs were transfected on Day 0 with LNPs for delivery of mRNA encoding dSpCas9-2xVP64 with a non-targeting (NT) gRNA or a gRNA targeting HEY1, TBX21, SPI1, or RUNX1. Results are shown for Day 4 post-transfection. FIG. 9C shows flow cytometry plots for assessing expression of CD7 and CD5. HPCs were transfected on Day 0 and Day 4 with LNPs for delivery of mRNA encoding dSpCas9-2xVP64 with a non-targeting (NT) gRNA or a gRNA targeting LEF1 or MYB. Results are shown for Day 7 post-transfection.

[0133] FIGS. 10A-10J show flow cytometry plots for assessing expression of cell surface markers of HPCs transfected on Day 0 and Day 4 with LNPs for delivery of mRNA encoding dSpCas9-2xVP64 and multiplexed gRNAs targeting different combinations of three genes. Results are shown for Day 7 post-transfection. FIG. 10A shows expression of CD45Ra and CD34, CD14 and IL3Ra, CD5 and CD7, and ckit and CD56 for positive control cells cultured in wells coated with Lymphoid Coating Material (LCM). FIG. 10B shows expression of CD45Ra and CD34, CD 14 and IL3Ra, CD5 and CD7, and ckit and CD56 for negative control cells transfected with a non-targeting (NT) gRNA. FIG. 10C shows expression of CD45Ra and CD34, CD 14 and IL3Ra, CD5 and CD7, and ckit and CD56 for cells transfected with gRNAs targeting RUNX3, SPI1, and IL7Ra. FIG. 10D shows expression of CD45Ra and CD34, CD 14 and IL3Ra, CD5 and CD7, and ckit and CD56 for cells transfected with gRNAs targeting RUNX1, HEY1, and TBX21. FIG. 10E shows expression of CD45Ra and CD34, CD14 and IL3Ra, CD5 and CD7, and ckit and CD56 for cells transfected with gRNAs targeting HEY1, IL7Ra, andTBX21. FIG. 10F shows expression of CD45Ra and CD34, CD14 and IL3Ra, CD5 and CD7, and ckit and CD56 for cells transfected with gRNAs targeting MYB, IL7Ra, and LEF1. FIG. 10G shows expression of CD45Ra and CD34, CD14 and IL3Ra, CD5 and CD7, and ckit and CD56 for cells transfected with gRNAs targeting RUNX3, IL7a, and MYB. FIG. 10H shows expression of CD45Ra and CD34, CD 14 and IL3Ra, CD5 and CD7, and ckit and CD56 for cells transfected with gRNAs targeting RUNX3, IL7a, and RUNX1. FIG. 101 shows expression of CD45Ra and CD34, CD 14 and IL3Ra, CD5 and CD7, and ckit and CD56 for cells transfected with gRNAs targeting RUNX3, IL7a, and TBX21. FIG. 10J shows expression of CD45Ra and CD34, CD 14 and IL3Ra, CD5 and CD7, and ckit and CD56 for cells transfected with gRNAs targeting RUNX3, IL7a, and LEF1.

[0134] FIGS. 11A-11C show plots quantifying cell surface marker expression, as assessed by flow cytometry, for cells derived from HPCs. Cells were transfected on Day 0 and Day 4 with LNPs for delivery of mRNA encoding dSpCas9-2xVP64 and multiplexed gRNAs targeting the combinations of genes summarized in Table E5 and subsequently cultured in Natural Killer (NK) cell induction media. HPCs cultured with Lymphoid Coating Material (LCM) prior to culturing in NK cell induction media served a positive control (POS). Cells were sequentially gated for live cells, CD45+EGFRt+, CD56+, and CD16+, DNAM-1+ or NKG2D+. Results are shown for Day 14 post-transfection as percent of parent gate. FIG. 11A shows percentage of CD45+EGFRt+, CD56+, CD16+, DNAM-1+ and NKG2D+ cells, as assessed by flow cytometry, for cells transfected with gRNA combinations that targeted RUNX3, IL7Ra, and TBX21 on Day 0 (Group 1). FIG. 11B shows percentage of CD45+EGFRt+, CD56+, CD16+, DNAM-1+ and NKG2D+ cells, as assessed by flow cytometry, for cells transfected with gRNA combinations that targeted RUNX3, IL7Ra, and CBFB on Day 0 (Group 2). FIG. 11C shows percentage of CD45+EGFRt+, CD56+, CD16+, DNAM-1+ and NKG2D+ cells, as assessed by flow cytometry, for cells transfected with gRNA combinations that targeted RUNX3 and IL7Ra, on Day 0 (Group 3).

[0135] FIG. 12A shows exemplary data for a cytotoxicity co-culture assay using H1975 tumor cells and induced Natural Killer (iNK) cells. iNK cells were derived from HPCs transfected on Day 0 and Day 4 with LNPs for delivery of mRNA encoding dSpCas9-2xVP64 and multiplexed gRNAs targeting the combinations of genes summarized in Table E5 and subsequently cultured in Natural Killer (NK) cell induction media. Results are shown for cells transfected with gRNA combinations that targeted RUNX3 and IL7Ra, on Day 0 (combinations 3-1 through 3-6). HPCs cultured with Lymphoid Coating Material (LCM) prior to culturing in NK cell induction media served as a positive control (POS). The H1975 tumor cell line was cultured alone (target alone) as a negative control. FIG. 12B shows a zoomed-in version of FIG. 12A to highlight the gene combinations that resulted in robust tumor killing capacity similar to the positive control cells.

Detailed Description

[0136] Provided herein are DNA-targeting systems for promoting transcriptional activation of genes for promoting differentiation of hematopoietic progenitor cells (HPCs) into a differentiated population of cells, such as into lymphoid progenitor cells or lymphoid cells. In some embodiments, the DNA-targeting systems are based on epiediting systems that promote the transcriptional activation of lymphoid cell differentiation (LCD) genes as described. The DNA-binding systems include DNA-targeting modules that comprise a DNA-binding domain that binds to a target site for a gene as described, and (ii) at least one transcriptional activator effector domain. In some embodiments, the DNA-targeting systems include a plurality of DNA- targeting modules that each target transcriptional activation to increase transcription of different genes for multiplexed transcriptional activation.

[0137] Provided herein is a DNA-targeting system in which the DNA-targeting system comprises one or more DNA-targeting modules, each composed of a fusion protein comprising: (a) a DNA-binding domain that binds to a target site for one or more lymphoid cell differentiation (LCD) genes; and (b) at least one transcriptional activation domain that increases transcription of the one or more LCD genes. In some embodiments, the one or more LCD are genes that lead to differentiation of hematopoietic progenitor cells (HPCs) into lymphoid cells. In some embodiments, the increased transcription of the one or more LCD genes lead to differentiation of HPCs into lymphoid cells. In some embodiments, the one or more LCD genes are transcription factor genes. Results herein demonstrate that targeting such genes for transcriptional activation using the DNA-targeting systems herein results in an increase in expression of the transcription factor for modulating and promoting differentiation of stem cells, such as HPCs, into lymphoid cells. The differentiation into lymphoid cells can be characterized by expression of CD45, CD7, CD5 and/or CD56. In some embodiments, the one or more transcription factor genes are genes that lead to upregulation of CD45, CD7, CD5, and/or CD56.

[0138] In some aspects, the LCD genes are genes that are activated by Notch signaling, and targeting one or more such genes by provided DNA-targeting systems can bypass the need for an extrinsic Notch signal to facilitate stem cell differentiation. Such DNA-targeting systems may provide a useful alternative to protocols that rely on the provision of extrinsic signals, such as Notch ligands. The DNA-targeting systems provide several potential advantages, including reduced cost, reduced hands-on time for cell-culture, and greater ability to scale up differentiation protocols to larger numbers of cells.

[0139] In some embodiments, the one or more LCD genes is selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3 and BCL1 IB. In some embodiments, the one or more LCD genes is selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the one or more LCD genes is selected from the group consisting of TCF7, GATA3 and BCL1 IB. In some embodiments, the target site may be within a regulatory region, such as a promoter or enhancer of a LCD gene.

[0140] In some embodiments, the DNA-targeting systems are synthetic transcription factors that can increase (or upregulate) transcription of a gene in a targeted manner. In some embodiments, the DNA-binding domain of the DNA-targeting system is a nuclease-inactive Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein (e.g., a dCas protein) or variant thereof complexed with a guide RNA (gRNA). Also provided are gRNAs for targeting to a target site in a LCD gene or a regulatory DNA element thereof. Also provided are CRISPR-Cas/gRNA combinations thereof composed of the gRNA and a nuclease inactivated Cas, such as a dCas9. Also provided herein are polynucleotides encoding the DNA- targeting system or the fusion protein of the DNA-targeting system, and vectors and cells containing the same. Also provided herein are methods of using the DNA-targeting system for activating transcription of LCD genes in stem cells and promoting lymphoid cell differentiation.

[0141] In some embodiments, the DNA-targeting system includes a single DNA-targeting module for targeting activation or increased expression of a single gene. In some embodiments, the gene is RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, or HEY1. In some embodiments, the gene is TCF7, GATA3 or BCL1 IB. In some embodiments, the DNA- targeting system includes a plurality of DNA-targeting modules, in which each DNA-targeting module is for targeting activation or increased expression of a different gene. In some embodiments, the DNA-targeting systems are multiplexed DNA-targeting systems, i.e. targeted to target sites for more than one gene. Hence, the terms DNA-targeting system may include a multiplexed DNA targeting system that includes more than one DNA-targeting module. A multiplexed DNA targeting system comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 DNA-targeting modules. In some embodiments, the plurality of DNA-targeting modules target activation of 2 or 3 genes, selected from RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the plurality of DNA-targeting modules target activation of 2 or 3 genes, selected from TCF7, GATA3, and BCL11B.

[0142] In some embodiments, the plurality of DNA-targeting modules comprises a first set and second set of DNA-targeting modules, in which each set of DNA-targeting modules is for targeting increased activation or increased expression of a different set of genes. In some embodiments, the plurality of DNA-targeting modules comprises a first set and second set of DNA-targeting modules, in which each set of DNA-targeting modules is for targeting increased activation or increased expression of an overlapping set of genes. In some embodiments, the first and second set of DNA-targeting modules each target 2, 3, 4, 5, or 6 six genes selected from RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the first set of DNA-targeting modules target activation of 2 or 3 genes, selected from RUNX3, IL7Ra, TBX21, and CBFB. In some embodiments, the second set of DNA targeting modules targets activation of 2, 3, 4, 5, or 6 genes, selected from RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

[0143] In some embodiments, any two DNA-targeting modules of a DNA-targeting system comprise separate (i.e. non-overlapping) components. In some embodiments, different DNA- targeting modules of a DNA-targeting system comprise separate (i.e. non-overlapping) components. For example, a DNA-targeting system may comprise a first DNA-targeting module comprising a first fusion protein comprising a DNA-binding domain (e.g. a ZFN or TALE-based DNA-binding domain) that targets a first target site, and a second DNA-targeting module comprising a second fusion protein comprising a second DNA-binding domain (e.g. a ZFN or TALE-based DNA-binding domain) that targets a second target site.

[0144] In some embodiments, any two DNA-targeting modules of a DNA-targeting system may comprise shared (i.e. overlapping) components. In some embodiments, different DNA- targeting modules of a DNA-targeting system comprise shared (i.e. overlapping) components. For example, in one aspect, a DNA-targeting system may comprise a first DNA-targeting module comprising (a) a fusion protein comprising a Cas protein and a transcriptional effector (e.g. activation) domain, and (b) a first gRNA that complexes with the Cas protein and targets a first target site, and a second DNA-targeting module comprising (a) the fusion protein of the first DNA-targeting module, and (b) a second gRNA that complexes with the Cas protein and targets a second target site. It will be understood that providing two or more different gRNAs for a given Cas protein allows the same Cas protein to be targeted to the target sites of the two or more gRNAs. Conversely, different Cas protein variants (e.g. SpCas9 and SaCas9) are compatible with different gRNA scaffold sequences and PAMs. Thus, it is possible to engineer a single DNA-targeting system comprising multiple non-overlapping CRISPR/Cas-based DNA- targeting modules.

[0145] The provided embodiments relate to compositions and methods for promoting lymphoid cell differentiation through transcriptional activation of one or more LCD genes. In some embodiments, the methods can be used in connection with adoptive cell therapies. In some embodiments, activating transcription of the one or more LCD genes modulates the expression of cell-surface makers. In some embodiments, expression of a cell-surface marker is decreased, such as CD34 which is characteristic of hematopoietic progenitor cells. In some embodiments, expression of a cells surface marker is increased, such as CD45 which is characteristic of lymphoid cells. In some embodiments, expression of a cell surface marker is increased, such as CD7 and/or CD5 which are characteristic of T-cells. In some embodiments, expression of a cell-surface marker is increased, such as CD56 which is characteristic of natural killer (NK) cells. In some embodiments, activation of the one or more LCD genes promotes stem cell differentiation into lymphoid cells without the provision of extrinsic signaling ligands.

[0146] The ability to differentiate various cell types from progenitor cells such as induced pluripotent stem cells (iPSCs) has tremendous therapeutic potential, including in regenerative medicine and adoptive cell therapy. Current protocols for differentiating stem cells into useful cell types and lineages, such as lymphoid cells, include providing extrinsic signals to stimulate differentiation. For example, iPSCs are cultured in the presence of the Notch ligands (e.g. Deltalike protein 4 (DLL4) or Delta-like protein 1 (DLL1)), which can be provided immobilized on beads, coated on a surface, or expressed by feeder cells. The Delta ligands stimulate Notch signaling by inducing a mechanical shear force that catalyzes the release of the Notch receptor intracellular domain, which upregulates Notch target genes. Upregulation of Notch target genes (e.g., TCF7, GATA3, and BCL11B) promotes differentiation of stem cells, for example into lymphoid cells, such as NK cells or T cells.

[0147] However, provision of extrinsic Notch ligands such as DLL4 presents several challenges. Not only are the ligands costly and difficult to source, but the ligands must also be bound to a surface to induce the mechanical shear force necessary to stimulate Notch signaling. Since the ligands need to be coated on the surface of beads or microwells during cell culture, this makes scaling to larger cultivation systems (e.g., large-scale bioreactors) very difficult. Moreover, depleting the coated material from the culture poses an additional challenge.

[0148] The provided embodiments can be used to directly upregulate Notch target genes (i.e., “lymphoid cell differentiation (LCD) genes”) to promote the differentiation of stem cells into lymphoid cells thereby eliminating the need to provide extrinsic signaling ligands. In particular, the provided embodiments provide for DNA-targeting systems and methods that can be used to upregulate LCD gene expression in stem cells and promote lymphoid cell differentiation. Results herein demonstrate the surprising result that the introduction of the provided DNA-targeting systems into stem cells activates transcription of LCD genes to biologically relevant levels. This approach circumvents the challenges associated with providing stem cells with extrinsic signaling ligands to stimulate differentiation. Moreover, the targeted gene activation does not modify DNA at the sequence level, thereby avoiding safety concerns with gene editing approaches. The ability to stimulate lymphoid cell differentiation using targeted gene activation provides an advantageous approach for producing highly functional cells for immunotherapy at scale.

[0149] All sequences of U.S. provisional applications No. 62/532,347 filed August 11, 2023 (SEQ ID NOs: 1-91) and U.S. provisional application No. 63/669,228 filed July 9, 2024 (SEQ ID NOs: 1-122), are incorporated by reference in their entireties.

[0150] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0151] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. DNA-TARGETING SYSTEMS

[0152] In some embodiments, provided are DNA-targeting systems capable of specifically targeting a target site for one or more lymphoid cell differentiation (LCD) genes, and activating transcription of the one or more LCD genes. In some embodiments, the target site for an LCD gene is a target site in the gene or a regulatory DNA element thereof. In provided embodiments, for each LCD gene that is targeted, the DNA-targeting system includes a fusion protein that comprises a DNA-binding domain that binds to the target site for the gene, and at least one effector domain for activating transcription of the gene. In some embodiments, the provided DNA-targeting systems increase transcription of the one or more LCD genes in a stem cell. In some embodiments, transcriptional activation of gene expression by the DNA-targeting systems provided herein can promote lymphoid progenitor cell differentiation. In some embodiments, transcriptional activation of gene expression by the DNA-targeting systems provided herein can promote lymphoid cell differentiation. In some embodiments, transcriptional activation of target genes by the DNA-targeting systems provided herein can promote expression of CD45, CD7, CD5, and/or CD56.

[0153] In some embodiments, the at least one effector domain is a transcriptional activation domain for increasing transcription of the one or more LCD genes (e.g. activates or increases transcription of the one or more genes as compared to transcription of the gene in the absence of the DNA-targeting system), such as any effector domain for transcriptional activation described in Section I.E. In some embodiments, the effector domain is a transcriptional activator effector domain, and the one or more LCD genes are genes that when activated lead to increased expression of CD45, CD7, CD5, and/or CD56 in the cell. In some embodiments, the effector domain is a transcriptional activator effector domain, and the one or more genes are selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the effector domain is a transcriptional activator effector domain, and the one or more genes are selected from the group consisting of: TCF7, GATA3 and BCL11B.

[0154] In some embodiments, the effector domain directly or indirectly leads to increased transcription of the gene. In some embodiments, the effector domain induces, catalyzes or leads to transcription activation. In some embodiments, the effector domain induces transcription activation. In some aspects, the effector domain comprises: a VP64 domain, a p65 activation domain, a p300 domain, an Rta domain, a CBP domain, a VPR domain, a VPH domain, an HSF1 domain, a TET protein domain, optionally wherein the TET protein is TET1, a SunTag domain, or a domain, portion, variant, or truncation of any of the foregoing. In some embodiments, the effector domain is VP64. In some embodiments, the effector domain includes a NCOA3 domain, a FOXO3 domain or is a fusion of NCOA3 and FOXO3 domains. In some embodiments, such a fusion may further include a VP64 domain.

[0155] In some embodiments, the DNA-targeting system includes a fusion protein comprising (a) at least one DNA-binding domain capable of being targeted to the target site; and (b) at least one effector domain capable of modulating transcription of the gene. In some embodiments, the at least one effector domain is a transcriptional activation domain. The fusion protein can be any suitable fusion protein, for example as described in Section I.F.

[0156] In some embodiments, the DNA-binding domain comprises or is derived from a CRISPR associated (Cas) protein, a zinc finger protein (ZFP), a transcription activator-like effector (TALE), meganuclease, homing endonuclease, LScel enzyme, or variants thereof. In some embodiments, the DNA-binding domain comprises a catalytically inactive (e.g. nucleaseinactive or nuclease-inactivated) variant of any of the foregoing. In some embodiments, the DNA-binding domain comprises a deactivated Cas9 (dCas9) protein or variant thereof that is a catalytically inactivated so that it is inactive for nuclease activity and is not able to cleave the DNA. The DNA-binding domain can be any suitable DNA-binding domain, for example as described in Sections I.C and I.D.

[0157] In some embodiments, the DNA-binding domain comprises or is derived from a Cas protein or variant thereof, such as a nuclease-inactive Cas or dCas (e.g. dCas9, and the DNA- targeting system comprises one or more guide RNAs (gRNAs), such as a combination of gRNAs (e.g. two gRNAs or three gRNAs). In some embodiments, the gRNA comprises a spacer sequence that is capable of targeting and/or hybridizing to the target site. In some embodiments, the gRNA is capable of complexing with the Cas protein or variant thereof. In some aspects, the gRNA directs or recruits the Cas protein or variant thereof to the target site. The gRNA can be any suitable gRNA, for example as described in section I.C.2.

[0158] In some embodiments, the DNA-targeting system is for increasing transcription of one or more genes, such as any described in Section I.B., and the fusion protein of a DNA- targeting module thereof is a dCas9-VP64 fusion protein, such as a dCas9-2xVP64 fusion protein. In some embodiments, the fusion protein is any as described herein, for example in Section I.F.

[0159] Exemplary components and features of the DNA-targeting systems are provided below in the following subsections.

A. DNA-Targeting Modules and Multiplexed DNA-Targeting Systems

[0160] In some embodiments, the DNA-targeting system contains one or more DNA- targeting modules, where each DNA-targeting module of the system is a component of the DNA-targeting system that is independently capable of targeting one target site for a target gene. In some embodiments, each DNA-targeting module includes (a) a DNA-binding domain capable of being targeted to the target site, and (b) an effector domain for modulating transcription of the gene. In some embodiments, the DNA-targeting system comprises a single DNA-targeting module for targeted transcriptional modulation of a single gene.

[0161] In some embodiments, the DNA-binding domain of the at least one DNA-targeting module comprises: a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein; a zinc finger protein (ZFP); a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an LScel enzyme or a variant thereof, optionally wherein the DNA- binding domain comprises a catalytically inactive variant of any of the foregoing, wherein when the DNA-binding domain of each fusion protein comprises a Cas protein, the DNA-targeting system further comprises one or more gRNAs, each capable of targeting the Cas protein to a target site.

[0162] In some embodiments, a DNA-targeting module is a CRISPR/Cas-based DNA- targeting module. In some embodiments, in a CRISPR/Cas-based DNA-targeting module, the DNA-binding domain of the fusion protein is a Cas protein or variant thereof (e.g. a dCas protein, such as dCas9) and the DNA-targeting module further comprises a gRNA for targeting the DNA-binding domain to the target site.

[0163] In some embodiments, a DNA-targeting module is a zinc finger protein (ZFP) -based DNA-targeting module. In some embodiments, in a ZFP-based DNA-targeting module, the DNA-binding domain of the fusion protein is an engineered zinc finger protein (eZFP).

[0164] In some embodiments, a DNA-targeting module is a transcription activator-like effector (TALE) -based DNA-targeting module. In some embodiments, in a TALE-based DNA- targeting module, the DNA-binding domain of the fusion protein is an engineered TALE.

[0165] In some embodiments, the DNA-targeting system includes a plurality of DNA- targeting modules, in which each DNA-targeting module targets a different target site. In some embodiments, one or more target sites are for different genes. In some embodiments, one or more target sites are for the same gene. In some embodiments, the plurality of DNA-targeting modules includes a first set and a second set of DNA-targeting modules, in which each set of DNA-targeting modules targets different target sites. In some embodiments, the plurality of DNA-targeting modules includes a first set and a second set of DNA-targeting modules, in which each set of DNA-targeting modules targets overlapping target sites. In some embodiments, the DNA-targeting system is a multiplexed DNA-targeting system, i.e. is targeted to target sites for more than one gene. Hence, the term DNA-targeting system may include a multiplexed epigenetic-modifying DNA targeting system that includes more than one DNA- targeting module. In some embodiments, a multiplexed epigenetic-modifying DNA targeting system comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, or more DNA-targeting modules. In some embodiments, a multiplexed epigenetic-modifying DNA-targeting system comprises 2 DNA-targeting modules. In some embodiments, a multiplexed epigenetic-modifying DNA-targeting system comprises 3 DNA-targeting modules. In some embodiments, a multiplexed epigenetic-modifying DNA- targeting system comprises 4 DNA-targeting modules. In some embodiments, a multiplexed epigenetic-modifying DNA-targeting system comprises 5 DNA-targeting modules. In some embodiments, a multiplexed epigenetic -modifying DNA-targeting system comprises 6 DNA- targeting modules.

[0166] In some embodiments, any two DNA-targeting modules of a DNA-targeting system can comprise separate (i.e. non-overlapping) components. For example, a DNA-targeting system may comprise a first DNA-targeting module comprising a first fusion protein with a DNA- binding domain (e.g. a ZFN or TALE-based DNA-binding domain) that targets a first target site, and a second DNA-targeting module comprising a second fusion protein with a second DNA- binding domain (e.g. a ZFN or TALE-based DNA-binding domain) that targets a second target site.

[0167] In some embodiments, any two DNA-targeting modules of a DNA-targeting system can comprise shared (i.e. overlapping) components. For example, a DNA-targeting system may comprise: i) a first DNA-targeting module comprising (a) a fusion protein comprising a Cas protein and an effector domain, and (b) a first gRNA that complexes with the Cas protein and targets a first target site, and ii) a second DNA-targeting module comprising (a) the fusion protein of the first DNA-targeting module, and (b) a second gRNA that complexes with the Cas protein and targets a second target site. It will be understood that providing two or more different gRNAs for a given Cas protein allows the Cas protein to be targeted to the target sites of the two or more gRNAs. Conversely, different Cas protein variants (e.g. SpCas9 and SaCas9) are compatible with different gRNA scaffold sequences and PAMs, as described herein. Thus, it is possible to engineer a single DNA-targeting system comprising multiple non-overlapping CRISPR/Cas-based DNA-targeting modules.

[0168] In some aspects, provided herein is an epigenetic-modifying DNA-targeting system comprising a plurality of DNA-targeting modules for modulating transcription of one or more genes. In some embodiments, the plurality of DNA-targeting modules comprises a first DNA- targeting module for modulating transcription of a first gene of the one or more genes, and a second DNA-targeting module for modulating transcription of a second gene of the one or more genes. In some embodiments, the plurality of DNA-targeting modules comprises a first set of DNA-targeting modules for modulating transcription of a first set of genes, and a second set of DNA-targeting modules for modulating transcription of a second set of genes. In some embodiments, the first set of genes and the second set of genes comprise different genes. In some embodiments, the first set of genes and the second set of genes comprise overlapping genes. In some embodiments, each DNA-targeting module comprises a fusion protein comprising: (a) a DNA-binding domain for targeting a target site of the target gene for the DNA-targeting module, and (b) at least one effector domain. In some embodiments, each DNA- targeting module comprises a transcriptional activator effector domain for increasing transcription of the one or more genes.

B. Target Genes and Target Sites For Promoting Lymphoid Cell Differentiation

[0169] In some aspects, provided herein are target sites in one or more lymphoid cell differentiation (LCD) genes in which modulation of one or more genes promotes lymphoid cell differentiation. In embodiments as described, the one or more LCD genes are transcription factor genes. In some embodiments, the target site is targeted using any of the provided DNA- targeting systems.

[0170] In some embodiments, the target site is in a gene in which increased expression of the gene promotes lymphoid cell differentiation, such as any one or more of the target genes described herein. In some embodiments, provided herein are target sites for one or more genes for which increased transcription promotes lymphoid cell differentiation. In some embodiments, the target site is a target site in an LCD gene selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3 and BCL11B. In some embodiments, the target site is a target site in an LCD gene selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the target site is a target site in an LCD gene selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the target site is a target site in an LCD gene selected from the group consisting of TCF7, GATA3, and BCL1 IB.

[0171] In some embodiments, the target site is targeted by a DNA-targeting system, such as by a DNA-targeting module of the DNA-targeting system, such as any described herein. In some embodiments, the target site for a gene is in the gene or a regulatory DNA element thereof. In some embodiments, provided herein are multiplexed DNA-targeting systems that target a combination of at least two LCD genes or regulatory DNA elements thereof described herein.

[0172] In some embodiments, the DNA-targeting system targets to or binds to a target site in a gene, such as any described herein. In some embodiments, the target site is located in the gene and/or a regulatory DNA element of the gene. In some embodiments, a regulatory DNA element is a sequence to which a gene regulatory protein may bind and affect transcription of the gene. In some embodiments, the regulatory DNA element is a cis, trans, distal, proximal, upstream, or downstream regulatory DNA element of a gene. In some embodiments, the regulatory DNA element is a promoter or enhancer of the gene. In some embodiments, the target site is located within a promoter, enhancer, exon, intron, untranslated region (UTR), 5’ UTR, or 3’ UTR of the gene. In some embodiments, the regulatory DNA element is a promoter. In some embodiments, a promoter is a nucleotide sequence to which RNA polymerase binds to begin transcription of the gene. In some embodiments, a promoter is a nucleotide sequence located within about 25bp, 50bp, 75bp, lOObp, about 500bp, about lOOObp, or more, of a transcriptional start site of the gene. In some embodiments, a promoter is within 550bp of a transcriptional start site of the gene. In some embodiments the target site is located within a sequence of unknown or known function that is suspected of being able to control expression of a gene.

[0173] In some embodiments, the DNA-targeting system targets to or binds within about 25bp, within about 50bp, within about 75bp, within about lOObp, within about 250bp, within about 500bp, within about, 750bps, or within about lOOObp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds about 25bp- lOOObp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 25bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 50bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 75bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about lOObp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 250bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA- targeting system targets to or binds within about 500bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 750bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about lOOObp upstream of the transcriptional start site of the LCD gene. In some embodiments, the target site is within the promoter of the LCD gene. [0174] In some embodiments, the DNA-targeting system targets to or binds within about 20bp, within about 50bp, within about lOObp, within about 200bp, within about 300bp, within about 400bp, within about 550bp, or within about 600bps upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds about 20bp-600bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 20bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 50bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about lOObp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 200bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 300bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA- targeting system targets to or binds within about 400bp upstream of the transcriptional start site of the LCD gene In some embodiments, the DNA-targeting system targets to or binds within about 550bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about 600bp upstream of the transcriptional start site of the LCD gene. In some embodiments, the DNA-targeting system targets to or binds within about lOOObp upstream of the transcriptional start site of the LCD gene.

[0175] In some embodiments, delivery of the DNA-targeting system increases transcription of one or more transcription factor genes leading to increased expression of CD45, CD7, CD5, and/or CD56 on the target cell. In some embodiments, delivery of the DNA-targeting system increases transcription of one or more LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B. In some embodiments, delivery of the DNA-targeting system increases transcription of one or more LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, delivery of the DNA- targeting system increases transcription of any combination of three different LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, delivery of the DNA-targeting system increases transcription of RUNX3 and IL7Ra, and one or more LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, delivery of the DNA-targeting system increases transcription of RUNX3, IL7Ra, and TBX21, and one or more LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, delivery of the DNA-targeting system increases transcription of RUNX3, IL7Ra, and CBFB, and one or more LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, delivery of the DNA- targeting system increases transcription of LCD genes in any of the combinations listed in Table E5. In some embodiments, delivery of the DNA-targeting system increases transcription of one or more LCD genes selected from the group consisting of: TCF7, GATA3, and BCL1 IB.

[0176] In some embodiments, the DNA-targeting system targets a target site. In some embodiments, the DNA-targeting system targets a target site for one or more LCD genes, wherein the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3 and BCL11B. In some embodiments, the DNA-targeting system targets a target site for one or more LCD genes, wherein the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the DNA- targeting system targets a target site for 2 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the DNA- targeting system targets a target site for 3 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the DNA- targeting system targets a target site for 4 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the DNA- targeting system targets a target site for 5 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the DNA- targeting system targets a target site for 6 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1.

[0177] In some embodiments, the DNA-targeting system targets a target site for any combination of three different LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the DNA- targeting system targets a target site for a first LCD gene, a second LCD gene and a third LCD gene. In some embodiments the first, second, and third LCD gene are independently selected from the group consisting of RUNX3, IL7Ra, TBX21, LEF1, MYB, RUNX1, SPI1, and HEY1. In some embodiments, the first and second LCD gene are RUNX3 and IL7Ra and the third LCD gene is selected from the group consisting of: TBX21, LEF1, MYB, RUNX1, SPI1, and HEYL

[0178] In some embodiments, the DNA-targeting system targets a target site for 2 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the DNA-targeting system targets a target site for 3 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the DNA- targeting system targets a target site for 4 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the DNA-targeting system targets a target site for 5 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the DNA-targeting system targets a target site for 6 or more of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the DNA-targeting system targets a target site for each of the following LCD genes: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

[0179] In some embodiments, the DNA-targeting system targets a target site for RUNX3, IL7Ra, and TBX21, and one or more LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the DNA-targeting system targets a target site for RUNX3, IL7Ra, and CBFB, and one or more LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the DNA-targeting system targets a target site for RUNX3 and IL7Ra, and one or more LCD genes selected from the group consisting of: RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, the DNA-targeting system targets a target site for any of the combinations of LCD genes listed in Table E5.

[0180] In some embodiments, the DNA-targeting system targets a target site for each of the following LCD genes: RUNX3 and IL7Ra. In some embodiments, the DNA-targeting system targets a target site for each of the following LCD genes: RUNX3, IL7Ra, and CBFB. In some embodiments, the DNA-targeting system targets a target site for each of the following LCD genes: RUNX3, IL7Ra, and TBX21. In some embodiments, the DNA-targeting system targets a target site for each of the following LCD genes: RUNX3, CBFB, LEF1, MYB, TBX21,and IL7Ra. In some embodiments, the DNA-targeting system targets a target site for each of the following LCD genes: RUNX3, CBFB, LEF1, MYB, and TBX21. In some embodiments, the DNA-targeting system targets a target site for each of the following LCD genes: RUNX3, CBFB, LEF1, and MYB. In some embodiments, the DNA-targeting system targets a target site for each of the following LCD genes: LEF1, MYB, and TBX21. In some embodiments, the DNA-targeting system targets a target site for each of the following LCD genes: LEF1 and TBX21. In some embodiments, the DNA-targeting system targets a target site for each of the following LCD genes: LEF1 and MYB.

[0181] In some embodiments, the DNA-targeting system targets a target site for one or more LCD genes, wherein the one or more LCD genes are selected from the group consisting of TCF7, GATA3, and BCL11B. In some embodiments, the DNA-targeting system targets a target site for at least a first gene and a second gene, wherein the first and second gene are independently selected from the group consisting of TCF7, GATA3, and BCL11B. In some embodiments, the DNA-targeting system targets a target site for TCF7, GATA3, and BCL1 IB. In some embodiments, the DNA-targeting system targets a target site for one or more genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B. In some embodiments, the target site comprises a sequence selected from any one of SEQ ID NOS: 1-18 and 93-106, or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOS: 1-18 and 93-106 that is 15, 16, 17, 18 or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence described herein above. In some embodiments, the target site is the sequence set forth in any one of SEQ ID NOS: 1-18 and 93-106.

[0182] In some embodiments, the DNA-targeting system targets a target site for RUNX3. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 99. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 100. [0183] In some embodiments, the DNA-targeting system targets a target site for IL7Ra. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 103, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 103, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 103.

[0184] In some embodiments, the DNA-targeting system targets a target site for TBX21. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 104, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 104, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 104.

[0185] In some embodiments, the DNA-targeting system targets a target site for CBFB. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 106, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 106, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 106.

[0186] In some embodiments, the DNA-targeting system targets a target site for LEF1. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 105, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 105, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 105.

[0187] In some embodiments, the DNA-targeting system targets a target site for MYB. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 93 or SEQ ID NO: 94, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 93 or SEQ ID NO: 94, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 93 or SEQ ID NO: 94. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 93. In some embodiments, the target site comprises the sequence set forth in SEQ

ID NO: 94.

[0188] In some embodiments, the DNA-targeting system targets a target site for RUNX1. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 96, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 96, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 96. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 95. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 96.

[0189] In some embodiments, the DNA-targeting system targets a target site for SPI1. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 101. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 102.

[0190] In some embodiments, the DNA-targeting system targets a target site for HEY1. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 98, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 98, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 98. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 97. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 98.

[0191] In some embodiments, the DNA-targeting system targets a target site for TCF7. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 1-6, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 1-6, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 1-6. In some embodiments, the DNA-targeting system targets a target site for TCF7 comprising the sequence set forth in SEQ ID NO:1.

[0192] In some embodiments, the DNA-targeting system targets a target site for GATA3. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 7- 12, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 7-12, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 7-12. In some embodiments, the DNA-targeting system targets a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 11.

[0193] In some embodiments, the DNA-targeting system targets a target site for BCL1 IB. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 13-18, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 13-18, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in any of SEQ ID NOs: 13-18. In some embodiments, the DNA-targeting system targets a target site for BCL1 IB comprising the sequence set forth in SEQ ID NO: 16.

[0194] In some embodiments, the DNA-targeting system targets a target site for TCF7 comprising the sequence set forth in SEQ ID NO:1, a target site for GATA3 comprising the sequence set forth in SEQ ID NO: 11, and a target site for BCL1 IB comprising the sequence set forth in SEQ ID NO: 16.

[0195] In some embodiments, the target site for RUNX3 is located within 550bp of human genome assembly GRCh38 (hg38) genomic coordinates chrl: 24,930,276. In some embodiments the target site for RUNX3 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl: 24,930,911- 24,929,930. In some embodiments the target site for RUNX3 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl: 24,930,661- 24,930,180. In some embodiments the target site for RUNX3 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl: 24,930,511- 24,930,330. In some embodiments the target site for RUNX3 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl: 24,930,461- 24,930,380. In some embodiments the target site for RUNX3 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl: 24,930,411- 24,930,430. In some embodiments the target site for RUNX3 is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrl: 24,965,172- 24,965,191.

[0196] In some embodiments, the target site for IL7Ra is located within 550bp of human genome assembly GRCh38 (hg38) genomic coordinates chr5: 35,856,891. In some embodiments the target site for IL7Ra is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr5: 35,857,258- 35,856,277. In some embodiments the target site for IL7Ra is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr5: 35,857,008- 35,856,527. In some embodiments the target site for IL7Ra is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr5: 35,856,858- 35,856,677. In some embodiments the target site for IL7Ra is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr5: 35,856,808- 35,856,727. In some embodiments the target site for IL7Ra is located within the genomic coordinates human genome assembly GRCh38 (hg38) chr5: 35,856,758- 35,856,777.

C. CRISPR/Cas-Based DNA-Targeting Systems and DNA-Binding Domains

[0197] In some embodiments, any of the provided DNA-targeting systems are based on CRISPR/Cas systems, i.e. CRISPR/Cas-based DNA-targeting systems, that are able to bind to a target site for a target gene as described, or to a combination of target sites, e.g. for a combination of target genes as escribed. In some embodiments, the CRISPR/Cas DNA-binding domain is nuclease inactive, such as includes a dCas (e.g. dCas9) so that the system binds to the target site for a target gene without mediating nucleic acid cleavage at the target site. The CRISPR/Cas-based DNA-targeting systems may be used to modulate expression of a target gene in a cell, such as a T cell. In some embodiments, the target gene may include any as described herein, including any described above in Section I.B. In some embodiments, the target site for the target gene may include any as described herein, including any described above in Section I.B. In some embodiments, the CRISPR/Cas-based DNA-targeting system can include any known Cas enzyme, and generally a nuclease-inactive or dCas. In some embodiments, the CRISPR/Cas-based DNA-targeting system includes a fusion protein of a nuclease-inactive Cas protein or a variant thereof and an effector domain, and at least one gRNA. In some embodiments, the effector domain increases transcription of the one or more genes (e.g. the effector domain is a transcriptional activator, such as any described in Section I.E).

[0198] The CRISPR system (also known as CRISPR/Cas system, or CRISPR-Cas system) refers to a conserved microbial nuclease system, found in the genomes of bacteria and archaea, that provides a form of acquired immunity against invading phages and plasmids. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), refers to loci containing multiple repeating DNA elements that are separated by non-repeating DNA sequences called spacers. Spacers are short sequences of foreign DNA that are incorporated into the genome between CRISPR repeats, serving as a “memory” of past exposures. Spacers encode the DNA-targeting portion of RNA molecules that confer specificity for nucleic acid cleavage by the CRISPR system. CRISPR loci contain or are adjacent to one or more CRISPR-associated (Cas) genes, which can act as RNA-guided nucleases for mediating the cleavage, as well as non-protein coding DNA elements that encode RNA molecules capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage.

[0199] In Type II CRISPR/Cas systems with the Cas protein Cas9, two RNA molecules and the Cas9 protein form a ribonucleoprotein (RNP) complex to direct Cas9 nuclease activity. The CRISPR RNA (crRNA) contains a spacer sequence that is complementary to a target nucleic acid sequence (target site), and that encodes the sequence specificity of the complex. The transactivating crRNA (tracrRNA) base-pairs to a portion of the crRNA and forms a structure that complexes with the Cas9 protein, forming a Cas/RNA RNP complex.

[0200] Naturally occurring CRISPR/Cas systems, such as those with Cas9, have been engineered to allow efficient programming of Cas/RNA RNPs to target desired sequences in cells of interest, both for gene-editing and modulation of gene expression. The tracrRNA and crRNA have been engineered to form a single chimeric guide RNA molecule, commonly referred to as a guide RNA (gRNA), for example as described in WO 2013/176772, WO 2014/093661, WO 2014/093655, Jinek, M. et al. Science 337(6096):816-21 (2012), or Cong, L. et al. Science 339(6121): 819-23 (2013). The spacer sequence of the gRNA can be chosen by a user to target the Cas/gRNA RNP complex to a desired locus, e.g. a desired target site in the target gene.

[0201] Cas proteins have also been engineered to be catalytically inactivated or nuclease inactive to allow targeting of Cas/gRNA RNPs without inducing cleavage at the target site. Mutations in Cas proteins can reduce or abolish nuclease activity of the Cas protein, rendering the Cas protein catalytically inactive. Cas proteins with reduced or abolished nuclease activity are referred to as deactivated Cas (dCas), or nuclease-inactive Cas (iCas) proteins, as referred to interchangeably herein. An exemplary deactivated Cas9 (dCas9) derived from .S', pyogenes contains silencing mutations of the RuvC and HNH nuclease domains (D10A and H840A), for example as described in WO 2013/176772, WO 2014/093661, Jinek, M. et al. Science 337(6096):816-21 (2012), and Qi, L. et al. Cell 152(5): 1173-83 (2013). Exemplary dCas variants derived from the Casl2 system (i.e. Cpfl) are described, for example in WO 2017/189308 and Zetsche, B. et al. Cell 163(3):759-71 (2015). Conserved domains that mediate nucleic acid cleavage, such as RuvC and HNH endonuclease domains, are readily identifiable in Cas orthologues, and can be mutated to produce inactive variants, for example as described in Zetsche, B. et al. Cell 163(3):759-71 (2015).

[0202] dCas-fusion proteins with transcriptional and/or epigenetic regulators have been used as a versatile platform for ectopically regulating gene expression in target cells. These include fusion of a Cas with an effector domain, such as a transcriptional activator or transcriptional repressor. For example, fusing dCas9 with a transcriptional activator such as VP64 (a polypeptide composed of four tandem copies of VP 16, a 16 amino acid transactivation domain of the Herpes simplex virus) can result in robust induction of gene expression. Alternatively, fusing dCas9 with a transcriptional repressor such as KRAB (Kruppel associated box) can result in robust repression of gene expression. A variety of dCas-fusion proteins with effector domains can be engineered for regulation of gene expression, for example as described in WO 2014/197748, WO 2016/130600, WO 2017/180915, WO 2021/226555, WO 2013/176772, WO 2014/152432, WO 2014/093661, WO 2021/247570, Adli, M. Nat. Commun. 9, 1911 (2018), Perez-Pinera, P. et al. Nat. Methods 10, 973-976 (2013), Mali, P. et al. Nat. Biotechnol. 31, 833-838 (2013), Maeder, M. L. et al. Nat. Methods 10, 977-979 (2013), Gilbert, L. A. et al. Cell 154(2):442-451 (2013), and Nunez, J.K. et al. Cell 184(9):2503-2519 (2021).

[0203] In some aspects, provided is a DNA-targeting system comprising a fusion protein comprising a DNA-binding domain comprising a nuclease-inactive Cas protein or variant thereof, and an effector domain for increasing transcription or inducing transcriptional activation (i.e. a transcriptional activator) when targeted to a target gene in a cell (e.g. a T cell). In some embodiments, the dCas protein is any suitable dCas protein, such as any described herein. In some embodiments, the dCas protein is a dCas9 protein, such dSpCas9 or dSaCas9. In some embodiments, the at least one effector domain is any suitable transcriptional activator effector domain, such as any described in Section I.E, such as VP64. In some embodiments, the at least one effector domain is VP64. In some embodiments, the fusion protein is a dCas9-VP64 fusion protein, for example as described in Section I.F. In such embodiments, the DNA-targeting system also includes one or more gRNAs (e.g. as described in Section I.C.2.), provided in combination or as a complex with the dCas protein or variant thereof, for targeting of the DNA- targeting system to the target site of the target gene. In some embodiments, the fusion protein is guided to a specific target site sequence of the target gene by the guide RNA, wherein the effector domain mediates targeted epigenetic modification to increase or activate transcription of the target gene. In some embodiments, a combination of gRNAs guides the fusion protein to a combination of target site sequences in a combination of genes, wherein the effector domain mediates targeted epigenetic modification to increase or activate transcription of the combination of target genes. Any of a variety of effector domains that increase or activate transcription can be used as described further below.

/. CP/SPP/Cas-Dased DNA-Dinding Domains

[0204] In some aspects, the DNA-binding domain comprises a CRISPR-associated (Cas) protein or variant thereof, or is derived from a Cas protein or variant thereof. In particular embodiments here, the Cas protein is nuclease-inactive (i.e. is a dCas protein).

[0205] In some embodiments, the Cas protein is derived from a Class 1 CRISPR system (i.e. multiple Cas protein system), such as a Type I, Type III, or Type IV CRISPR system. In some embodiments, the Cas protein is derived from a Class 2 CRISPR system (i.e. single Cas protein system), such as a Type II, Type V, or Type VI CRISPR system. In some embodiments, the Cas protein is from a Type V CRISPR system. In some embodiments, the Cas protein is derived from a Cas 12 protein (i.e. Cpfl) or variant thereof, for example as described in WO 2017/189308 and Zetsche, B. et al. Cell. 163(3):759-71 (2015). In some embodiments, the Cas protein is derived from a Type II CRISPR system. In some embodiments, the Cas protein is derived from a Cas9 protein or variant thereof, for example as described in WO 2013/176772, WO 2014/152432, WO 2014/093661, WO 2014/093655, Jinek, M. et al. Science 337(6096):816-21 (2012), Mali, P. et al. Science 339(6121):823-6 (2013), Cong, L. et al. Science 339(6121):819-23 (2013), Perez-Pinera, P. et al. Nat. Methods 10, 973-976 (2013), or Mali, P. et al. Nat. Biotechnol. 31, 833-838 (2013). Various CRISPR/Cas systems and associated Cas proteins for use in gene editing and regulation have been described, for example in Moon, S.B. et al. Exp. Mol. Med. 51, 1-11 (2019), Zhang, F. Q. Rev. Biophys. 52, E6 (2019), and Makarova K.S. et al. Methods Mol. Biol. 1311:47-75 (2015).

[0206] In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule, or variant thereof. In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule of .S'. pyogenes, S. thermophilus, S. aureus, C. jejuni, N. meningitidis, F. novicida, S. canis, S. auricularis, or variant thereof. In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of .S', aureus. In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of .S', pyogenes.

[0207] Non-limiting examples of Cas9 orthologs from other bacterial strains include but are not limited to: Cas proteins identified in Acaryochloris marina MBIC 11017; Acetohalobium arabaticum DSM 5501; Acidithiobacillus caldus; Acidithiobacillus ferrooxidans ATCC 23270; Alicyclobacillus acidocaldarius LAA1; Alicyclobacillus acidocaldarius subsp. acidocaldarius DSM 446; Allochromatium vinosum DSM 180; Ammonifex degensii KC4; Anabaena variabilis ATCC 29413; Arthrospira maxima CS-328; Arthrospira platensis str. Paraca; Arthrospira sp. PCC 8005; Bacillus pseudomycoides DSM 12442; Bacillus selenitireducens MLS 10; Burkholderiales bacterium 1_1_47; Caldicelulosiruptor becscii DSM 6725; Candidatus Desulforudis audaxviator MP104C; Caldicellulosiruptor hydrothermalis 108; Clostridium phage c-st; Clostridium botulinum A3 str. Loch Maree; Clostridium botulinum Ba4 str. 657;

Clostridium difficile QCD-63q42; Crocosphaera watsonii WH 8501; Cyanothece sp. ATCC 51142; Cyanothece sp. CCY0110; Cyanothece sp. PCC 7424; Cyanothece sp. PCC 7822; Exiguobacterium sibiricum 255-15; Finegoldia magna ATCC 29328; Ktedonobacter racemifer DSM 44963; Lactobacillus delbrueckii subsp. bulgaricus PB2003/044-T3-4; Lactobacillus salivarius ATCC 11741; Listeria innocua; Lyngbya sp. PCC 8106; Marinobacter sp. ELB 17; Methanohalobium evestigatum Z-7303; Microcystis phage Ma-LMMOl; Microcystis aeruginosa NIES-843; Microscilla marina ATCC 23134; Microcoleus chthonoplastes PCC 7420; Neisseria meningitidis; Nitrosococcus halophilus Nc4; Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111; Nodularia spumigena CCY9414; Nostoc sp. PCC 7120; Oscillatoria sp. PCC 6506; Pelotomaculum_thermopropionicum SI; Petrotoga mobilis SJ95; Polaromonas naphthalenivorans CJ2; Polaromonas sp. JS666; Pseudoalteromonas haloplanktis TAC125;

Streptomyces pristinaespiralis ATCC 25486; Streptomyces pristinaespiralis ATCC 25486; Streptococcus thermophilus; Streptomyces viridochromogenes DSM 40736; Streptosporangium roseum DSM 43021; Synechococcus sp. PCC 7335; and Thermosipho africanus TCF52B (Chylinski et al., RNA Biol., 2013; 10(5): 726-737).

[0208] In some aspects, the Cas protein is a variant that lacks nuclease activity (i.e. is a dCas protein). In some embodiments, the Cas protein is mutated so that nuclease activity is reduced or eliminated. Such Cas proteins are referred to as deactivated Cas or dead Cas (dCas) or nucleaseinactive Cas (iCas) proteins, as referred to interchangeably herein. In some embodiments, the variant Cas protein is a variant Cas9 protein that lacks nuclease activity or that is a deactivated Cas9 (dCas9, or iCas9) protein.

[0209] In some embodiments, the Cas9 protein or a variant thereof is derived from a Staphylococcus aureus Cas9 (SaCas9) protein or a variant thereof. In some embodiments, the SaCas9 protein comprises the sequence set forth in SEQ ID NO: 83 or SEQ ID NO: 197, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant Cas9 is a Staphylococcus aureus dCas9 protein (dSaCas9) that comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO:83. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO:84, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0210] In some embodiments, the Cas9 protein or variant thereof is derived from a Streptococcus pyogenes Cas9 (SpCas9) protein or a variant thereof. In some embodiments, the SpCas9 protein comprises the sequence set forth in SEQ ID NO: 63 or SEQ ID NO: 195, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant Cas9 is a Streptococcus pyogenes dCas9 (dSpCas9) protein that comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO:63. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 64 or SEQ ID NO: 196, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

2. Guide RlVAs (g GVAs)

[0211] In some embodiments, the Cas protein (e.g. dCas9) is provided in combination or as a complex with one or more guide RNA (gRNA). In some embodiments, the gRNA is capable of complexing with the Cas protein or variant thereof. In some embodiments, the gRNA comprises a gRNA spacer sequence (also known as a spacer sequence or a guide sequence) that is capable of hybridizing to the target site or is complementary to the target site, such as any target site described herein, for example, any target site in a genome. In some embodiments, the gRNA comprises a scaffold sequence that complexes with or binds to the Cas protein. In some embodiments, a gRNA specific to a target locus of interest (e.g. a regulatory DNA element of an LCD gene) is used to recruit an RNA-guided protein (e.g. a Cas protein) or variant thereof or a fusion protein comprising such RNA-guided protein (e.g., a Cas polypeptide), to the target site. In some aspects, the gRNA is a nucleic acid that promotes the specific targeting or homing of the gRNA/Cas RNP complex to the target site of the target gene, such as any described above in Section I.B. In some embodiments, a target site of a gRNA may be referred to as a protospacer.

[0212] Provided herein are gRNAs, such as gRNAs that target or bind to a target site for a lymphoid cell differentiation (LCD) gene, such as in a target gene or regulatory DNA element thereof, such as any described herein, for example in Section I.B. Provided herein are gRNAs, such as gRNAs that target or can bind to a regulatory DNA element of a LCD gene. In some embodiments, the gRNAs bind to a target site that is located in the LCD gene and/or a regulatory DNA element of the LCD gene. In some embodiments, the gRNAs bind to a target site that is located in the LCD gene. In some embodiments, the gRNAs bind to a target site that is located in a regulatory DNA element of the LCD gene.

[0213] In some embodiments, the gRNA is capable of complexing with the Cas protein or variant thereof. In some embodiments, any of the provided gRNA sequences is complexed with or is provided in combination with a fusion protein comprising Cas9. In some embodiments, the Cas9 is a dCas9. In some embodiments, the dCas9 is a dSpCas9, such as a dSpCas9. The Cas9 can be any as described herein such as in Section I.C.l. In some embodiments, the gRNA comprises a gRNA spacer sequence (i.e. a spacer sequence or a guide sequence) that is capable of hybridizing to the target site, or that is complementary to the target site, such as any target site described herein. In some embodiments, the gRNA comprises a scaffold sequence that complexes with or binds to the Cas protein.

[0214] In some aspects, a “gRNA molecule” is a nucleic acid that promotes the specific targeting or homing of a gRNA molecule/Cas9 molecule complex to a target nucleic acid, such as a locus on the genomic DNA of a cell. In general, a spacer sequence of the guide RNA, is any polynucleotide sequences comprising at least a sequence portion that has sufficient complementarity with a target polynucleotide sequence, such as the at an LCD gene locus in humans, to hybridize with the target sequence at the target site and direct sequence- specific binding of the CRISPR complex to the target sequence. In some embodiments, in the context of formation of a CRISPR complex, “target sequence” is to a sequence to which a spacer sequence is designed to have complementarity, where hybridization between the target sequence and a spacer sequence of the guide RNA promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. Generally, a spacer sequence is selected to reduce the degree of secondary structure within the spacer sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.

[0215] In some embodiments, a guide RNA (gRNA) specific to a target locus of interest (e.g. at an LCD gene locus) is used with RNA-guided nucleases or variants thereof, e.g., nuclease-inactive Cas variants, to target the provided DNA-targeting system to the target site or target position. Methods for designing gRNAs and exemplary spacer sequences are known. Exemplary gRNA structures that can be associated with particular RNA-guided nucleases or variants thereof, e.g., nuclease-inactive Cas variants, with particular domains and scaffold regions, are also known. In some aspects, gRNA molecules comprise a scaffold sequence, e.g., sequences that can be complexed with the Cas protein.

[0216] In some embodiments, the scaffold is specific for a Cas protein. In some embodiments, the scaffold is specific for SpCas9 or a dSpCas9. In some embodiments the scaffold comprises the nucleic acid sequence set forth in SEQ ID NO: 55 and 56. In some embodiments the scaffold comprises the nucleic acid sequence set forth in SEQ ID NO: 121 and 122. In some embodiments, the scaffold is specific for a SaCas9 or a dSaCas9. In some embodiments, the scaffold comprises the nucleic acid sequence set forth in SEQ ID NO: 90 and 91.

[0217] In some embodiments, the gRNAs provided herein are chimeric gRNAs. In general, gRNAs can be unimolecular (i.e. composed of a single RNA molecule), or modular (comprising more than one, and typically two, separate RNA molecules). Modular gRNAs can be engineered to be unimolecular, wherein sequences from the separate modular RNA molecules are comprised in a single gRNA molecule, sometimes referred to as a chimeric gRNA, synthetic gRNA, or single gRNA. In some embodiments, the chimeric gRNA is a fusion of two noncoding RNA sequences: a crRNA sequence and a tracrRNA sequence, for example as described in WO 2013/176772, or Jinek, M. et al. Science 337(6096):816-21 (2012). In some embodiments, the chimeric gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the Type II Effector system, wherein the naturally occurring crRNA:tracrRNA duplex acts as a guide for the Cas9 protein.

[0218] A guide RNA can comprise at least a spacer sequence that hybridizes to a target nucleic acid sequence of interest, and a CRISPR repeat sequence. In Type II systems, the gRNA also comprises a second RNA called the tracrRNA sequence. In the Type II guide RNA (gRNA), the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex. In the Type V guide RNA (gRNA), the crRNA forms a duplex. In both systems, the duplex can bind a site-directed polypeptide, such that the guide RNA and site-direct polypeptide form a complex. The gRNA can provide target specificity to the complex by virtue of its association with the site-directed polypeptide. The gRNA thus can direct the activity of the site- directed polypeptide.

[0219] In some aspects, the spacer sequence of a gRNA is a polynucleotide sequence comprising at least a portion that has sufficient complementarity with the target site to hybridize with the target site in the target gene and direct sequence- specific binding of a Cas/gRNA complex to the sequence of the target site. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization. In some embodiments, the gRNA comprises a spacer sequence that is complementary, e.g., at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% (e.g., fully complementary), to the target site. The strand of the target nucleic acid comprising the target site sequence may be referred to as the “complementary strand” of the target nucleic acid.

[0220] In some aspects, a gRNA targets a target site in double- stranded DNA. Thus, in some aspects, the sequence of the target site may be defined by the sequence that the gRNA spacer hybridizes to, or by the sequence complementary to the sequence that the gRNA spacer hybridizes to. In some aspects, the sequence of the target site may be defined by the sequence that the gRNA spacer displaces in order to hybridize to the DNA. In some embodiments, the sequence of the target site is the sequence that the gRNA hybridizes to.

[0221] In some embodiments, the gRNA spacer sequence is between about 14 nucleotides (nt) and about 26 nt, or between 16 nt and 22 nt in length. In some embodiments, the gRNA spacer sequence is 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt or 22 nt, 23 nt, 24 nt, 25 nt, or 26 nt in length. In some embodiments, the gRNA spacer sequence is 18 nt, 19 nt, 20 nt, 21 nt or 22 nt in length. In some embodiments, the gRNA spacer sequence is 20 nt in length. [0222] In some embodiments the gRNA is a concatenation of two non-coding RNA sequences: a crRNA sequence and a tracrRNA sequence. The gRNA may target a desired DNA sequence by exchanging the sequence encoding a 20 bp protospacer which confers targeting specificity through complementary base pairing with the desired DNA target. gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the Type II CRISPR/Cas system (e.g., Cas9). This duplex, which may include, for example, a 42-nucleotide crRNA and a 75- nucleotide tracrRNA, acts as a guide for the Cas9 protein to cleave the target nucleic acid. The CRISPR/Cas9-based system may include two or more gRNAs, wherein the two or more gRNAs target different DNA sequences. The target DNA sequences may be overlapping or nonoverlapping. The target DNA sequences may be located within or near the same gene or different genes. The target sequence or protospacer is followed by a PAM sequence at the 3' end of the protospacer. Different Type II systems have differing PAM requirements. For example, the Streptococcus pyogenes Type II system uses an “NGG” sequence, where “N” can be any nucleotide.

[0223] A target site of a gRNA may be referred to as a protospacer. In some aspects, the spacer is designed to target a protospacer with a specific protospacer-adjacent motif (PAM), i.e. a sequence immediately adjacent to the protospacer that contributes to and/or is required for Cas binding specificity. Different CRISPR/Cas systems have different PAM requirements for targeting. For example, in some embodiments, 5. pyogenes Cas9 uses the PAM 5’-NGG-3’ (SEQ ID NO: 85), where N is any nucleotide. In some embodiments, the PAM of a gRNA for complexing with a Type V CRISPR/Cas system, such as with Casl2a (also known as Cpfl) or variant thereof uses TTTV (SEQ ID NO: 178), where V is A, C, or G. In some embodiments, 5. aureus Cas9 uses the PAM 5’- NNGRRT-3’ (SEQ ID NO: 86), where N is any nucleotide, and R is G or A. In some embodiments, N. meningitidis Cas9 uses the PAM 5'-NNNNGATT-3’ (SEQ ID NO: 179), where N is any nucleotide. In some embodiments, C. jejuni Cas9 uses the PAM 5'-NNNNRYAC-3' (SEQ ID NO: 180), where N is any nucleotide, R is G or A, and Y is C or T. In some embodiments, S. thermophilus uses the PAM 5’-NNAGAAW-3’ (SEQ ID NO: 181), where N is any nucleotide and W is A or T. In some embodiments, F. Novicida Cas9 uses the PAM 5’-NGG-3’ (SEQ ID NO: 85), where N is any nucleotide. In some embodiments, T. denticola Cas9 uses the PAM 5’-NAAAAC-3’ (SEQ ID NO: 182), where N is any nucleotide. In some embodiments, Casl2a (also known as Cpfl) from various species, uses the PAM 5’- TTTV-3’ (SEQ ID NO: 178). In some embodiments, Cas proteins may use or be engineered to use different PAMs from those listed above. For example, mutated SpCas9 proteins may use the PAMs 5’-NGG-3’ (SEQ ID NO:85), 5’-NGAN-3’ (SEQ ID NO: 183), 5’-NGNG-3’ (SEQ ID NO: 184), 5’-NGAG-3’ (SEQ ID NO: 154), or 5’-NGCG-3’ (SEQ ID NO: 185), where N is any nucleotide. In some embodiments, the protospacer-adjacent motif (PAM) of a gRNA for complexing with .S'. pyogenes Cas9 or variant thereof is NGG, as set forth in SEQ ID NO: 85. In some embodiments, the PAM of a gRNA for complexing with .S'. aureus Cas9 or variant thereof is NNGRRT, as set forth in SEQ ID NO: 86. Methods for designing or identifying gRNA spacer sequences and/or protospacer sequences in a particular region, are known. gRNA spacer sequences and/or protospacer sequences can be determined based on the type of Cas protein used and the associated PAM sequence.

[0224] A spacer sequence may be selected to reduce the degree of secondary structure within the spacer sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.

[0225] In some embodiments, the gRNA (including the guide sequence) will comprise the base uracil (U), whereas DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, in some embodiments, it is believed that the complementarity of the guide sequence with the target sequence contributes to specificity of the interaction of the gRNA molecule/Cas molecule complex with a target nucleic acid. It is understood that in a guide sequence and target sequence pair, the uracil bases in the guide sequence will pair with the adenine bases in the target sequence.

[0226] In some embodiments, the gRNA comprises modified nucleotides, e.g. for increased stability. In some embodiments, one, more than one, or all of the nucleotides of a gRNA can have a modification, e.g., to render the gRNA less susceptible to degradation and/or improve bio-compatibility. By way of non-limiting example, the backbone of the gRNA can be modified with a phosphorothioate, or other modification(s). In some cases, a nucleotide of the gRNA can comprise a 2’ modification, e.g., a 2-acetylation, e.g., a 2’ methylation, or other modification(s).

[0227] Methods for designing gRNAs and exemplary targeting domains can include those described in, e.g., International PCT Pub. Nos. WO 2014/197748, WO 2016/130600, WO 2017/180915, WO 2021/226555, WO 2013/176772, WO 2014/152432, WO 2014/093661, WO 2014/093655, WO 2015/089427, WO 2016/049258, WO 2016/123578, WO 2021/076744, WO 2014/191128, WO 2015/161276, WO 2017/193107, and WO 2017/093969.

[0228] In some aspects, the gRNA comprises scaffold sequences. In some aspects, the scaffold sequence (in some cases including a crRNA sequence and/or a tracrRNA sequence) will be different depending on the Cas protein. In some aspects, different CRISPR/Cas systems have different gRNA scaffold sequences for associating with Cas protein. In some embodiments, an exemplary scaffold sequence for S. aureus Cas9 comprises a sequence set forth in SEQ ID NO:91, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:91. In some embodiments, an exemplary scaffold sequence for S. aureus Cas9 comprises a sequence set forth in SEQ ID NO:91. In some embodiments, an exemplary scaffold sequence for S. pyogenes Cas9 comprises a sequence set forth in SEQ ID NO:56, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:56. In some embodiments, an exemplary scaffold sequence for S. pyogenes Cas9 comprises a sequence set forth in SEQ ID NO:56.

[0229] In some embodiments, an exemplary scaffold sequence for Acidaminococcus sp. Casl2a comprises a sequence set forth in SEQ ID NO: 186, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 186. In some embodiments, an exemplary scaffold sequence for CasPhi-2 comprises a sequence set forth in SEQ ID NO: 187, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 187. In some embodiments, an exemplary scaffold sequence for UnlCasl2fl comprises a sequence set forth in SEQ ID NO:188, the sequence “GGAATGAAC” (SEQ ID NO: 190), or the sequence “TTTTATTTT” (SEQ ID NO: 189), or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 188, 189 or 190. In some embodiments, an exemplary scaffold sequence for UnlCasl2fl comprises a sequence set forth in SEQ ID NO: 188 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 188. In some embodiments, an exemplary scaffold sequence for UnlCasl2fl comprises a sequence set forth in SEQ ID NO: 190, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 190. In some embodiments, an exemplary scaffold sequence for UnlCasl2fl comprises a sequence set forth in SEQ ID NO: 189, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 189. In some embodiments, an exemplary scaffold sequence for C. jejuni Cas9 comprises a sequence set forth in SEQ ID NO: 192, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 192. In some embodiments, an exemplary scaffold sequence for Casl2k comprises a sequence set forth in SEQ ID NO: 193, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 193. In some embodiments, an exemplary scaffold sequence for CasMini comprises a sequence set forth in SEQ ID NO: 130, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 194.

[0230] In some aspects, the gRNA can target the DNA-targeting system to direct the activities of an associated polypeptide (e.g., fusion protein, DNA-targeting system, effector domain, etc.) to a specific target site within a target nucleic acid (e.g., regulatory DNA element of a LCD gene locus).

[0231] In some embodiments, a gRNA provided herein targets a target site for an LCD gene or regulatory element thereof for transcriptional activation. In some embodiments, a gRNA provided herein targets a target site for an LCD gene or regulatory element thereof for transcriptional activation. In some embodiments, the target site is located on an LCD gene. In some embodiments, the target site is located in a regulatory DNA element of the gene. In some embodiments, a regulatory DNA element is a sequence to which a gene regulatory protein may bind and affect transcription of the gene. In some embodiments, a regulatory DNA element is a sequence to which a gene regulatory protein may bind and affect transcription of an LCD gene. Exemplary target sites and combinations of target sites for gRNA of the DNA-binding systems, including multiplexed DNA-binding systems, include any described in Section I.B.

[0232] In some embodiments, a gRNA provided herein targets a target site for a gene for transcriptional activation, such as any target site or gene described in Section I.B. In some embodiments, a gRNA provided herein targets a target site for an LCD gene, wherein the gene is selected from: RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B.

[0233] In some embodiments, the gRNA targets a target site that comprises a sequence selected from any one of SEQ ID NOs:l-18 and 93-106, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs:l-18 and 93-106 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in any one of SEQ ID NOs: 1-18 and 93-106.

[0234] In some embodiments, the gRNA comprises a spacer sequence selected from any one of SEQ ID NOs: 19-36 and 107-120, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOs: 19-36 and 107-120 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOS: 19-36 and 107-120.

[0235] In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 37-54 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 37-54. In some embodiments, the gRNA is set forth in any one of SEQ ID NOs: 37-54.

[0236] In some embodiments, the gRNA comprises modified nucleotides. In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 65-82 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 65-82. In some embodiments, the gRNA is set forth in any one of SEQ ID NOs: 65-82.

[0237] In some embodiments, the gRNA targets a target site for TCF7. In some embodiments, the gRNA targets the target site that comprises a sequence selected from any one of SEQ ID NOs: 1-6, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs: 1-6 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in any one of SEQ ID NOs: 1-6. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:1, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 1, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 1 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:1. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:2, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 2, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 2 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:2. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:3, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 3, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 3 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:3. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:4, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 4, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 4 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:4. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:5, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 5, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 5 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:5. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:6, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 6, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 6 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:6.

[0238] In some embodiments, the gRNA targeting a target site for TCF7 comprises a spacer sequence selected from any one of SEQ ID NOs: 19-24, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOs: 19-24 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOS: 19-24. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 19, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 19. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 19. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 19. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:20, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:20. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:20. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:20. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:21, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:21. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:21. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:21. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:22, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:22. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:22. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:22. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:23, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:23. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:23. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:23. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:24, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:24. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:24. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:24.

[0239] In some embodiments, the gRNA targeting a target site for TCF7 comprises a sequence selected from any one of SEQ ID NOs: 37-42 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 37-42. In some embodiments, the gRNA is set forth in any one of SEQ ID NOs: 37-42. In some embodiments, the gRNA targeting a target site for TCF7 comprises the sequence set forth in SEQ ID NO: 37 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 37. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 37. In some embodiments, the gRNA is set forth in SEQ ID NO: 37. In some embodiments, the gRNA targeting a target site for TCF7 comprises the sequence set forth in SEQ ID NO: 38 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 38. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 38. In some embodiments, the gRNA is set forth in SEQ ID NO: 38. In some embodiments, the gRNA targeting a target site for TCF7 comprises the sequence set forth in SEQ ID NO: 39 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 39. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 39. In some embodiments, the gRNA is set forth in SEQ ID NO: 39. In some embodiments, the gRNA targeting a target site for TCF7 comprises the sequence set forth in SEQ ID NO: 40 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 40. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 40. In some embodiments, the gRNA is set forth in SEQ ID NO: 40. In some embodiments, the gRNA targeting a target site for TCF7 comprises the sequence set forth in SEQ ID NO: 41 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 41. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 41. In some embodiments, the gRNA is set forth in SEQ ID NO: 41. In some embodiments, the gRNA targeting a target site for TCF7 comprises the sequence set forth in SEQ ID NO: 42 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 42. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 42. In some embodiments, the gRNA is set forth in SEQ ID NO: 42.

[0240] In some embodiments, the gRNA targeting a target site for TCF7 comprises modified nucleotides. In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 65-70 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 65-70. In some embodiments, the gRNA is set forth in any one of SEQ ID NOs: 65-70. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 65 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 65. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 65. In some embodiments, the gRNA is set forth in SEQ ID NO:65. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 66 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 66. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 66. In some embodiments, the gRNA is set forth in SEQ ID NO:66. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 67 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 67. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 67. In some embodiments, the gRNA is set forth in SEQ ID NO:67. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 68 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 68. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 68. In some embodiments, the gRNA is set forth in SEQ ID NO:68. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 69 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 69. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 69. In some embodiments, the gRNA is set forth in SEQ ID NO:69. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 70 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 70. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 70. In some embodiments, the gRNA is set forth in SEQ ID NO:70.

[0241] In some embodiments, the gRNA targets a target site for GATA3. In some embodiments, the gRNA targets the target site that comprises a sequence selected from any one of SEQ ID NOs:7-12, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs:7-12 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in any one of SEQ ID NOs: 7-12. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:7, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 7, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 7. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 7 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:7. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:8, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 8, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 8. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 8 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:8. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:9, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 9, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 9. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 9 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:9. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 10, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 10, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 10 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 10. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 11, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 11, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 11 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:11. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 12, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 12, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 12 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 12.

[0242] In some embodiments, the gRNA targeting a target site for GATA3 comprises a spacer sequence selected from any one of SEQ ID NOs:25-30, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOs:25-30 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOS:25-30. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:25, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:25. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:25. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:25. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:26, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:26. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:26. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:26. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:27, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:27. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:27. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:27. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:28, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:28. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:28. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:28. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:29, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:29. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:29. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:29. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:30, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:30. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:30. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:30.

[0243] In some embodiments, the gRNA targeting a target site for GATA3 comprises a sequence selected from any one of SEQ ID NOs: 43-48 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 43-48. In some embodiments, the gRNA is set forth in any one of SEQ ID NOs: 43-48. In some embodiments, the gRNA targeting a target site for GATA3 comprises the sequence set forth in SEQ ID NO: 43 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 43. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 43. In some embodiments, the gRNA is set forth in SEQ ID NO: 43. In some embodiments, the gRNA targeting a target site for GATA3 comprises the sequence set forth in SEQ ID NO: 44 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 44. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 44. In some embodiments, the gRNA is set forth in SEQ ID NO: 44. In some embodiments, the gRNA targeting a target site for GATA3 comprises the sequence set forth in SEQ ID NO: 45 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 45. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 45. In some embodiments, the gRNA is set forth in SEQ ID NO: 45. In some embodiments, the gRNA targeting a target site for GATA3 comprises the sequence set forth in SEQ ID NO: 46 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 46. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 46. In some embodiments, the gRNA is set forth in SEQ ID NO: 46. In some embodiments, the gRNA targeting a target site for GATA3 comprises the sequence set forth in SEQ ID NO: 47 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 47. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 47. In some embodiments, the gRNA is set forth in SEQ ID NO: 47. In some embodiments, the gRNA targeting a target site for GATA3 comprises the sequence set forth in SEQ ID NO: 48 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 48. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 48. In some embodiments, the gRNA is set forth in SEQ ID NO: 48.

[0244] In some embodiments, the gRNA targeting a target site for GATA3 comprises modified nucleotides. In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 71-76 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 71-76. In some embodiments, the gRNA is set forth in any one of SEQ ID NOs: 71-76. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 71 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 71. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 71. In some embodiments, the gRNA is set forth in SEQ ID NO:71. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 72 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 72. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 72. In some embodiments, the gRNA is set forth in SEQ ID NO:72. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 73 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 73. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 73. In some embodiments, the gRNA is set forth in SEQ ID NO:73. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 74 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 74. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 74. In some embodiments, the gRNA is set forth in SEQ ID NO:74. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 75 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 75. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 75. In some embodiments, the gRNA is set forth in SEQ ID NO:75. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 76 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 76. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 76. In some embodiments, the gRNA is set forth in SEQ ID NO:76.

[0245] In some embodiments, the gRNA targets a target site for BCL1 IB. In some embodiments, the gRNA targets the target site that comprises a sequence selected from any one of SEQ ID NOs:13-18, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs:13-18 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in any one of SEQ ID NOs:13-18. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 13, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 13, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 13. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 13 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 13. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 14, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 14, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 14 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 14. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 15, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 15, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 15. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 15 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 15. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 16, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 16, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 16. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 16 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 16. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 17, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 17, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 17. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 17 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 17. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 18, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 18, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 18. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 18 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 18.

[0246] In some embodiments, the gRNA targeting a target site for BCL1 IB comprises a spacer sequence selected from any one of SEQ ID NOs:31-36, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOs:31-36 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOS:31-36. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:31, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:31. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:31. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:31. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:32, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:32. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:32. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:32. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:33, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:33. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:33. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:33. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:34, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:34. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:34. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:34. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:35, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:35. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:35. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:35. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:36, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:36. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:36. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:36.

[0247] In some embodiments, the gRNA targeting a target site for BCL1 IB comprises a sequence selected from any one of SEQ ID NOs: 49-54 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 49-54. In some embodiments, the gRNA is set forth in any one of SEQ ID NOs: 49-54. In some embodiments, the gRNA targeting a target site for BCL1 IB comprises the sequence set forth in SEQ ID NO: 49 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 49. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 49. In some embodiments, the gRNA is set forth in SEQ ID NO: 49. In some embodiments, the gRNA targeting a target site for BCL11B comprises the sequence set forth in SEQ ID NO: 50 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 50. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 50. In some embodiments, the gRNA is set forth in SEQ ID NO: 50. In some embodiments, the gRNA targeting a target site for BCL1 IB comprises the sequence set forth in SEQ ID NO: 51 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 51. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 51. In some embodiments, the gRNA is set forth in SEQ ID NO: 51. In some embodiments, the gRNA targeting a target site for BCL11B comprises the sequence set forth in SEQ ID NO: 52 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 52. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 52. In some embodiments, the gRNA is set forth in SEQ ID NO: 52. In some embodiments, the gRNA targeting a target site for BCL11B comprises the sequence set forth in SEQ ID NO: 53 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 53. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 53. In some embodiments, the gRNA is set forth in SEQ ID NO: 53. In some embodiments, the gRNA targeting a target site for BCL1 IB comprises the sequence set forth in SEQ ID NO: 54 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 54. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 54. In some embodiments, the gRNA is set forth in SEQ ID NO: 54.

[0248] In some embodiments, the gRNA targeting a target site for BCL1 IB comprises modified nucleotides. In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 77-82 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 77-82. In some embodiments, the gRNA is set forth in any one of SEQ ID NOs: 77-82. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 77 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 77. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 77. In some embodiments, the gRNA is set forth in SEQ ID NO:77. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 78 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 78. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 78. In some embodiments, the gRNA is set forth in SEQ ID NO:78. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 79 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 79. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 79. In some embodiments, the gRNA is set forth in SEQ ID NO:79. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 80 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 80. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 80. In some embodiments, the gRNA is set forth in SEQ ID NO:80. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 81 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 81. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 81. In some embodiments, the gRNA is set forth in SEQ ID NO:81. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 82 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 82. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 82. In some embodiments, the gRNA is set forth in SEQ ID NO:82.

[0249] In some embodiments, the gRNA targets a target site for MYB. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO:93 or SEQ ID NO:94, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO:93 or SEQ ID NO:94 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO:93 or SEQ ID NO:94. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:93, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:93, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:93. In some embodiments, the target site is a contiguous portion of SEQ ID NO:93 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:93. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:94, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:94, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:94. In some embodiments, the target site is a contiguous portion of SEQ ID NO:94 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:94.

[0250] In some embodiments, the gRNA targeting a target site for MYB comprises a spacer sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 107 or SEQ ID NO: 108 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ D NO: 107 or SEQ ID NO: 108. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 107, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 107. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 107. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 107. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 108, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 108. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 108. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 108.

[0251] In some embodiments, the gRNA targeting a target site for MYB 1 comprises a sequence comprising a spacer sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for MYB1 comprises modified nucleotides.

[0252] In some embodiments, the gRNA targets a target site for RUNX1. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO:95 or SEQ ID NO:96, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO:95 or SEQ ID NO:96 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO:95 or SEQ ID NO:96. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:95, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:95, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:95. In some embodiments, the target site is a contiguous portion of SEQ ID NO:95 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:95. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:96, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:96, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 96. In some embodiments, the target site is a contiguous portion of SEQ ID NO:96 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:96.

[0253] In some embodiments, the gRNA targeting a target site for RUNX1 comprises a spacer sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 109 or SEQ ID NO: 110 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ D NO: 109 or SEQ ID NO: 110. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 109, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 109. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 109. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 109. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 110, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 110. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 110. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 110.

[0254] In some embodiments, the gRNA targeting a target site for RUNX1 comprises a sequence comprising a spacer sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for RUNX1 comprises modified nucleotides.

[0255] In some embodiments, the gRNA targets a target site for HEY1. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO:97 or SEQ ID NO:98, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO:97 or SEQ ID NO:98 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO:97 or SEQ ID NO:98. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:97, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:97, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:97. In some embodiments, the target site is a contiguous portion of SEQ ID NO:97 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:97. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:98, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:98, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:98. In some embodiments, the target site is a contiguous portion of SEQ ID NO:98 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:98.

[0256] In some embodiments, the gRNA targeting a target site for HEY 1 comprises a spacer sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:111 or SEQ ID NO: 112 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ D NO: 111 or SEQ ID NO: 112. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 111, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 111. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 111. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 111. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 112, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 112. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 112.

[0257] In some embodiments, the gRNA targeting a target site for HEY 1 comprises a sequence comprising a spacer sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for HEY1 comprises modified nucleotides.

[0258] In some embodiments, the gRNA targets a target site for RUNX3. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO:99 or SEQ ID NO: 100 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO:99 or SEQ ID NO: 100. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:99, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO:99, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:99. In some embodiments, the target site is a contiguous portion of SEQ ID NO:99 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO:99. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 100, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 100. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 100 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 100.

[0259] In some embodiments, the gRNA targeting a target site for RUNX3 comprises a spacer sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 113 or SEQ ID NO: 114 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ D NO: 113 or SEQ ID NO: 114. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 113, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 113. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 113. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 113. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 114, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 114. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 114. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 114.

[0260] In some embodiments, the gRNA targeting a target site for RUNX3 comprises a sequence comprising a spacer sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for RUNX3 comprises modified nucleotides.

[0261] In some embodiments, the gRNA targets a target site for SPI1. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 101 or SEQ ID NO: 102 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO: 101 or SEQ ID NO: 102. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 101, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 101, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 101. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 101 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 101. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 102, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 102, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 102. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 102 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in SEQ ID NO: 102.

[0262] In some embodiments, the gRNA targeting a target site for RUNX3 comprises a spacer sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 115 or SEQ ID NO: 116 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ D NO: 115 or SEQ ID NO: 116. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 115, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 115. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 115. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 115. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 116, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 116. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 116. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 116.

[0263] In some embodiments, the gRNA targeting a target site for SPI1 comprises a sequence comprising a spacer sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for SPI1 comprises modified nucleotides.

[0264] In some embodiments, the gRNA targets a target site for ILR7a. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO: 103, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 103 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO: 103.

[0265] In some embodiments, the gRNA targeting a target site for ILR7a comprises a spacer sequence set forth in SEQ ID NO: 117, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 117 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 117.

[0266] In some embodiments, the gRNA targeting a target site for ILR7a comprises a sequence comprising a spacer sequence set forth in SEQ ID NO: 117 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for ILR7a comprises modified nucleotides.

[0267] In some embodiments, the gRNA targets a target site for TBX21. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO: 104, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 104 that is 14, 15,

16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO: 104.

[0268] In some embodiments, the gRNA targeting a target site for TBX21 comprises a spacer sequence set forth in SEQ ID NO: 118, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 118 that is 14, 15, 16,

17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 118.

[0269] In some embodiments, the gRNA targeting a target site for TBX21 comprises a sequence comprising a spacer sequence set forth in SEQ ID NO: 118 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for TBX21 comprises modified nucleotides.

[0270] In some embodiments, the gRNA targets a target site for LEF1. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO: 105, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 105 that is 14, 15,

16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO: 105.

[0271] In some embodiments, the gRNA targeting a target site for LEF1 comprises a spacer sequence set forth in SEQ ID NO: 119, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 119 that is 14, 15, 16,

17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 119.

[0272] In some embodiments, the gRNA targeting a target site for LEF1 comprises a sequence comprising a spacer sequence set forth in SEQ ID NO: 119 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for LEF1 comprises modified nucleotides.

[0273] In some embodiments, the gRNA targets a target site for CBFB. In some embodiments, the gRNA targets the target site that comprises a sequence set forth in SEQ ID NO: 106, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of SEQ ID NO: 106 that is 14, 15,

16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in SEQ ID NO: 106.

[0274] In some embodiments, the gRNA targeting a target site for CBFB comprises a spacer sequence set forth in SEQ ID NO: 120, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 120 that is 14, 15, 16,

17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 120.

[0275] In some embodiments, the gRNA targeting a target site for CBFB comprises a sequence comprising a spacer sequence set forth in SEQ ID NO: 120 and further comprises a scaffold sequence, such as any scaffold sequence disclosed herein. In some embodiments, the gRNA targeting a target site for CBFB comprises modified nucleotides.

[0276] In some embodiments, provided herein is a combination of gRNAs that each target a target site for a gene for transcriptional activation. In some embodiments, provided herein is a multiplexed DNA-targeting system comprising the combination of gRNAs.

[0277] In some embodiments, the combination of gRNAs comprises at least two gRNAs targeting at least two different genes for transcriptional activation. In some embodiments, the gRNAs target a combination of genes selected from: RUNX3, IL7Ra, TBX21, CBFB, EEF1, MYB, RUNX1, SPI1, HEY1,TCF7, GATA3, and BCE11B. In some embodiments, each gRNA of the combination of gRNAs is selected from any of the gRNAs described herein for targeted transcriptional activation. In some embodiments, the combination of gRNAs comprises a first gRNA targeted to a first gene and a second gRNA targeted to a second gene. In some embodiments, the combination of gRNAs comprises at least three gRNAs targeting at least three different genes. In some embodiments, the gRNAs are a combination of gRNAs that bind to target sites for any of the transcription factor genes or any of the combinations of transcription factor genes listed in Section I.B.

D. Other DNA-Binding Domains and DNA-Targeting Systems

[0278] In some of any of the provided embodiments, the DNA-binding domain comprises a zinc finger protein (ZFP); a transcription activator-like effector (TAEE); a meganuclease; a homing endonuclease; or an I-Scel enzyme or a variant thereof. In some embodiments, the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing. In some embodiments, the fusion protein of the DNA-targeting system, or one or more DNA-targeting modules thereof, comprises a DNA-binding domain described herein, such as a DNA-binding domain that is an engineered zinc finger protein (eZFP) or a TAEE.

[0279] In some embodiments, a ZFP, a zinc finger DNA binding protein, or zinc finger DNA binding domain, is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Among the ZFPs are artificial, or engineered ZFPs (eZFPs), comprising ZFP domains targeting specific DNA sequences, typically 9-18 nucleotides long, generated by assembly of individual fingers. ZFPs include those in which a single finger domain is approximately 30 amino acids in length and contains an alpha helix containing two invariant histidine residues coordinated through zinc with two cysteines of a single beta turn, and having two, three, four, five, or six fingers. Generally, sequence-specificity of a ZFP may be altered by making amino acid substitutions at the four helix positions (-1, 2, 3, and 6) on a zinc finger recognition helix. Thus, for example, the ZFP or ZFP-containing molecule is non-naturally occurring, e.g., is an eZFP that is engineered to bind to a target site of choice.

[0280] In some embodiments, zinc fingers are custom-designed (i.e. designed by the user), or obtained from a commercial source. Various methods for designing zinc finger proteins are available. For example, methods for designing zinc finger proteins to bind to a target DNA sequence of interest are described, for example in Liu, Q. et al., PNAS, 94(l l):5525-30 (1997); Wright, D.A. et al., Nat. Protoc., 1(3): 1637-52 (2006); Gersbach, C.A. et al., Acc. Chem. Res., 47(8):2309-18 (2014); Bhakta M.S. et al., Methods Mol. Biol., 649:3-30 (2010); and Gaj et al., Trends Biotechnol, 31(7):397-405 (2013). In addition, various web-based tools for designing zinc finger proteins to bind to a DNA target sequence of interest are publicly available. See, for example, the Zinc Finger Tools design web site from Scripps available on the world wide web at scripps.edu/barbas/zfdesign/zfdesignhome.php. Various commercial services for designing zinc finger proteins to bind to a DNA target sequence of interest are also available. See, for example, the commercially available services or kits offered by Creative Biolabs (world wide web at creative-biolabs.com/Design-and-Synthesis-of-Artificial-Zinc-Finger-Proteins.html), the Zinc Finger Consortium Modular Assembly Kit available from Addgene (world wide web at addgene.org/kits/zfc-modular-assembly/), or the CompoZr Custom ZFN Service from Sigma Aldrich (world wide web at sigmaaldrich.com/life-science/zinc-finger-nuclease- technology /custom- zfn.html).

[0281] In some embodiments, the fusion protein of the DNA-targeting system comprises an eZFP DNA-binding domain and an effector domain.

[0282] Transcription activator-like effectors (TALEs), are proteins naturally found in Xanthomonas bacteria. TALEs comprise a plurality of repeated amino acid sequences, each repeat having binding specificity for one base in a target sequence. Each repeat comprises a pair of variable residues in position 12 and 13 (repeat variable diresidue; RVD) that determine the nucleotide specificity of the repeat. In some embodiments, RVDs associated with recognition of the different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A and YG for recognizing T, TL for recognizing A, VT for recognizing A or G and SW for recognizing A. In some embodiments, RVDs can be mutated towards other amino acid residues in order to modulate their specificity towards nucleotides A, T, C and G and in particular to enhance this specificity. Binding domains with similar modular base-per-base nucleic acid binding properties can also be derived from different bacterial species. These alternative modular proteins may exhibit more sequence variability than TALE repeats.

[0283] In some embodiments, a “TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains/units. The repeat domains, each comprising a repeat variable diresidue (RVD), are involved in binding of the TALE to its cognate target DNA sequence. A single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. TALE proteins may be designed to bind to a target site using canonical or non-canonical RVDs within the repeat units. See, e.g., U.S. Pat. Nos. 8,586,526 and 9,458,205.

[0284] In some embodiments, the fusion protein of the DNA-targeting system comprises a TALE DNA-binding domain and an effector domain.

[0285] Zinc finger and TALE DNA-binding domains can be engineered to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger protein, by engineering of the amino acids in a TALE repeat involved in DNA binding (the repeat variable diresidue or RVD region), or by systematic ordering of modular DNA-binding domains, such as TALE repeats or ZFP domains. Therefore, engineered zinc finger proteins or TALE proteins are proteins that are non-naturally occurring. Non-limiting examples of methods for engineering zinc finger proteins and TALEs are design and selection. A designed protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP or TALE designs (canonical and non-canonical RVDs) and binding data. See, for example, U.S. Pat. Nos. 9,458,205; 8,586,526; 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

E. Effector Domains

[0286] In some aspects, the DNA-targeting systems provided herein further include one or more effector domains, such as a transcriptional activator effector domain. In some embodiments, provided herein is a DNA-targeting system comprising a fusion protein comprising: (a) a DNA-binding domain capable of being targeted to a target site in a gene or regulatory DNA element thereof, such as any DNA-binding domain described above in Section I.C or Section I.D, and (b) at least one effector domain. In some aspects, the effector domain is capable of increasing transcription of the gene, such as any of the genes described in Section I.B. In some aspects, the effector domain comprises a transcription activation domain.

[0287] In some aspects, the effector domain activates, induces, catalyzes, or leads to increased transcription of a gene when ectopically recruited to the gene or DNA regulatory element thereof. In some embodiments, the effector domain activates, induces, catalyzes, or leads to: transcription activation, transcription co-activation, transcription elongation, transcription de-repression, transcription factor release, polymerization, histone modification, histone acetylation, histone deacetylation, nucleosome remodeling, chromatin remodeling, reversal of heterochromatin formation, proteolysis, ubiquitination, deubiquitination, phosphorylation, dephosphorylation, DNA methylation, DNA demethylation, histone methylation, histone demethylation, or DNA base oxidation. In some embodiments, the effector domain activates, induces, catalyzes or leads to transcription activation, transcription coactivation, or transcription elongation. In some embodiments, the effector domain induces transcription activation. In some embodiments, the effector domain has one of the aforementioned activities itself (i.e. acts directly). In some embodiments, the effector domain recruits and/or interacts with a polypeptide domain that has one of the aforementioned activities (i.e. acts indirectly).

[0288] Gene expression of endogenous mammalian genes, such as human genes, can be achieved by targeting a fusion protein comprising a DNA-binding domain, such as a dCas9, and an effector domain, such as a transcription activation domain, to mammalian genes or regulatory DNA elements thereof (e.g. a promoter or enhancer) via one or more gRNAs. Any of a variety of effector domains for transcriptional activation (e.g. transcription activation domains) are known and can be used in accord with the provided embodiments. Transcription activation domains, as well as activation of target genes by Cas fusion proteins (with a variety of Cas molecules) and the transcription activation domains, are described, for example, in WO 2014/197748, WO 2016/130600 , WO 2017/180915, WO 2021/226555 , WO 2021/226077, WO 2013/176772 , WO 2014/152432, WO 2014/093661, WO 2024/015881, Adli, M. Nat. Commun. 9, 1911 (2018), Perez-Pinera, P. et al. Nat. Methods 10, 973-976 (2013), Mali, P. et al. Nat. Biotechnol. 31, 833-838 (2013), and Maeder, M. L. et al. Nat. Methods 10, 977-979 (2013), the disclosures of which are incorporated by reference in their entirety.

[0289] In some embodiments, a transcriptional activation domain comprises a domain of a protein selected from among VP64, p65, Rta, p300, CBP, VPR, VPH, HSF1, a TET protein (e.g. TET1), a partially or fully functional fragment or domain thereof, or a combination of any of the foregoing. In some embodiments, a transcriptional activator domain further comprises at least one domain of a protein selected from among FOXO3 and NCOA3, that exhibits transcriptional activation, is capable of inducing or activating transcription from a gene, is a functional transcriptional activation domain, and/or has a function of transcription activation. In some embodiments, a transcriptional activator domain further comprises at least one domain selected from among FOXO3 and NCOA3.

[0290] In some embodiments, the transcriptional activation domain comprises a VP64 domain. For example, dCas9-VP64 can be targeted to a target site by one or more gRNAs to activate a gene. VP64 is a polypeptide composed of four tandem copies of VP 16, a 16 amino acid transactivation domain of the Herpes simplex virus. VP64 domains, including in dCas fusion proteins, have been described, for example, in WO 2014/197748, WO 2013/176772, WO 2014/152432, and WO 2014/093661. In some embodiments, the transcriptional activation domain comprises at least one VP 16 domain, or a VP 16 tetramer (“VP64”) or a variant thereof. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:60, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:60, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:60. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:62, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:62, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO:62. [0291] In some embodiments, the transcriptional activation domain comprises a p65 activation domain (p65AD). p65AD is the principal transactivation domain of the 65kDa polypeptide of the nuclear form of the NF-KB transcription factor. An exemplary sequence of human transcription factor p65 is available at the Uniprot database under accession number Q04206. p65 domains, including in dCas fusion proteins, have been described, for example in WO 2017/180915 and Chavez, A. et al. Nat. Methods 12, 326-328 (2015). An exemplary p65 activation domain is set forth in SEQ ID NO: 123. In some embodiments, the transcriptional activation domain comprises SEQ ID NO: 123, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 123, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO: 123.

[0292] In some embodiments, the transcriptional activation domain comprises an R transactivator (Rta) domain. Rta is an immediate-early protein of Epstein-Barr virus (EBV), and is a transcriptional activator that induces lytic gene expression and triggers virus reactivation. The Rta domain, including in dCas fusion proteins, has been described, for example in WO 2017/180915 and Chavez, A. et al. Nat. Methods 12, 326-328 (2015). An exemplary Rta domain is set forth in SEQ ID NO: 124. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:80, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 124, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO: 124.

[0293] In some embodiments, the transcriptional activation domain comprises a CREB- binding protein (CBP) domain or a p300 domain. In some aspects, CBP refers to the CREB- binding protein encoded by the human CREBBP gene. CBP is a coactivator that interacts with cAMP-response element binding protein (CREB). In some aspects, p300 refers to the Histone acetyltransferase p300 protein encoded by the human EP300 gene, and is a coactivator closely related to CBP. CBP and p300 each interact with a variety of transcriptional activators to affect gene transcription (Gerritsen, M.E. et al. PNAS 94(7):2927-2932 (1997)). In some embodiments, the transcriptional activation domain comprises a p300 domain. p300 domains (such as the catalytic core of p300) including in dCas fusion proteins for gene activation, has been described, for example, in WO 2016/130600, WO 2017/180915, and Hilton, I.B. et al., Nat. Biotechnol. 33(5):510-517 (2015). An exemplary human CBP sequence is set forth in SEQ ID NO: 125. An exemplary human p300 sequence is set forth in SEQ ID NO: 126. An exemplary p300 domain is set forth in SEQ ID NO: 127. In some embodiments, the transcriptional activation domain comprises any one of SEQ ID NOS: 125- 127, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOS: 125- 127, or a portion thereof. In some embodiments, the transcriptional activation domain comprises SEQ ID NO: 127, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 127, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO: 127.

[0294] In some embodiments, the transcriptional activation domain comprises a HSF1 domain. In some aspects, HSF1 refers to the Heat shock factor protein 1 protein encoded by the human HSF1 gene. HSF1, including in dCas fusion proteins for gene activation, has been described, for example, in WO 2021/226555, WO 2015/089427, and Konermann et al. Nature 517(7536):583-8 (2015). An exemplary human HSF1 sequence is set forth in SEQ ID NO: 128. An exemplary HSF1 domain sequence is set forth in SEQ ID NO: 128. In some embodiments, the transcriptional activation domain comprises SEQ ID NO: 128 or SEQ ID NO: 129, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 128 or SEQ ID NO: 129, or a portion thereof. In some embodiments, the transcriptional activation domain comprises SEQ ID NO: 128, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 128, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO: 129.

[0295] In some embodiments, the transcriptional activation domain comprises the tripartite activator VP64-p65-Rta (also known as VPR). VPR comprises three transcription activation domains (VP64, p65, and Rta) fused by short amino acid linkers, and can effectively upregulate target gene expression. VPR, including in dCas fusion proteins for gene activation, has been described, for example, in WO 2021/226555 and Chavez, A. et al. Nat. Methods 12, 326-328 (2015). An exemplary VPR polypeptide is set forth in SEQ ID NO: 130. In some embodiments, the transcriptional activation domain comprises SEQ ID NO: 130, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 130 or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO: 130. [0296] In some embodiments, the transcriptional activation domain comprises VPH. VPH is a tripartite activator polypeptide comprising VP64, mouse p65, and HSF1. VPH, including in dCas fusion proteins for gene activation, has been described, for example, in WO 2021/226555. An exemplary VPH polypeptide is set forth in SEQ ID NO: 131. In some embodiments, the transcriptional activation domain comprises SEQ ID NO:131, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:131, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO: 131.

[0297] In some embodiments, the transcriptional activation effector domain has demethylase activity. The effector domain may include an enzyme that remove methyl (CH3-) groups from nucleic acids, proteins (in particular histones), and other molecules. The effector domain may covert the methyl group to hydroxymethylcytosine in a mechanism for demethylating DNA. Alternatively, the transcriptional activation domain can convert the methyl group to hydroxymethylcytosine in a mechanism for demethylating DNA. The effector domain can catalyze this reaction. For example, the transcriptional activation domain that catalyzes this reaction may comprise a domain from a TET protein, for example TET1 (Ten-eleven translocation methylcytosine dioxygenase 1). In some aspects, TET1 refers to the Methylcytosine dioxygenase TET1 protein encoded by the human TET1 gene. TET1 catalyzes the conversion of the modified genomic base 5-methylcytosine (5mC) into 5- hydroxymethylcytosine (5hmC) and plays a key role in active DNA demethylation. TET1, including in dCas fusion proteins for gene activation, has been described, for example, in WO 2021/226555. An exemplary human TET1 sequence is set forth in SEQ ID NO: 132. An exemplary TET1 catalytic domain is set forth in SEQ ID NO: 133. In some embodiments, the transcriptional activation domain comprises SEQ ID NO: 132 or SEQ ID NO: 133, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 132 or SEQ ID NO: 133, or a portion thereof. In some embodiments, the transcriptional activation domain comprises SEQ ID NO: 132, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 133, or a portion thereof. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO: 133.

[0298] In some embodiments, the effector domain may comprise a SunTag domain. SunTag is a repeating peptide array, which can recruit multiple copies of an antibody-fusion protein that binds the repeating peptide. The antibody-fusion protein may comprise an additional effector domain, such as a transcription activation domain (e.g. VP64), to induce increased transcription of the target gene. SunTag, including in dCas fusion proteins for gene activation, has been described, for example, in WO 2016/011070 and Tanenbaum, M. et al. Cell. 159(3):635-646 (2014). An exemplary SunTag effector domain includes a repeating GCN4 peptide having the amino acid sequence LLPKNYHLENEVARLKKLVGER (SEQ ID NO: 134) separated by linkers having the amino acid sequence GGSGG (SEQ ID NO: 135). In some embodiments, the effector domain comprises the sequence set forth in SEQ ID NO: 134, a domain thereof, a portion thereof, or a variant thereof, or an amino acid sequence that has at least 135%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing. In some embodiments, the SunTag effector domain recruits an antibody-fusion protein that comprises a transcriptional activator effector domain (e.g. VP64) and binds the GCN4 peptide, thereby activating transcription at the target site and acting as a transcriptional activator effector domain.

[0299] In some embodiments, a transcriptional activation domain comprises a FOXO3 domain, i.e. a domain from FOXO3. In some aspects, FOXO3 refers to the Forkhead box protein 03 encoded by the human FOXO3 gene. FOXO3 functions as a transcriptional activator that recognizes and binds to specific DNA sequences. An exemplary human FOXO3 sequence is set forth in SEQ ID NO: 136. An exemplary FOXO3 domain sequence is set forth in SEQ ID NO: 137 and SEQ ID NO: 138. In some embodiments, the transcriptional activation domain comprises a sequence set forth in any of SEQ ID NOS:136-138 or a domain or a portion thereof, such as a contiguous portion thereof of at least 10, 15, 20, 22, 25, 30, 35, 37, 40, 42, 45, 47, 49, 50, 55, 57, 60, 61, 62, 65, 70, 72, 75, 76, or 80 amino acids, such as at least 20 amino acids, or a variant thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in any of SEQ ID NOS: 136- 138 or a domain or a portion thereof, such as a contiguous portion thereof of at least 10, 15, 20, 22, 25, 30, 35, 37, 40, 42, 45, 47, 49, 50, 55, 57, 60, 61, 62, 65, 70, 72, 75, 76, or 80 amino acids, such as at least 20 amino acids, or a variant thereof. In some embodiments, the transcriptional activation domain is or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 136. In some embodiments, the transcriptional activation domain comprises a contiguous portion of SEQ ID NO: 136 that is at least 80 amino acids in length. In some embodiments, the transcriptional activation domain comprises SEQ ID NO: 137. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO: 137. An exemplary nucleotide sequence encoding the transcriptional activation domain set forth in SEQ ID NO: 137 is set forth in SEQ ID NO:222. In some embodiments, the transcriptional activation domain is or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 138. In some embodiments, the transcriptional activation domain comprises a contiguous portion of SEQ ID NO: 136 that is at least 42 amino acids in length. In some embodiments, the transcriptional activation domain comprises SEQ ID NO: 138. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO: 138.

[0300] In some embodiments, a transcriptional activation domain comprises a NCOA3 domain, i.e. a domain from NCOA3. In some aspects, NCOA3 refers to the Nuclear receptor coactivator 3 protein encoded by the human NCOA3 gene. NCOA3 functions as a transcriptional coactivator for steroid receptors and nuclear receptors. An exemplary human NCOA3 sequence is set forth in SEQ ID NO: 139. An exemplary NCOA3 domain sequence is set forth in SEQ ID NO: 140 and SEQ ID NO: 141. In some embodiments, the transcriptional activation domain comprises a sequence set forth in any of SEQ ID NOS: 141, 139, and 140 or a domain or a portion thereof, such as a contiguous portion thereof of at least 10, 15, 20, 22, 25, 30, 35, 37, 40, 42, 45, 47, 49, 50, 55, 57, 60, 61, 62, 65, 70, 72, 75, 76, or 80 amino acids, such as at least 20 amino acids, or a variant thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in any of SEQ ID NOS: 141, 139, and 140 or a domain or a portion thereof, such as a contiguous portion thereof of at least 10, 15, 20, 22, 25, 30, 35, 37, 40, 42, 45, 47, 49, 50, 55, 57, 60, 61, 62, 65, 70, 72, 75, 76, or 80 amino acids, such as at least 20 amino acids, or a variant thereof. In some embodiments, the transcriptional activation domain is or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 140. In some embodiments, the transcriptional activation domain comprises a contiguous portion of SEQ ID NO: 139 that is at least 80 amino acids in length. In some embodiments, the transcriptional activation domain comprises SEQ ID NO: 140. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO: 140. An exemplary nucleotide sequence encoding the transcriptional activation domain set forth in SEQ ID NO: 140 is set forth in SEQ ID NO: 185. In some embodiments, the transcriptional activation domain is or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:141. In some embodiments, the transcriptional activation domain comprises a contiguous portion of SEQ ID NO: 139 that is at least 49 amino acids in length. In some embodiments, the transcriptional activation domain comprises SEQ ID NO: 141. In some embodiments, the transcriptional activation domain is set forth in SEQ ID NO: 141.

[0301] In some embodiments, the transcriptional activation domain comprises a fusion of NCOA3 and FOXO3 domains as described herein, e.g. the NCOA3 domain set forth in SEQ ID NO: 141 and the FOXO3 domain set forth in SEQ ID NO: 138. In some embodiments, the transcriptional activation domain comprises a fusion of two NCOA3 domains and one FOXO3. The fusion protein contains these domains can be arranged in any order. In some embodiments, the transcriptional activation domain is arranged, from N terminus to C terminus, as follows: a first NCOA3 domain, a FOXO3 domain, and a second NCOA3 domain, also referred to as a NCOA3-FOXO3-NCOA3 domain (NFN). In some embodiments, the NFN domain is or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 142. In some embodiments, the NFN domain comprises SEQ ID NO: 142. In some embodiments, the NFN domain is SEQ ID NO: 142. In some embodiments, the domains are either directly linked to each other, or they are linked via a linker, such as a peptide linker.

[0302] In some embodiments, the fusion protein comprises a transcriptional activation domain that is a NFN domain. In some embodiments, the fusion protein further comprises an additional transcriptional activation domain that is a VP64 domain. In some embodiments, the NFN and VP64 domains are either directly linked to each other, or they are linked via a linker, or they are separated by a DNA-binding domain. In some embodiments, the NFN and VP64 domains are separated by a DNA-binding domain.

F. Fusion Proteins

[0303] In some aspects, the DNA-targeting systems provided herein include fusion proteins. In some embodiments, the fusion protein comprises: (a) a DNA-binding domain capable of being targeted to a target site for one or more genes, and (b) at least one transcriptional activator effector domain for increasing transcription of the one or more genes.

[0304] In some embodiments, the fusion protein comprises at least one of any of the DNA- binding domains described herein in Section I.C or Section I.D, and at least one of any of the effector domains described herein. In some embodiments, the fusion protein contains a CRISPR/Cas-based DNA-binding domain, such as described in Section I.C., and at least one effector domain for transcriptional activation, as described in section I.E. In some aspects, the fusion protein is targeted to a target site in a gene or regulatory element thereof, and leads to increased or activated transcription of the gene. In some aspects, the fusion protein is targeted to target sites in a combination of genes or regulatory elements thereof, and leads to increased or activated transcription of each of the genes.

[0305] In some embodiments, the DNA-binding domain and effector domain of the fusion protein are heterologous, i.e. the domains are from different species, or at least one of the domains is not found in nature. In some aspects, the fusion protein is an engineered fusion protein, i.e. the fusion protein is not found in nature.

[0306] In some embodiments, the at least one effector domain is fused to the N-terminus, the C-terminus, or both the N-terminus and the C-terminus, of the DNA-binding domain or a component thereof. The at least one effector domain may be fused to the DNA-binding domain directly, or via any intervening amino acid sequence, such as a linker sequence or a nuclear localization sequence (NLS).

[0307] In some embodiments, the fusion protein of a provided DNA-binding system, or a DNA-targeting module thereof, comprises, from N- to C-terminal order: a transcriptional activator effector domain and a DNA-binding domain. In some embodiments, the fusion protein of a provided DNA-binding system, or a DNA-targeting module thereof, comprises, from N- to C-terminal order: a DNA-binding domain and a transcriptional activator effector domain.

[0308] In some embodiments, the at least one effector domain of the fusion protein includes more than one effector domain. In some embodiments, the fusion protein includes 2, 3 or 4 effector domains, or more than 4 effector domains. In some embodiments, at least two of the effector domains of the fusion protein are different. In some embodiments, each of the effector domains of the fusion protein are different. In some embodiments, the at least one effector domain includes two effector domains in which the two effector domains are different. In some embodiments, the effector domains and the DNA-binding domain can be arranged in any order.

[0309] In some embodiments, the at least one effector domain of the fusion protein includes two different effector domains. The two different effector domains and the DNA-binding domain can be arranged in any order. In some embodiments, each of the effector domains are N-terminal to the DNA-binding domain in which a first effector domain is fused to the N- terminus of the second effector domain and the second effector domain is fused to the N- terminus of the DNA-binding domain. In some embodiments, the fusion protein of a provided DNA-binding system, or a DNA-targeting module thereof, comprises from N- to C-terminal order: a first effector domain, a second effector domain and the DNA binding domain. In some embodiments, each of the effector domains are C-terminal to the DNA-binding domain in which a first effector domain is fused to the C-terminus of the DNA-binding domain and the second effector domain is fused to the C-terminus of the first effector domain. In some embodiments, the fusion protein of a provided DNA-binding system, or a DNA-targeting module thereof, comprises from N- to C-terminal order: a DNA-binding domain, a first effector domain, and a second effector domain. In some embodiments, the DNA-binding domain is between the effector domains, in which one effector domain is fused to the N-terminus of the DNA-binding domain and the other effector domain is fused to the C-terminus of the DNA-binding domain. In some embodiments, the fusion protein of a provided DNA-binding system, or a DNA- targeting module thereof, comprises from N- to C-terminal order: a first effector domain, a DNA-binding domain, and a second effector domain. In some embodiments, one or more of the components may be fused to each other directly, or via any intervening amino acid sequence, such as via a linker sequence or a nuclear localization sequence (NLS).

[0310] In some embodiments, the fusion protein comprises one or more linkers. In some embodiments, the linker is a peptide linker. In some embodiments, the one or more linkers connect the DNA-binding domain or a component thereof to the at least one effector domain. A linker may be included anywhere in the polypeptide sequence of the fusion protein, for example, between the effector domain and the DNA-binding domain or a component thereof. A linker may be of any length and designed to promote or restrict the mobility of components in the fusion protein. A linker may comprise any amino acid sequence of about 2 to about 100, about 5 to about 80, about 10 to about 60, or about 20 to about 50 amino acids. A linker may comprise an amino acid sequence of at least about 2, 3, 4, 5, 10, 15, 20, 25, or 30 amino acids. A linker may comprise an amino acid sequence of less than about 100, 90, 80, 70, 60, 50, or 40 amino acids. A skilled artisan can readily choose an appropriate linker for the connection of two domains. In some embodiments, the linker is a flexible linker. Flexible linkers are generally composed of small, non-polar or polar residues such as glycine, serine or threonine. A linker may include sequential or tandem repeats of an amino acid sequence that is 2 to 20 amino acids in length. Linkers may be rich in amino acids glycine (G), serine (S), and/or alanine (A). Linkers may include, for example, a GS linker. An exemplary linker is represented by the sequence GGGGS (SEQ ID NO: 143). A linker may comprise repeats of a sequence, for example as represented by the formula (GGGGS)n, wherein n is an integer that represents the number of times the GGGGS sequence is repeated (e.g. between 1 and 10 times). The number of times a linker sequence is repeated can be adjusted to optimize the linker length and achieve appropriate separation of the functional domains. For example, in some embodiments, the linker is the (GGGGS)n linker, whereby n is an integer of 1 to 10. Other examples of linkers may include, for example, GGGGG (SEQ ID NO: 144), GGAGG (SEQ ID NO: 145), GGGGSSS (SEQ ID NO: 146), or GGGGAAA (SEQ ID NO: 147).

[0311] In some embodiments, artificial linker sequences can be used. In some embodiments, the linker is EASGSGRASPGIPGSTR (SEQ ID NO: 148). In some embodiments, the linker is linker is GIHGVPAA (SEQ ID NO: 149). In some embodiments, the linker is SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 150). In some embodiments, the linker is KRPAATKKAGQAKKKKASDAKSLTAWS (SEQ ID NO: 151).

[0312] In some embodiments, the linker is an XTEN linker. In some aspects, an XTEN linker is a recombinant polypeptide (e.g., an unstructured recombinant peptide) lacking hydrophobic amino acid residues. Exemplary XTEN linkers are described in, for example, Schellenberger et al., Nature Biotechnology 27, 1186-1190 (2009) or WO 2021/247570. In some embodiments, a linker comprises the sequence set forth in SEQ ID NO: 152, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 152. In some aspects, the linker comprises the sequence set forth in SEQ ID NO: 154, or a contiguous portion of SEQ ID NO: 152 of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 amino acids. In some aspects, the linker consists of the sequence set forth in SEQ ID NO: 152, or a contiguous portion of SEQ ID NO: 152 of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 amino acids. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO: 152. In some embodiments, the linker consists of the sequence set forth in SEQ ID NO: 152. In some embodiments, a linker comprises the sequence set forth in SEQ ID NO: 153, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing. In some aspects, the linker comprises the sequence set forth in SEQ ID NO: 153, or a contiguous portion of SEQ ID NO: 153 of at least 5, 10, orl5 amino acids. In some aspects, the linker consists of the sequence set forth in SEQ ID NO: 153, or a contiguous portion of SEQ ID NO: 153 of at least 5, 10 or 15 amino acids. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO: 153. In some embodiments, the linker consists of the sequence set forth in SEQ ID NO: 153.

[0313] Appropriate linkers may be selected or designed based rational criteria known in the art, for example as described in Chen et al. Adv. Drug Deliv. Rev. 65(10): 1357-1369 (2013). In some embodiments, a linker comprises a linker described in WO 2021/247570.

[0314] In some embodiments, the fusion protein of the DNA-targeting system, or a DNA- targeting module thereof, comprises one or more nuclear localization signals (NLS). In some embodiments, a fusion protein described herein comprises one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies. Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO 155): the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 156); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 157) or RQRRNELKRSP (SEQ ID NO: 158); the hRNPAl M9 NLS having the sequence NQSSNEGPMKGGNEGGRSSGPYGGGGQYEAKPRNQGGY (SEQ ID NO: 159); the sequence RMRIZEKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 160) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 161) and PPKKARED (SEQ ID NO: 162) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 163) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 164) of mouse c- abl IV; the sequences DRLRR(SEQ ID NO: 165) and PKQKKRK (SEQ ID NO: 166) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 167) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 168) of the mouse Mxl protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 169) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 170) of the steroid hormone receptors (human) glucocorticoid. The NLS may comprise a portion of any of the foregoing. In general, the one or more NLSs are of sufficient strength to drive accumulation of the fusion protein in a detectable amount in the nucleus of a eukaryotic cell. In general, strength of nuclear localization activity may derive from the number of NLSs in the fusion protein, the particular NLS(s) used, or a combination of these factors. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the fusion protein, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g. a stain specific for the nucleus such as DAPI). Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of the fusion protein (e.g. an assay for altered gene expression activity in a cell transformed with the DNA-targeting system comprising the fusion protein), as compared to a control condition (e.g. an untransformed cell).

[0315] In some embodiments, the NLS is linked to the N-terminus or the C-terminus of the DNA-binding domain via a linker. In some embodiments, the NLS is linked to the N-terminus or the C-terminus of an effector domain via a linker. The linker may be any linker as described above. In some embodiments, the linker is GIHGVPAA (SEQ ID NO: 149). In some embodiments, the NLS and linker has the sequence PKKKRKVGIHGVPAA (SEQ ID NO: 171).

[0316] In some configurations, the N- or C-terminus of the fusion protein can be linked to a moiety for detection and/or purification. In some aspects, the moiety is or includes a Flag tag DYKDDDDK (SEQ ID NO: 172), a 3xFlag tag MDYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 173), an HA tag YPYDVPDYA (SEQ ID NO: 174) or a His tag, such as HHHHHH (SEQ ID NO: 175).

[0317] In some embodiments, the fusion protein is a dCas-VP64 fusion protein, such as dSpCas9-2xVP64, which is a fusion of dSpCas9 fused to two copies of VP64. In some embodiments, the fusion protein is dSpCas9-2xVP64. In some embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO:58, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO:58. In some embodiments, the fusion protein is encoded by the nucleotide sequence set forth in SEQ ID NO:57.

/. Split Fusion Proteins

[0318] In some embodiments, the fusion protein is a split protein, i.e. comprises two or more separate polypeptide domains that interact or self-assemble to form a functional fusion protein. In some aspects, the split fusion protein comprises a dCas9 and an effector domain. In some aspects, the fusion protein comprises a split dCas9-effector domain fusion protein. [0319] In some embodiments, the split fusion protein is assembled from separate polypeptide domains comprising trans-splicing inteins. Inteins are internal protein elements that self-excise from their host protein and catalyze ligation of flanking sequences with a peptide bond. In some embodiments, the split fusion protein is assembled from a first polypeptide comprising an N-terminal intein and a second polypeptide comprising a C-terminal intein. In an exemplary embodiment, the N terminal intein is the N terminal Npu Intein set forth in SEQ ID NO: 176 In some embodiments, the C terminal intein is the C terminal Npu intein set forth in SEQ ID NO: 177

[0320] In some embodiments, the split fusion protein comprises a split dCas9-effector domain fusion protein assembled from two polypeptides. In an exemplary embodiment, the first polypeptide comprises an effector domain catalytic domain and an N-terminal fragment of dSpCas9, followed by an N terminal Npu Intein (effector domain-dSpCas9-573N), and the second polypeptide comprises a C terminal Npu Intein, followed by a C-terminal fragment of dSpCas9 (dSpCas9-573C). The N- and C-terminal fragments of the fusion protein are split at position 573Glu of the SpCas9 molecule, with reference to SEQ ID NO: 63 (corresponding to residue 572Glu of the dSpCas9 molecule set forth in SEQ ID NO: 196). In some aspects, the N- terminal Npu Intein (SEQ ID NO: 176) and C-terminal Npu Intein (set forth in SEQ ID NO: 177) may self-excise and ligate the two fragments, thereby forming the full-length dSpCas9-effector domain fusion protein when expressed in a cell.

[0321] In some embodiments, the polypeptides of a split protein may interact non-covalently to form a complex that recapitulates the activity of the non-split protein. For example, two domains of a Cas enzyme expressed as separate polypeptides may be recruited by a gRNA to form a ternary complex that recapitulates the activity of the full-length Cas enzyme in complex with the gRNA, for example as described in Wright et al. PNAS 112(10):2984-2989 (2015). In some embodiments, assembly of the split protein is inducible (e.g. light inducible, chemically inducible, small-molecule inducible).

[0322] In some aspects, the two polypeptides of a split fusion protein may be delivered and/or expressed from separate vectors, such as any of the vectors described herein. In some embodiments, the two polypeptides of a split fusion protein may be delivered to a cell and/or expressed from two separate AAV vectors, i.e. using a split AAV-based approach, for example as described in WO 2017/197238. [0323] Approaches for the rationale design of split proteins and their delivery, including Cas proteins and fusions thereof, are described, for example, in WO 2016/114972, WO 2017/197238, Zetsche, et al. Nat. Biotechnol. 33(2): 139-42 (2015), Wright et al. PNAS 112(10):2984-2989 (2015), Truong, et al. Nucleic Acids Res. 43, 6450-6458 (2015), and Fine et al. Sci. Rep. 5, 10777 (2015).

IL POLYNUCLEOTIDES, VECTORS, AND RELATED METHODS FOR DELIVERY

[0324] In some aspects, provided are polynucleotides encoding any of the DNA-targeting systems described herein in Section I or a portion or a component of any of the foregoing. In some aspects, the polynucleotides can encode any of the components of the DNA-targeting systems, and/or any nucleic acid or proteinaceous molecule necessary to carry out aspects of the methods of the disclosure. In particular embodiments, provided are polynucleotides encoding any of the fusion proteins described herein, for example in Section I.F. Also provided herein are polynucleotides encoding any of the gRNAs described herein, for example in Section I.C.

[0325] In some embodiments, provided are polynucleotides comprising the gRNAs described herein. In some embodiments, the gRNA is transcribed from a genetic construct (i.e. vector or plasmid) in the target cell. In some embodiments, the gRNA is produced by in vitro transcription and delivered to the target cell. In some embodiments, the gRNA comprises one or more modified nucleotides for increased stability. In some embodiments, the gRNA is delivered to the target cell pre-complexed as a RNP with the fusion protein.

[0326] In some embodiments, a provided polynucleotide encodes a fusion protein as described herein that includes (a) a DNA-binding domain capable of being targeted to a target site of a target gene as described; and (b) at least one effector domain capable of increasing transcription of the gene. In some embodiments, the fusion protein includes a fusion protein of a Cas protein or variant thereof and at least one effector domain capable of increasing transcription of a gene. In some embodiments, the Cas is a dCas, such as dCas9. In some embodiments, the dCas9 is a dSpCas9. Examples of such domains and fusion proteins include any as described in Section I.

[0327] In some embodiments, the polynucleotide encodes a dCas-VP64 fusion protein, such as dSpCas9-2xVP64. In some embodiments, the polynucleotide comprises the sequence set forth in SEQ ID NO:57, or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the polynucleotide is set forth in SEQ ID NO:57. In some embodiments, the polynucleotide encodes an amino acid sequence comprising SEQ ID NO:58, or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the polynucleotide encodes the amino acid sequence set forth in SEQ ID NO:58.

[0328] In some embodiments, the polynucleotide is RNA or DNA. In some embodiments, the polynucleotide, such as a polynucleotide encoding a provided fusion protein, is mRNA. The mRNA can be 5' capped and/or 3' poly adenylated. In another embodiment, a polynucleotide provided herein, such as a polynucleotide encoding a provided fusion protein, is DNA. The DNA can be present in a vector.

[0329] Also provided herein is a vector that contains any of the provided polynucleotides. In some embodiments, the vector comprises a genetic construct, such as a plasmid or an expression vector.

[0330] In some embodiments, the expression vector comprising the sequence encoding the fusion protein of a DNA-targeting system provided herein can further comprise a polynucleotide sequence encoding at least one gRNA. The sequence encoding the gRNA can be operably linked to at least one transcriptional control sequence for expression of the gRNA in the cell. For example, DNA encoding the gRNA can be operably linked to a promoter sequence that is recognized by RNA polymerase III (Pol III). Examples of suitable Pol III promoters include, but are not limited to, mammalian U6, U3, Hl, and 7SL RNA promoters.

[0331] In some embodiments, provided is a vector containing a polynucleotide that encodes a fusion protein comprising a DNA-binding domain comprising a dCas and at least one effector domain capable of modulating (e.g. increasing or decreasing) transcription of a gene, and a polynucleotide(s) encoding at least one gRNA. In some embodiments, the dCas is a dCas9, such as dSpCas9. In some embodiments, the polynucleotide encodes a fusion protein that includes a dSpCas9 set forth in SEQ ID NO:64. In some embodiments, the polynucleotide encoding at least one gRNA encodes a gRNA as described in Section I.C.2. For example, the polynucleotide can encode a gRNA comprising a spacer sequence selected from any one of SEQ ID NOS: 19- 36, or a contiguous portion thereof of at least 14 nt. In some embodiments, the polynucleotide encodes the fusion protein and the at least one gRNA.

[0332] In some embodiments, the polynucleotide as provided herein can be codon optimized for efficient translation into protein in the eukaryotic cell or animal of interest. For example, codons can be optimized for expression in humans, mice, rats, hamsters, cows, pigs, cats, dogs, fish, amphibians, plants, yeast, insects, and so forth. Programs for codon optimization are available as freeware. Commercial codon optimization programs are also available.

[0333] In some embodiments, a polynucleotide described herein can comprise one or more transcription and/or translation control elements. Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. can be used in the expression vector.

[0334] Non-limiting examples of suitable eukaryotic promoters (i.e., promoters functional in a eukaryotic cell) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, human elongation factor-1 promoter (EFl), a hybrid construct comprising the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter (CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase- 1 locus promoter (PGK), and mouse metallothionein-I.

[0335] For expressing small RNAs, including guide RNAs used in connection with the DNA-targeting systems, various promoters such as RNA polymerase III promoters, including for example U6 and Hl, can be advantageous. Descriptions of and parameters for enhancing the use of such promoters are known in the art, and additional information and approaches are regularly being described; see, e.g., Ma, H. et al., Molecular Therapy — Nucleic Acids 3, el61 (2014) doi:10.1038/mtna.2014.12.

[0336] The expression vector can also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector can also comprise appropriate sequences for amplifying expression. The expression vector can also include nucleotide sequences encoding non-native tags (e.g., histidine tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to the site-directed polypeptide, thus resulting in a fusion protein.

[0337] A promoter can be an inducible promoter (e.g., a heat shock promoter, tetracycline- regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor- regulated promoter, etc.). The promoter can be a constitutive promoter (e.g., CMV promoter, UBC promoter). In some cases, the promoter can be a spatially restricted and/or temporally restricted promoter (e.g., a tissue specific promoter, a cell type specific promoter (e.g. a T cell specific promoter), etc.). [0338] Expression vectors contemplated include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus) and other recombinant vectors. Other vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pXTl, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Other vectors can be used so long as they are compatible with the host cell.

[0339] In some embodiments, the vector is a viral vector, such as an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, or a gammaretroviral vector. In some embodiments In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is selected from among an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is a non-viral vector, for example a lipid nanoparticle, a liposome, an exosome, or a cell penetrating peptide. In some embodiments, the vector comprises one vector, or two or more vectors.

[0340] In some embodiments, a vector described herein is or comprises a lipid nanoparticle (LNP). Among provided embodiments, is a lipid nanoparticle that contains any of the provided polynucleotides for delivery of an epigenetic-modifying DNA-targeting system. In some embodiments, the LNP contains a polynucleotide that encodes a fusion protein as provided herein that includes (a) a DNA-binding domain capable of being targeted to a target site for one or more genes; and (b) at least one effector domain. In some embodiments, the DNA-binding domain is a Cas (e.g. dCas) and the LNP further includes a gRNA. In some embodiments, the polynucleotide encoding the fusion protein is an mRNA and the gRNA is provided as an RNA.

[0341] In some embodiments, any of the epigenetic-modifying DNA-targeting systems, gRNAs, Cas-gRNA combinations, polynucleotides, fusion proteins, or components thereof described herein, are incorporated in lipid nanoparticles (LNPs), such as for delivery. In some embodiments, the lipid nanoparticle is a vector for delivery. In some embodiments, the nanoparticle may comprise at least one lipid. The lipid may be selected from, but is not limited to, C24, LP-01, C14-4, dLin-DMA, dLin-K-DMA, 98N12- 5, C12-200, dLin-MC3-DMA, dLin- KC2-DMA, DODMA, PLGA, PEG, PEG-DMG and PEGylated lipids. In another aspect, the

I ll lipid may be a cationic lipid such as, but not limited to, dLin-DMA, dLin-D-DMA, dLin-MC 3 - DMA, dLin-KC2-DMA and DODMA. Typically, the LNPs are composed of two or more lipids, such as 3, 4 or 5 lipids. In some embodiments, at least one lipid is either ionizable cationic or cationic. In some embodiments, the lipid may be an ionizable lipid such as, but not limited to, C24, LP-01, and C14-4. Exemplary ionizable lipids include, but are not limited to, those described in published PCT applications WO 2022/081750, WO 2015/095340, WO 2021/077067, WO 2021/077066, and WO 2023/056282, the disclosures of which are incorporated by reference in their entirety, particularly with respect to the described ionizable lipids and related lipid nanoparticles.

[0342] Lipid nanoparticles can be used for the delivery of encapsulated or associated (e.g., complexed) therapeutic agents, including nucleic acids and proteins, such as those encoding and/or comprising CRISPR/Cas systems. See, e.g., US Patent No. 10,723,692, US Patent No. 10,941,395, and WO 2015/035136.

[0343] In some embodiments, the provided methods involve use of a lipid nanoparticle (LNP) comprising mRNA, such as mRNA encoding a protein component of any of the provided DNA-targeting systems, for example any of the fusion proteins provided herein. In some embodiments, the mRNA can be produced using methods known in the art such as in vitro transcription. In some embodiments of the method, the mRNA comprises a 5' cap. In some embodiments, the 5’ cap is an altered nucleotide on the 5’ end of primary transcripts such as messenger RNA. In some aspects, the 5’ caps of the mRNA improves one or more of RNA stability and processing, mRNA metabolism, the processing and maturation of an RNA transcript in the nucleus, transport of mRNA from the nucleus to the cytoplasm, mRNA stability, and efficient translation of mRNA to protein. In some embodiments, a 5’ cap can be a naturally- occurring 5’ cap or one that differs from a naturally-occurring cap of an mRNA. A 5’ cap may be any 5' cap known to a skilled artisan. In certain embodiments, the 5' cap is selected from the group consisting of an Anti-Reverse Cap Analog (ARCA) cap, a 7-methyl-guanosine (7mG) cap, a CleanCap® analog, a vaccinia cap, and analogs thereof. For instance, the 5’ cap may include, without limitation, an anti-reverse cap analogs (ARCA) (US7074596), 7-methyl- guanosine, CleanCap® analogs, such as Cap 1 analogs (Trilink; San Diego, CA), or enzymatically capped using, for example, a vaccinia capping enzyme or the like. In some embodiments, the mRNA may be polyadenylated. The mRNA may contain various 5’ and 3’ untranslated sequence elements to enhance expression of the encoded protein and/or stability of the mRNA itself. Such elements can include, for example, posttranslational regulatory elements such as a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the mRNA comprises at least one nucleoside modification. The mRNA may contain modifications of naturally-occurring nucleosides to nucleoside analogs. Any nucleoside analogs known in the art are envisioned. Such nucleoside analogs can include, for example, those described in US 8,278,036. In certain embodiments of the method, the nucleoside modification is selected from the group consisting of a modification from uridine to pseudouridine and uridine to Nl- methyl pseudouridine. In particular embodiments of the method the nucleoside modification is from uridine to pseudouridine.

[0344] In some embodiments, LNPs useful for in the present methods comprise a cationic lipid selected from dLin-DMA ( 1 ,2-dilinoleyloxy-3 -dimethylaminopropane) , dLin-MC3 -DM A (dilinoleylmethyl-4-dimethylaminobutyrate), dLin-KC2-DMA (2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane), DODMA (1,2- dioleyloxy-N,N-dimethyl-3- aminopropane), SS-OP (Bis[2-(4-{2-[4-(cis-9 octadecenoyloxy )phenylacetoxy]ethyl}piperidinyl)ethyl] disulfide), and derivatives thereof. dLin-MC3-DMA and derivatives thereof are described, for example, in WO 2010/144740. DODMA and derivatives thereof are described, for example, in US 7,745,651 and Mok et al. (1999), Biochimica et Biophysica Acta, 1419(2): 137-150. dLin-DMA and derivatives thereof are described, for example, in US 7,799,565. dLin-KC2-DMA and derivatives thereof are described, for example, in US 9,139,554. SS-OP (NOF America Corporation, White Plains, NY) is described, for example, at https://www.nofamerica.com/store/index.php?dispatch=products.view&product_id=962. Additional and non-limiting examples of cationic lipids include methylpyridiyl-dialkyl acid (MPDACA), palmitoyl-oleoyl-nor-arginine (PONA), guanidino-dialkyl acid (GUADACA), 1,2- di-0-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2- dioleoyl-3- trimethylammonium-propane (DOTAP), Bis{2-[N-methyl-N-(a-D- tocopherolhemisuccinatepropyl)amino]ethyl} disulfide (SS-33/3AP05), Bis{2-[4-(a-D- tocopherolhemisuccinateethyl)piperidyl] ethyl} disulfide (SS33/4PE15), Bis{2-[4-(cis-9- octadecenoateethyl)-l-piperidinyl] ethyl} disulfide (SS18/4PE16), and Bis{2-[4-(cis,cis-9,12- octadecadienoateethyl)-l-piperidinyl] ethyl} disulfide (SS18/4PE13). In further embodiments, the lipid nanoparticles also comprise one or more non-cationic lipids and a lipid conjugate. [0345] In some embodiments, the molar concentration of the cationic lipid is from about 20% to about 80%, from about 30% to about 70%, from about 40% to about 60%, from about 45% to about 55%, or about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% of the total lipid molar concentration, wherein the total lipid molar concentration is the sum of the cationic lipid, the non-cationic lipid, and the lipid conjugate molar concentrations. In certain embodiments, the lipid nanoparticles comprise a molar ratio of cationic lipid to any of the polynucleotides of from about 1 to about 20, from about 2 to about 16, from about 4 to about 12, from about 6 to about 10, or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20.

[0346] In some embodiments, the lipid nanoparticles can comprise at least one non-cationic lipid. In particular embodiments, the molar concentration of the non-cationic lipids is from about 20% to about 80%, from about 30% to about 70%, from about 40% to about 70%, from about 40% to about 60%, from about 46% to about 50%, or about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 48.5%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% of the total lipid molar concentration. Non-cationic lipids include, in some embodiments, phospholipids and steroids.

[0347] In some embodiments, phospholipids useful for the lipid nanoparticles described herein include, but are not limited to, l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- Didecanoyl-sn-glycero-3- phosphocholine (DDPC), l,2-Dierucoyl-sn-glycero-3- phosphate(Sodium Salt) (DEPA-NA), l,2-Dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2- Dierucoyl-sn-glycero-3- phosphoethanolamine (DEPE), l,2-Dierucoyl-sn-glycero-3[Phospho- rac-(l-glycerol)(Sodium Salt) (DEPG-NA), l,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC), 1,2-Dilauroyl-sn- glycero-3-phosphate(Sodium Salt) (DLPA-NA), 1,2-Dilauroyl-sn- glycero-3-phosphocholine (DLPC), l,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE),

1.2-Dilauroyl-sn- glycero-3[Phospho-rac-(l-glycerol...)(Sodium Salt) (DLPG-NA), 1,2- Dilauroyl-sn-glycero- 3[Phospho-rac-(l-glycerol)(Ammonium Salt) (DLPG-NH4), 1,2- Dilauroyl-sn-glycero-3- phosphoserine(Sodium Salt) (DLPS-NA), l,2-Dimyristoyl-sn-glycero-3- phosphate(SodiumSalt) (DMPA-NA), l,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC),

1.2-Dimyristoyl- sn-glycero-3-phosphoethanolamine (DMPE), 1,2-Dimyristoyl-sn-glycero- 3[Phospho-rac-(l- glycerol)(Sodium Salt) (DMPG-NA), l,2-Dimyristoyl-sn-glycero-3[Phospho- rac-(l- glycerol)(Ammonium Salt) (DMPG-NH4), l,2-Dimyristoyl-sn-glycero-3[Phospho-rac-(l- glycerol)(Sodium/ Ammonium Salt) (DMPG-NH4/NA), l,2-Dimyristoyl-sn-glycero-3- phosphoserine(Sodium Salt) (DMPS-NA), l,2-Dioleoyl-sn-glycero-3-phosphate(Sodium Salt) (DOPA-NA), l,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dioleoyl-sn- glycero-3- phosphoethanolamine (DOPE), l,2-Dioleoyl-sn-glycero-3[Phospho-rac-(l- glycerol)(Sodium Salt) (DOPG-NA), l,2-Dioleoyl-sn-glycero-3-phosphoserine(Sodium Salt) (DOPS-NA), 1,2- Dipalmitoyl-sn-glycero-3-phosphate(Sodium Salt) (DPPA-NA), 1,2- Dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1 ,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 ,2- Dipalmitoyl-sn-glycero- 3[Phospho-rac-(l-glycerol)(Sodium Salt) (DPPG-NA), 1,2-Dipalmitoyl- sn-glycero- 3[Phospho-rac-(l-glycerol)(Ammonium Salt) (DPPG-NH4), 1,2-Dipalmitoyl-sn- glycero-3- phosphoserine(Sodium Salt) (DPPS-NA), l,2-Distearoyl-sn-glycero-3- phosphate(Sodium Salt) (DSPA-NA), l,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2- Distearoyl-sn-glycero-3[Phospho-rac-(l-glycerol)(Sodium Salt) (DSPG-NA), 1,2- Distearoyl- sn-glycero-3[Phospho-rac-(l-glycerol)(Ammonium Salt) (DSPG-NH4), 1,2- Distearoyl-sn- glycero-3-phosphoserine(Sodium Salt) (DSPS-NA), Egg-PC (EPC), Hydrogenated Egg PC (HEPC), Hydrogenated Soy PC (HSPC), l-Myristoyl-sn-glycero-3- phosphocholine (LY S OPCM YRIS TIC ) , l-Palmitoyl-sn-glycero-3-phosphocholine (LYSOPCPALMITIC), 1- Stearoyl-sn-glycero-3-phosphocholine (LYSOPC STEARIC), 1- Myristoyl-2-palmitoyl-sn- glycero3-phosphocholine (MPPC), l-Myristoyl-2-stearoyl-sn-glycero- 3 -phosphocholine (MSPC), l-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1- Palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC), l-Palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), l-Palmitoyl-2-oleoyl-sn-glycero-3[Phospho-rac-(l- glycerol)] (Sodium Salt) (POPG-NA), l-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PS PC), 1- Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), l-Stearoyl-2-oleoyl- sn-glycero-3- phosphocholine (SOPC), and l-Stearoyl-2-palmitoyl-sn-glycero-3- phosphocholine (SPPC). In particular embodiments, the phospholipid is DSPC. In particular embodiments, the phospholipid is DOPE. In particular embodiments, the phospholipid is DOPC.

[0348] In some embodiments, the non-cationic lipids comprised by the lipid nanoparticles include one or more steroids. Steroids useful for the lipid nanoparticles described herein include, but are not limited to, cholestanes such as cholesterol, cholanes such as cholic acid, pregnanes such as progesterone, androstanes such as testosterone, and estranes such as estradiol. Further steroids include, but are not limited to, cholesterol (ovine), cholesterol sulfate, desmosterol-d6, cholesterol-d7, lathosterol-d7, desmosterol, stigmasterol, lanosterol, dehydrocholesterol, dihydrolanosterol, zymosterol, lathosterol, zymosterol-d5, 14-demethyl-lanosterol, 14-demethyl- lanosterol-d6, 8(9)- dehydrocholesterol, 8(14)-dehydrocholesterol, diosgenin, DHEA sulfate, DHEA, lanosterol- d6, dihydrolanosterol-d7, campesterol-d6, sitosterol, lanosterol-95, Dihydro FF-MAS-d6, zymostenol-d7, zymostenol, sitostanol, campestanol, campesterol, 7- dehydrodesmosterol, pregnenolone, sitosterol-d7, Dihydro T-MAS, Delta 5-avenasterol, Brassicasterol, Dihydro FF-MAS, 24-methylene cholesterol, cholic acid derivatives, cholesteryl esters, and glycosylated sterols. In particular embodiments, the lipid nanoparticles comprise cholesterol.

[0349] In some embodiments, the lipid nanoparticles comprise a lipid conjugate. Such lipid conjugates include, but are not limited to, ceramide PEG derivatives such as C8 PEG2000 ceramide, C16 PEG2000 ceramide, C8 PEG5000 ceramide, C16 PEG5000 ceramide, C8 PEG750 ceramide, and C16 PEG750 ceramide, phosphoethanolamine PEG derivatives such as 16:0 PEG5000PE, 14:0 PEG5000 PE, 18:0 PEG5000 PE, 18:1 PEG5000 PE, 16:0 PEG3000 PE, 14:0 PEG3000 PE, 18:0 PEG3000 PE, 18:1 PEG3000 PE, 16:0 PEG2000 PE, 14:0 PEG2000 PE, 18:0 PEG2000 PE, 18:1 PEG2000 PE 16:0 PEG1000 PE, 14:0 PEG1000 PE, 18:0 PEG1000 PE, 18:1 PEG 1000 PE, 16:0 PEG750 PE, 14:0 PEG750 PE, 18:0 PEG750 PE, 18:1 PEG750 PE, 16:0 PEG550 PE, 14:0 PEG550 PE, 18:0 PEG550 PE, 18:1 PEG550 PE, 16:0 PEG350 PE, 14:0 PEG350 PE, 18:0 PEG350 PE, and 18:1 PEG350, sterol PEG derivatives such as Chol- PEG600, and glycerol PEG derivatives such as DMG-PEG5000, DSG-PEG5000, DPG- PEG5000, DMG-PEG3000, DSG-PEG3000, DPG-PEG3000, DMG-PEG2000, DSG- PEG2000, DPG-PEG2000, DMG-PEG1000, DSG-PEG1000, DPG-PEG1000, DMG- PEG750, DSG- PEG750, DPG-PEG750, DMG-PEG550, DSG-PEG550, DPG-PEG550, DMG-PEG350, DSG- PEG350, and DPG-PEG350. In some embodiments, the lipid conjugate is a DMG-PEG. In some particular embodiments, the lipid conjugate is DMG- PEG2000. In some particular embodiments, the lipid conjugate is DMG-PEG5000.

[0350] It is within the level of a skilled artisan to select the cationic lipids, non-cationic lipids and/or lipid conjugates which comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other, such as based upon the characteristics of the selected lipid(s), the nature of the delivery to the intended target cells, and the characteristics of the nucleic acids and/or proteins to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus, the molar ratios of each individual component may be adjusted accordingly.

[0351] The lipid nanoparticles for use in the method can be prepared by various techniques which are known to a skilled artisan. Nucleic acid-lipid particles and methods of preparation are disclosed in, for example, U.S. Patent Publication Nos. 20040142025 and 20070042031.

[0352] In some embodiments, the lipid nanoparticles will have a size within the range of about 25 to about 500 nm. In some embodiments, the lipid nanoparticles have a size from about 50 nm to about 300 nm, or from about 60 nm to about 120 nm. The size of the lipid nanoparticles may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421 A150 (1981). A variety of methods are known in the art for producing a population of lipid nanoparticles of particular size ranges, for example, sonication or homogenization. One such method is described in U.S. Pat. No. 4,737,323.

[0353] In some embodiments, the lipid nanoparticles comprise a cell targeting molecule such as, for example, a targeting ligand (e.g., antibodies, scFv proteins, DART molecules, peptides, aptamers, and the like) anchored on the surface of the lipid nanoparticle that selectively binds the lipid nanoparticles to the targeted cell, such as any cell described herein, e.g. a T cell.

[0354] In some embodiments, the vector exhibits immune cell or T cell tropism.

[0355] In some aspects, provided herein are pluralities of vectors that comprise any of the vectors described herein, and one or more additional vectors comprising one or more additional polynucleotides encoding an additional portion or an additional component of any of the DNA- targeting systems described herein, any of the gRNAs described herein, any of the fusion proteins described herein, or a portion or a component of any of the foregoing.

[0356] Provided are pluralities of vectors, that include: a first vector comprising any of the polynucleotides described herein; and a second vector comprising any of the polynucleotides described herein.

[0357] In some aspects, vectors provided herein may be referred to as delivery vehicles. In some aspects, any of the DNA-targeting systems, components thereof, or polynucleotides disclosed herein can be packaged into or on the surface of delivery vehicles for delivery to cells. Delivery vehicles contemplated include, but are not limited to, nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycol particles, hydrogels, and micelles. As described in the art, a variety of targeting moieties can be used to enhance the preferential interaction of such vehicles with desired cell types or locations.

[0358] Methods of introducing a nucleic acid into a host cell are known in the art, and any known method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell. Suitable methods include, include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome- mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery, and the like. In some embodiments, the composition may be delivered by mRNA delivery and ribonucleoprotein (RNP) complex delivery. Direct delivery of the RNP complex, including the DNA-binding domain complexed with the sgRNA, can eliminate the need for intracellular transcription and translation and can offer a robust platform for host cells with low transcriptional and translational activity. The RNP complexes can be introduced into the host cell by any of the methods known in the art.

[0359] In some embodiments, the method of introducing a nucleic acid into a host cell is a method comprising transient delivery, such as described in Section I.B.

[0360] Nucleic acids or RNPs of the disclosure can be incorporated into a host using viruslike particles (VLP). VLPs contain normal viral vector components, such as envelope and capsids, but lack the viral genome. For instance, nucleic acids expressing the Cas and sgRNA can be fused to the viral vector components such as gag and introduced into producer cells. The resulting virus-like particles containing the sgRNA-expressing vectors can infect the host cell for efficient editing.

[0361] Introduction of the complexes, polypeptides, and nucleic acids of the disclosure can occur by protein transduction domains (PTDs). PTDs, including the human immunodeficiency virus- 1 TAT, herpes simplex virus- 1 VP22, Drsophila Antennapedia Antp, and the poluarginines, are peptide sequences that can cross the cell membrane, enter a host cell, and deliver the complexes, polypeptides, and nucleic acids into the cell.

[0362] Introduction of the complexes, polypeptides, and nucleic acids of the disclosure into cells can occur by viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery, and the like, for example as described in WO 2017/193107, WO 2016/123578, WO 2014/152432, WO 2014/093661, WO 2014/093655, or WO 2021/226555.

[0363] Various methods for the introduction of polynucleotides are well known and may be used with the provided methods and compositions. Exemplary methods include those for transfer of polynucleotides encoding the DNA targeting systems provided herein, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.

[0364] In some embodiments, polynucleotides can be cloned into a suitable vector, such as an expression vector or vectors. The expression vector can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable cell. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses.

[0365] In some embodiments, the vector can a vector of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), or the pEX series (Clontech, Palo Alto, Calif.). In some embodiments, animal expression vectors include pEUK- Cl, pMAM and pMAMneo (Clontech). In some embodiments, a viral vector is used, such as a lentiviral or retroviral vector. In some embodiments, the recombinant expression vectors can be prepared using standard recombinant DNA techniques. In some embodiments, vectors can contain regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based. In some embodiments, the vector can contain a nonnative promoter operably linked to the nucleotide sequence encoding the recombinant receptor. In some embodiments, the promoter can be a non- viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus. Other promoters known to a skilled artisan also are contemplated.

[0366] In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, or adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into cells (e.g. T cells) using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46;

Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557.

[0367] In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3: 102-109.

[0368] In some embodiments, the vector is a lentiviral vector. In some embodiments, the lentiviral vector is an integrase-deficient lentiviral vector. In some embodiments, the lentiviral vector is a recombinant lentiviral vector. In some embodiments, the lentivirus is selected or engineered for a desired tropism (e.g. for T cell or immune cell tropism). Methods of lentiviral production, transduction, and engineering are known, for example as described in Kasaraneni, N. et al. Sci. Rep. 8(1): 10990 (2018), Ghaleh, H.E.G. et al. Biomed. Pharmacother. 128:110276 (2020), and Milone, M.C. et al. Leukemia. 32(7):1529-1541 (2018). Additional methods for lentiviral transduction are described, for example in Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101: 1637- 1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.

[0369] In some embodiments, recombinant nucleic acids are transferred into cells (e.g. T cells) via electroporation {see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material into immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

III. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS OF DNA- TARGETING SYSTEMS OR ENCODING POLYNUCLEOTIDES OR VECTORS

[0370] In some aspects, provided herein are compositions, such as pharmaceutical compositions and formulations for administration, that include any of the DNA-targeting systems described herein, for example in Section I, or any of the polynucleotides or vectors encoding the same, for example as described in Section II. In some aspects, the pharmaceutical composition contains one or more DNA-targeting systems provided herein or a component thereof. In some aspects, the pharmaceutical composition comprises one or more vectors, e.g., viral vectors that contain polynucleotides that encode one or more components of the DNA- targeting systems provided herein. Such compositions can be used in accord with the provided methods, and/or with the provided articles of manufacture or compositions, such as in the prevention or treatment of diseases, conditions, and disorders, or in detection, diagnostic, and prognostic methods.

[0371] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject or a cell to which the formulation would be administered.

[0372] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be functional molecules as vehicles, adjuvants, carriers, or diluents.

[0373] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0374] In some aspects, the choice of carrier is determined in part by the particular agent and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn -protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).

[0375] In some embodiments, the pharmaceutically acceptable excipient may be a transfection facilitating agent, which may include surface active agents, such as immune- stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents.

[0376] In some embodiments, the transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid. In some embodiments, the transfection facilitating agent is poly-L-glutamate. In some embodiments, the transfection facilitating agent may also include surface active agents such as immune- stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the genetic construct. In some embodiments, the DNA vector encoding the DNA-targeting system may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA- liposome mixture (see for example WO9324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. In some embodiments, the transfection facilitating agent is a polyanion, polycation, including poly-L- glutamate (LGS), or lipid.

[0377] Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0378] Sterile injectable solutions can be prepared by incorporating the agent in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The formulations to be used for in vivo or ex vivo administration or use are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

[0379] The pharmaceutical composition in some embodiments contains components in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactic ally effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0380] In some embodiments, the composition can be administered to a subject by any suitable means, for example, by bolus infusion or by injection, e.g., by intravenous or subcutaneous injection. In some embodiments, a given dose is administered by a single bolus administration of the composition. In some embodiments, the composition is administered by multiple bolus administrations of the composition, for example, over a period of no more than 3 days, or by continuous infusion administration of the composition. In some embodiments, the composition is administered parenterally, for example by intravenous, intramuscular, subcutaneous, or intraperitoneal administration. In some embodiments, the composition is administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0381] In some embodiments, the composition is contacted with our introduced into cells (e.g. primary HPCs) from a subject ex vivo, and the cells are subsequently administered to the same subject or to a different subject.

[0382] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of agent or agents, the type of cells or recombinant receptors, the severity and course of the disease, whether the agent or cells are administered for preventive or therapeutic purposes, previous therapy, the subject’s clinical history and response to the agent or the cells, and the discretion of the attending physician. The compositions are in some embodiments suitably administered to the subject at one time or over a series of treatments.

IV. METHODS OF LYMPHOID CELL DIFFERENTIATION

[0383] In some embodiments, the provided DNA-targeting systems provided herein can be used to differentiate a hematopoietic progenitor cell or a population of hematopoietic progenitor cells into a differentiated population of cells (e.g. lymphoid progenitor cells or lymphoid cells). In some embodiments, the differentiation is induced by transcriptional activation to stimulate the transcription factors RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1,TCF7, GATA3 and/or BCL1 IB, which are transcription factors triggered by the signal resulting from the Notch-DLL4/1 interaction. The differentiated population of cells are also referred to herein as “induced” cells as noted by the prefix “i” before the differentiated cell population. In provided embodiments, the “induced” or “i” population of cells exhibit a phenotype and function characteristic of such a cell type, such as present by a native (primary) population of such cells, for example a primary population of the cell type from a human subject, or a known population of cells differentiated by other accepted methods. For example, iLymphocytes in connection with the provided disclosure are a population of lymphocytes that have been differentiated and cultured by the provided methods; such provided iLymphocytes are characterized as, e.g., iNK cells or iT cells. For example, iNK cells in connection with the provided disclosure are a population of NK cells that have been differentiated and cultured by the provided methods; such provided iNK cells are characterized as being CD56+, DNAM+, NKP30+, NKG2D+ and CD16, and exhibit cytotoxic activity (see e.g., Example 8). For example, iT cells in connection with the provided disclosure are a population of T cells that have been differentiated and cultured by the provided methods; such provided iT cells are characterized as being CD4+ and CD8b+, and exhibit cytotoxic activity. Description of a cell type as an induced cell of a particular cell type, such as iNK cells, does not mean that all features of the cells (e.g., extent of gene expression, surface markers, etc..) are identical between such induced cells and primary cells of the cell type or cells of the cell type differentiated by other methods.

[0384] The methods provided herein include use of one or more DNA-targeting systems provided herein (e.g. as described in Section I), or polynucleotide or vector for delivery of same (e.g. as described in Section II) to a hematopoietic progenitor cell or a population of hematopoietic progenitor cells. In some embodiments, the DNA-targeting system (or polynucleotides or vectors for delivery of same) is contacted with a hematopoietic progenitor cell or a population of hematopoietic progenitor cells. In some embodiments, the contacting introduces the epigenome-modifying DNA-targeting system (or polynucleotides or vectors for delivery of same) into the hematopoietic progenitor cell, such as where it is able to translocate or localize to the nucleus of the hematopoietic progenitor cell or nuclei of hematopoietic progenitor cells of the population of hematopoietic progenitor cells. In some embodiments, the methods increase the expression of one or more of the described target genes in the hematopoietic progenitor cell or the population of hematopoietic progenitor cells, and induces differentiation into differentiated cells or a differentiated population of cells. Also provided herein is a population of differentiated cells produced by any of the provided methods. In some embodiments, the methods increase the expression of one or more of the described target genes in the hematopoietic progenitor cell or population of hematopoietic progenitor cells to induce differentiation into lymphoid progenitor cells or a population of lymphoid progenitor cells. In some embodiments, the lymphoid progenitor cells are induced common lymphoid progenitor cells (iCLPs). Also provided herein is a population of lymphoid progenitor cells produced by any of the provided methods.

[0385] In some embodiments, the methods provided herein increase transcription of the one or more lymphoid cell differentiation (LCD) genes RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and/or BCL11B in the population of hematopoietic progenitor cells. In some embodiments, the methods provided herein increase transcription of RUNX3 and IL7Ra in the population of hematopoietic progenitor cells. In some embodiments, the methods provided herein increase transcription of RUNX3, IL7Ra, and CBFB in the population of hematopoietic progenitor cells. In some embodiments, the methods provided herein increase transcription of RUNX3, IF7Ra, and TBX21 in the population of hematopoietic progenitor cells. In some embodiments, the methods provided herein increase transcription of RUNX3, CBFB, FEF1, MYB, TBX21, and IE7Ra in the population of hematopoietic progenitor cells. In some embodiments, the methods provided herein increase transcription of RUNX3, CBFB, EEF1, MYB, and TBX21 in the population of hematopoietic progenitor cells. In some embodiments, the methods provided herein increase transcription of RUNX3, CBFB, EEF1, and MYB in the population of hematopoietic progenitor cells. In some embodiments, the methods provided herein increase transcription of EEF1, MYB, and TBX21 in the population of hematopoietic progenitor cells. In some embodiments, the methods provided herein increase transcription of EEF1 and TBX21 in the population of hematopoietic progenitor cells. In some embodiments, the methods provided herein increase transcription of EEF1 and MYB in the population of hematopoietic progenitor cells.

[0386] In some embodiments, the methods provided herein increase transcription of TCF7, GATA3, and BCE1 IB in the population of hematopoietic progenitor cells. In some embodiments, the methods provided herein increase transcription of TCF7 and GATA3 in the population of hematopoietic progenitor cells. In some embodiments, the methods provided herein increase transcription of TCF7 and BCE11B in the population of hematopoietic progenitor cells. In some embodiments, the methods provided herein increase transcription of GATA3 and BCE1 IB in the population of hematopoietic progenitor cells. In some embodiments, the increase in transcription in the population of cells is relative to the transcription levels of the genes in a comparable population of cells that has not been contacted with or introduced with the DNA-targeting system (or polynucleotides or vectors for delivery of same) provided herein.

[0387] In some embodiments, provided herein are methods of differentiating a differentiated population of cells in which the method involves introducing the DNA-targeting system into a hematopoietic progenitor cell or a population of hematopoietic progenitor cells. In some embodiments, provided herein are methods of differentiating a differentiated population of cells in which the method involves introducing any of the provided Cas-gRNA combination into hematopoietic progenitor cell or a population of hematopoietic progenitor cells. In some embodiments, provided herein are methods of differentiating a differentiated population of cells in which the method involves introducing any of the provided polynucleotides into a hematopoietic progenitor cell or a population of hematopoietic progenitor cells. In some embodiments, provided herein are methods of differentiating a population of differentiated population of cells in which the method involves introducing any of the provided vectors into a hematopoietic progenitor cell or a population of hematopoietic progenitor cells.

[0388] In some embodiments, provided herein are methods of differentiating a population of lymphoid progenitor cells in which the method involves introducing the DNA-targeting system into a hematopoietic progenitor cell or a population of hematopoietic progenitor cells. In some embodiments, provided herein are methods of differentiating a population of lymphoid progenitor cells in which the method involves introducing any of the provided Cas-gRNA combinations into a hematopoietic progenitor cell or a population of hematopoietic progenitor cells. In some embodiments, provided herein are methods of differentiating a population of lymphoid progenitor cells in which the method involves introducing any of the provided polynucleotides into a hematopoietic progenitor cell or a population of hematopoietic progenitor cells. In some embodiments, provided herein are methods of differentiating a population of lymphoid progenitor cells in which the method involves introducing any of the provided vectors into a hematopoietic progenitor cell or a population of hematopoietic progenitor cells.

[0389] In some further embodiments, provided herein are methods of differentiating a population of lymphoid cells in which the method involves introducing the DNA-targeting system into a hematopoietic progenitor cell or a population of hematopoietic progenitor cells. In some embodiments, provided herein are methods of differentiating a population of lymphoid cells in which the method involves introducing any of the provided Cas-gRNA combinations into a hematopoietic progenitor cell or a population of hematopoietic progenitor cells. In some embodiments, provided herein are methods of differentiating a population of lymphoid progenitor cells in which the method involves introducing any of the provided polynucleotides into a hematopoietic progenitor cell or a population of hematopoietic progenitor cells. In some embodiments, provided herein are methods of differentiating a population of lymphoid progenitor cells in which the method involves introducing any of the provided vectors into a hematopoietic progenitor cell or a population of hematopoietic progenitor cells. [0390] In some embodiments, the epigenome-modifying DNA-targeting system (or polynucleotides or vectors for delivery of same) can be cultured with a hematopoietic progenitor cell or a population of hematopoietic progenitor cells under conditions in which the epigenomemodifying DNA-targeting system (or polynucleotides or vectors for delivery of same) are introduced into or delivered to the hematopoietic progenitor cell or hematopoietic progenitor cells in the population of hematopoietic progenitor cells.

[0391] In some embodiments, the methods can be carried out in vitro. In some embodiments, the methods can be carried out ex vivo on cells isolated from a subject. In some embodiments, the methods can be carried out in vivo in which the DNA-targeting system (or polynucleotides or vectors for delivery of same) are administered to a subject.

[0392] In some embodiments, the introducing is by transient delivery of the DNA-targeting system (or polynucleotides or vectors for delivery of same) into the hematopoietic progenitor cell or the population of hematopoietic progenitor cells. In some embodiments, the DNA- targeting system is transiently expressed and/or transiently present in the hematopoietic progenitor cell or the population of hematopoietic progenitor cells. In some embodiments, the transient delivery is by electroporation, transfection, or transduction. Various methods of introducing protein and nucleic acid systems to cells are known to a skilled artisan. In some embodiments, the DNA-targeting systems are delivered in a vector. In some embodiments, the vector is a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector. In some embodiments, the vector is a lipid nanoparticle (LNP). Among provided embodiments, there is provided a lipid nanoparticle that contains any of the provided polynucleotides for delivery of an epigenetic DNA-targeting system. In some embodiments, the LNP contains a polynucleotide that encodes a fusion protein as provided herein that includes (a) a DNA-binding domain capable of being targeted to a target site of a target gene as described; and (b) at least one effector domain capable of increasing transcription of the gene (e.g., a transcriptional activation domain). In some embodiments, the DNA-binding domain is a Cas (e.g. dCas) and the LNP further includes a gRNA. In some embodiments, the polynucleotide encoding the fusion protein is an mRNA and the gRNA is provided as an RNA.

[0393] In some embodiments, the DNA-targeting systems are introduced (e.g. by transient delivery) to the hematopoietic progenitor cell or the population of hematopoietic progenitor cells one time or multiple times during a culture for differentiating cells. In some embodiments, the introducing involves a single delivery of the DNA-targeting system to the hematopoietic progenitor cell or the population of hematopoietic progenitor cells. In some embodiments, the introducing involves multiple deliveries of the DNA-targeting system to the hematopoietic progenitor cell or the population of hematopoietic progenitor cells. In some such embodiments, a provided DNA-targeting system is introduced to the cells intermittently several times during the culture of the cells. In some embodiments, the DNA-targeting system is introduced at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 times during a differentiation culture. In some embodiments, the DNA-targeting system is introduced at least 2 individual times during a differentiation culture.

[0394] In some embodiments, the introduction of the DNA-targeting system to cells during a differentiation culture is performed according to a set interval of time. In some embodiments, the DNA-targeting system is introduced to hematopoietic progenitor cell or the population of hematopoietic progenitor cells at an initial or first time of culture (e.g. Day 0) and then this is repeated once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days, once every 13 days, once every 14 days, once every 15 days, once every 16 days, once every 17 days, once every 18 days, once every 19 days, or once every 20 days. In some embodiments, the DNA-targeting system is introduced to hematopoietic progenitor cell or the population of hematopoietic progenitor cells at an initial or first time of culture (e.g. Day 0) and once every other day. In some embodiments, the DNA- targeting system is introduced to hematopoietic progenitor cell or the population of hematopoietic progenitor cells at an initial or first time of culture (e.g. Day 0) and one every three days. In some embodiments, the DNA-targeting system is introduced to hematopoietic progenitor cell or the population of hematopoietic progenitor cells at an initial or first time of culture (e.g. Day 0) and once every four day. In some embodiments, the delivery of the DNA- targeting system to the cells is repeated 2-6 times, such as 2-4 times, during the culture. In some embodiments, the delivery of the DNA-targeting system to the cells is repeated 2 times during the culture. In some embodiments, the delivery of the DNA-targeting system to the cells is repeated 3 times during the culture

[0395] In some embodiments, the introducing comprises delivery of a DNA-targeting system comprising at least two DNA-targeting modules where each DNA-targeting module is delivered at a different time. In some embodiments, the at least two DNA-targeting modules is a first set of DNA-targeting modules and second set of DNA-targeting modules that are introduced at different times. In some embodiments, the first and second set for DNA-targeting modules target transcriptional activation of different combinations of genes. The first and second set of DNA-targeting modules can be any as described herein, such as in Seciton I. In some embodiments, the second set of DNA-targeting modules is delivered 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 14 days after the delivery of the first set of DNA-targeting modules. In some embodiments, the second set of DNA-targeting modules is delivered 2 days after the delivery of the first set of DNA-targeting modules. In some embodiments, the second set of DNA-targeting modules is delivered 3 days after the delivery of the first set of DNA-targeting modules. In some embodiments, the second set of DNA-targeting modules is delivered 4 days after the delivery of the first set of DNA-targeting modules.

[0396] In some embodiments, the first set of DNA-targeting modules targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, and CBFB. In some embodiments, the second set of DNA-targeting modules targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, first set of DNA-targeting modules targets: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; or RUNX3, IL7Ra, and TBX21. In some embodiments, the second set of DNA- targeting modules targets: RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB. In some embodiments, the first set of DNA-targeting modules targets RUNX3 and IL7RA and the second set of DNA-targeting modules targets LEF1 and TBX21. In some embodiments, the first set of DNA-targeting modules targets RUNX3 and IL7RA and the second set of DNA-targeting modules targets RUNX3, CBFB, LEF1 and MYB. In some embodiments, the first set of DNA-targeting modules targets RUNX3 and IL7RA and the second set of DNA-targeting modules targets LEF1 and MYB. In some embodiments, the first set of DNA-targeting modules targets RUNX3 and IL7RA and the second set of DNA- targeting modules targets LEF1, MYB and TBX21. In some embodiments, the first set of DNA- targeting modules targets RUNX3 and IL7RA and the second set of DNA-targeting modules targets RUNX3, CBFB, LEF1, MYB and TBX21. In some embodiments, the first set of DNA- targeting modules targets RUNX3 and IL7RA and the second set of DNA-targeting modules targets RUNX3, CBFB, LEF1, MYB, TBX21 and IL7Ra.

[0397] In some embodiments, the introducing comprises delivery of a DNA-targeting system comprising at least two DNA-targeting modules where each DNA-targeting module is delivered at a different time. In some embodiments, the at least two DNA-targeting modules is a first DNA-targeting module comprising a first set of gRNAs and a second DNA-targeting module comprising a second set of gRNAs that are introduced at different times. In some embodiments, the second DNA-targeting module is delivered 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 14 days after the delivery of the first DNA-targeting module. In some embodiments, the second DNA-targeting module is delivered 2 days after the delivery of the first DNA-targeting module. In some embodiments, the second DNA-targeting module is delivered 3 days after the delivery of the first DNA-targeting module. In some embodiments, the second DNA-targeting module is delivered 4 days after the delivery of the first DNA-targeting module.

[0398] In some embodiments, the first DNA targeting module targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, and CBFB. In some embodiments, the second DNA-targeting module targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, first DNA-targeting module targets: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; or RUNX3, IL7Ra, and TBX21. In some embodiments, the second DNA-targeting module targets: RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB. In some embodiments, the first DNA-targeting module targets RUNX3 and IL7RA and the second DNA-targeting module targets LEF1 and TBX21. In some embodiments, the first DNA-targeting module targets RUNX3 and IL7RA and the second DNA-targeting module targets RUNX3, CBFB, LEF1 and MYB. In some embodiments, the first DNA-targeting module targets RUNX3 and IL7RA and the second DNA-targeting module targets LEF1 and MYB. In some embodiments, the first DNA-targeting module targets RUNX3 and IL7RA and the second DNA-targeting module targets LEF1, MYB and TBX21. In some embodiments, the first DNA-targeting module targets RUNX3 and IL7RA and the second DNA-targeting module targets RUNX3, CBFB, LEF1, MYB and TBX21. In some embodiments, the first DNA- targeting module targets RUNX3 and IL7RA and the second DNA-targeting module targets RUNX3, CBFB, LEF1, MYB, TBX21 and IL7Ra.

[0399] In some embodiments, the first set of gRNAs targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, and CBFB. In some embodiments, the second set of gRNAs targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. In some embodiments, first set of gRNAs targets: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; or RUNX3, IL7Ra, and TBX21. In some embodiments, the second set of gRNAs targets: RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB. In some embodiments, the first set of gRNAs targets RUNX3 and IL7RA and the second set of gRNAs targets LEF1 and TBX21. In some embodiments, the first set of gRNAs targets RUNX3 and IL7RA and the second set of gRNAs targets RUNX3, CBFB, LEF1 and MYB. In some embodiments, the first set of gRNAs targets RUNX3 and IL7RA and the second set of gRNAs targets LEF1 and MYB. In some embodiments, the first set of gRNAs targets RUNX3 and IL7RA and the second set of gRNAs targets LEF1, MYB and TBX21. In some embodiments, the first set of gRNAs targets RUNX3 and IL7RA and the second set of gRNAs module targets RUNX3, CBFB, LEF1, MYB and TBX21. In some embodiments, the first set of gRNAs targets RUNX3 and IL7RA and the second set of gRNAs targets RUNX3, CBFB, LEF1, MYB, TBX21 and IL7Ra

[0400] In some embodiments, the population of hematopoietic progenitor cells (HPCs) are differentiated from iPSCs. In some embodiments, the HPCs are induced hematopoietic progenitor cells (iHPCs). In some embodiments, the HPCs are primary hematopoietic progenitor cells. In some embodiments, the population of cells are or include hematopoietic stem cells (HSCs). In some embodiments, the population of HSCs are differentiated from iPSCs. In some embodiments, the population of HPCs is a population of induced hematopoietic progenitor cells (iHPCs).

[0401] In some embodiments, an iPSC cells can be obtained by introducing a specific factor (nuclear reprogramming factor) into a mammalian somatic cell or an undifferentiated stem cell to reprogram them. A skilled artisan is familiar with various types of iPSCs and methods for obtaining the same. In some embodiments, iPSCs are established by introducing the four factors Oct3/4, Sox2, Klf4, and c-Myc into mouse fibroblasts, such as described by Yamanaka et al. (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676). In some embodiments, iPSC cells can be derived from human cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872). In some embodiments, the iPSCs may be Nanog-iPS cells established by introducing the four factors, then selecting them by using the expression of Nanog as an index (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317). In some embodiments, iPS cells can be prepared by a method free of C-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106). In some embodiments, iPS cells can be established by introducing six factors by a virus-free method (Okita K et al. Nat. Methods 2011 May; 8(5): 409-12, Okita K et al. Stem Cells. 31(3): 458-66). In some embodiments, iPSCs can be established by introducing the four factors OCT3/4, SOX2, NANOG, and LIN28, such as described by Thomson et al. (Yu J., Thomson J A. et al., Science (2007) 318: 1917-1920). In some embodiments, iPSCs can be prepared by methods described in Daley et al. (Park I H, Daley G Q. et al., Nature (2007) 451: 141-146). In some embodiments, iPSCs can be prepared by methods described in Sakurada et al. (JP 2008-307007 A). Other methods for inducing or obtaining iPSCs are well known, including any described in any published literature (for example, Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol 3, Issue 5, 568-574; Kim J B., Scholer H R., et al., Nature, (2008) 454, 646-650; and Huangfu D., Melton, D A., et al., Nature Biotechnology, (2008) 26, No 7, 795-797), or patent (for example, JP 2008-307007 A, JP 2008-283972 A, US2008-2336610, US2009-047263, W02007-069666, W02008-118220, WO2008-124133, W02008-151058, W02009-006930, W02009-006997, and W02009-007852). In some embodiments, the cells can be an iPSC cell line. Examples of human iPS cell lines include RIKEN's HiPS-RIKEN-lA line, HiPS-RIKEN- 2A line, HiPS-RIKEN-12A line, and Nips-B2 line, and Kyoto University's 253G1 line, 201B7 line, 409B2 line, 454E2 line, 606A1 line, 610B1 line, and 648A1 line.

[0402] In provided embodiments, the provided methods can relate to differentiation of hematopoietic stem and progenitor cell (HSPC) or a population of HSPCs into a differentiated population of cells. Thus, the methods are not limited to use with HPCs but can also be used for differentiation of hematopoietic stem cells (HSCs) into a differentiated population of cells. In some embodiments, while the methods are described as being carried out for differentiation of hematopoietic progenitor cells into a differentiated cell population, the methods can alternatively be carried out with hematopoietic stem cells. Thus, any reference to provided methods involving a hematopoietic progenitor cell or a population of hematopoietic progenitor cells in the provided disclosure can instead be a hematopoietic stem cell or a population of hematopoietic stem cells. In some embodiments, a hematopoietic stem cell (HSC) is a multipotent stem cell that can differentiate into blood cells, including lymphocytes. In some embodiments, a hematopoietic progenitor cell (HPC) is a cell that has the ability to differentiate into blood cells but does not have the ability to self-renew as much as a stem cell. In some embodiments, a hematopoietic stem cell (HSC) is a multipotent stem cell that can differentiate into blood cells, including lymphocytes. In some embodiments, a hematopoietic progenitor cell (HPC) is a cell that has the ability to differentiate into blood cells but does not have the ability to self-renew as much as a stem cell. In humans, HSCs and HPCs are mainly present in bone marrow, but are also present in peripheral blood and cord blood, and can be collected from each site. In provided embodiments, the hematopoietic stem cell may be a cell isolated from living tissues such as bone marrow, blood, or the like, or may be a cell prepared from an ES cell or an iPS cell. Both hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are cells that are CD34-positive and CD3-negative as cell markers (CD34+CD3- cells). Whether a cell is a hematopoietic stem cell can be confirmed by the fact that it survives when transplanted and grafted to an animal, removed again, and then transplanted to another individual, which means that it has the ability to self-renew, in other words, that it is a “stem cell”, not a “progenitor cell”.

[0403] In some embodiments, HPCs are cultured with a provided DNA-targeting system (or polynucleotides or vectors for delivery of same) under conditions to introduce DNA-targeting system (or polynucleotides or vectors for delivery of same) into stem cells of the population to promote lymphoid differentiation. In some embodiments, the HPCs express CD34. In some embodiments, the HPCs express at least two markers from the group consisting of CD43, CD34, CD31, CD41, CD235 and CD45. In some embodiments, HPCs express one or more of the cell surface markers selected from the group consisting of CD34, CD43, CD7, DLL4, CD 144, and CD235. In some embodiments, the HPCs express CD144, CD34, CD45, and CD7. In some embodiments, the HPCs express CD144, CD34, CD45, and CD7.

[0404] In some embodiments, the methods lead to differentiation of a differentiated population of cells. In some embodiments, the methods lead to differentiation of lymphoid progenitor cells or a population of lymphoid progenitor cells in the population. In some embodiments, the lymphoid progenitor cells or population of lymphoid progenitor cells express one or more of the markers selected from the group consisting of CD5, CD45RA, CD 10, CD123, CD34, CD7, CD127, CD117, CD14, and CD56. In some embodiments, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50% or more of the cells in the population of lymphoid progenitor cells are positive for one or more of the markers selected from the group consisting of CD5, CD45RA, CD 10, CD 123, CD34, CD7, CD 127, CD 117, CD 14, and CD56. In some embodiments, the methods further include isolating or enriching cells from the population of lymphoid progenitor cells that are positive for one or more of the markers selected from the group consisting of CD5, CD45RA, CD 10, CD 123, CD34, CD7, CD 127, CD 117, CD 14, and CD56. In some embodiments, the differentiated lymphoid progenitor cells express CD5. In some embodiments, the differentiated lymphoid progenitor cells express CD7. In some embodiments, the differentiated lymphoid progenitor cells express CD5 and CD7.

[0405] In some embodiments, the differentiated lymphoid cells express one or more of the markers selected from the group consisting of CD8, CD7, CD45, CD5, CD4 and CD3. In some embodiments, the methods differentiate the population of stem cells into a population of cells that are enriched for cells that express one or more of the markers selected from the group consisting of CD8, CD7, CD45, CD5, CD4 and CD3. In some embodiments, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50% or more of the cells in the population of cells are positive for one or more of the markers selected from the group consisting of CD8, CD7, CD45, CD5, CD4 and CD3. In some embodiments, the method further include isolating or enriching cells from the population of differentiated cells that are positive for one or more of the markers selected from the group consisting of CD8, CD7, CD45, CD5, CD4 and CD3. In some embodiments, the differentiated lymphoid cells are T lymphocytes.

[0406] In some embodiments, the differentiated lymphoid cells express CD56. In some embodiments, the methods differentiate the population of stem cells into a population of cells that are enriched for cells that express CD56. In some embodiments, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50% or more of the cells in the population of cells are positive for CD56. In some embodiments, the method further include isolating or enriching cells from the population of differentiated cells that are positive for the marker CD56.

[0407] In some embodiments, the differentiated lymphoid cells express one or more of the markers selected from the group consisting of CD8, CD7, CD45, CD5, CD4 and CD3. In some embodiments, the methods differentiate the population of stem cells into a population of cells that are enriched for cells that express one or more of the markers selected from the group consisting of CD8, CD7, CD45, CD5, CD4 and CD3. In some embodiments, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50% or more of the cells in the population of cells are positive for one or more of the markers selected from the group consisting of CD8, CD7, CD45, CD5, CD4 and CD3. In some embodiments, the method further include isolating or enriching cells from the population of differentiated cells that are positive for one or more of the markers selected from the group consisting of CD8, CD7, CD45, CD5, CD4 and CD3. In some embodiments, the differentiated lymphoid cells are T lymphocytes.

[0408] In some embodiments, the differentiated lymphoid cells express CD56. In some embodiments, the methods differentiate the population of stem cells into a population of cells that are enriched for cells that express CD56. In some embodiments, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50% or more of the cells in the population of cells are positive for CD56. In some embodiments, the method further include isolating or enriching cells from the population of differentiated cells that are positive for the marker CD56

[0409] In some embodiments, the methods lead to differentiation of a population of lymphoid cells (LCs; also called lymphocytes). In some embodiments, the methods lead to differentiation of a population of lymphoid progenitor cells into LCs. In some embodiments, the LCs are T cells. In some embodiments, the LCs are natural killer (NK) cells. In some embodiments, the LCs are induced lymphocyte cells (iLymphocytes). In some embodiments, the iLymphocytes are induced T (iT) cells. In some embodiments, the iLymphocytes are induced NK (iNK) cells.

[0410] In some embodiments, the lymphoid cells or iLymphocytes express one or more of the markers selected from the group consisting of CD45, CD56, DNAM1, NKG2D, NKP30, CD16, CD2, CD3, CD4, CD5, CD7 and CD8b,. In some embodiments, the methods differentiate the population of hematopoietic progenitor cells into a population of lymphoid cells or iLymphocytes that are enriched for cells that express one or more of the markers selected from the group consisting of CD45, CD56, DNAM1, NKG2D, NKP30, CD16, CD2, CD3, CD4, CD5, CD7 and CD8b. In some embodiments, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50% or more of the cells in the population of lymphoid cells or iLymphocytes are positive for one or more of the markers selected from the group consisting of CD45, CD56, DNAM1, NKG2D, NKP30, CD16, CD2, CD3, CD4, CD5, CD7 and CD8b. In some embodiments, the method further includes isolating or enriching cells from the population of lymphoid cells or iLymphocytes that are positive for one or more of the markers selected from the group consisting of CD45, CD56, DNAM1, NKG2D, NKP30, CD16, CD2, CD3, CD4, CD5, CD7 and CD8b. [0411] In some embodiments, the LCs are T cells or iT cells. In some embodiments, the T cells or iT cells express CD2, CD3, CD4, CD8b, or any combination thereof. In some embodiments, the iT cells express CD5 or CD7. In some embodiments, the iT cells express CD4 or CD8b. In some embodiments, the iT cells express CD4 or CD8b. In some embodiments, the methods differentiate the population of hematopoietic progenitor cells into a population of iT cells that are enriched for cells that express CD4 and CD8b. In some embodiments, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50% or more of the cells in the population of iT cells are positive for CD4 and CD8b. In some embodiments, the methods further include isolating or enriching cells from the population of differentiated cells that are positive for the markers CD4 and CD 8b.

[0412] In some embodiments, the LCs are NK cells or iNK cells. In some embodiments, the NK cells or iNK cells express CD56, DNAM1, NKG2D, NKP30, and/or CD16, or any combination thereof. In some embodiments, the NK cells or iNK cells do not express CD3. In some embodiments, the iNK cells are CD56+ and CD3-. In some embodiments, the iNK cells are CD56+CD3- and further express one or more markers selected from the group consisting of DNAM1, NKG2D, NKP30, and CD16. In some embodiments, the methods differentiate the population of hematopoietic progenitor cells into a population of iNK cells that are enriched for cells that are CD56+CD3-. In some embodiments, the methods differentiate the population of hematopoietic progenitor cells into a population of iNK cells that are enriched for cells that are CD56+CD3- and further express one or more markers selected from the group consisting of DNAM1, NKG2D, NKP30, and CD16. In some embodiments, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50% or more of the cells in the population of iNK cells are CD56+/CD3- and further express one or more markers selected from the group consisting of DNAM1, NKG2D, NKP30, and CD 16. In some embodiments, the methods further include isolating or enriching cells from the population of differentiated cells that are CD56+ and/or CD3-.

[0413] In some embodiments, the method further include isolating or enriching cells from the population of differentiated cells. Any of a variety of immunoaffinity-based methods can be used to isolate or enrich cells. In some aspects, isolating or enriching the cells is by magnetic- activated cell sorting (MACS). In some aspects, isolating or enriching the cells is by flow cytometry. In some embodiments, the isolating or enriching is based on positive selection for markers expressed on the differentiated cell population and/or based on negative selection for markers not expressed on the differentiated cell population. In some embodiments, the isolating or enriching is based on positive selection for markers expressed on the lymphoid progenitor cells and/or based on negative selection for markers not expressed on the lymphoid progenitor cells. In some embodiments, the isolating or enriching is based on positive selection for markers expressed on the lymphoid cells and/or based on negative selection for markers not expressed on the lymphoid cells.

[0414] Also provided herein is a differentiated population of cells produced by any of the provided methods. Also provided herein is population of lymphoid progenitor cells produced by any of the provided methods. Also provided herein is population of differentiated lymphoid cells produced by any of the provided methods. Also provided herein is a pharmaceutical composition comprising the population of population of cells, the lymphoid progenitor cells, or the differentiated lymphoid cells.

[0415] In some embodiments, the lymphoid progenitor cells and/or differentiated lymphoid cells provided by methods and compositions of certain aspects can be used in a variety of applications. These include but are not limited to transplantation or implantation of the cells in vivo. In some embodiments, the lymphoid progenitor cells and/or differentiated lymphoid cells can be administered to a subject for treating a disease or condition. In some embodiments, differentiated lymphoid cells exhibit cytotoxic killing activity for target cells associated with the disease or condition, and can thereby treat the disease or condition. A skilled artisan is readily familiar with cytotoxic cell therapies and methods of preparing and using the same. The provided differentiated lymphoid cells can be used in any of such methods.

V. DEFINITIONS

[0416] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. [0417] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting” and/or “consisting essentially of’ aspects and variations.

[0418] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0419] The term “about” as used herein refers to the usual error range for the respective value readily known. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”. In some embodiments, “about” may refer to ±25%, ±20%, ±15%, ±10%, ±5%, or ±1%.

[0420] As used herein, recitation that nucleotides or amino acid positions “correspond to” nucleotides or amino acid positions in a disclosed sequence, such as set forth in the Sequence listing, refers to nucleotides or amino acid positions identified upon alignment with the disclosed sequence to maximize identity using a standard alignment algorithm, such as the GAP algorithm. By aligning the sequences, corresponding residues can be identified, for example, using conserved and identical amino acid residues as guides. In general, to identify corresponding positions, the sequences of amino acids are aligned so that the highest order match is obtained (see, e.g. : Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carrillo et al. (1988) SIAM J Applied Math 48: 1073).

[0421] A “gene,” includes a DNA region encoding a gene product. Thus, the gene typically refers to coding and/or transcribed sequences. The sequence of a gene is typically present at a fixed chromosomal position or locus on a chromosome in the cell.

[0422] A “regulatory element” or “DNA regulatory element,” which terms are used interchangeably herein, in reference to a gene refers to DNA regions which regulate the production of a gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a regulatory element includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.

[0423] As used herein, a “target site” or “target nucleic acid sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule (e.g. a DNA- binding domain disclosed herein) will bind, provided sufficient conditions for binding exist.

[0424] The term “expression” with reference to a gene or “gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or can be a protein produced by translation of an mRNA. For instance, expression includes the transcription and/or translation of a particular nucleotide sequence drive by its promoter. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation. Hence, reference to expression or gene expression includes protein (or polypeptide) expression or expression of a transcribable product of or a gene such as mRNA. The protein expression may include intracellular expression or surface expression of a protein. Typically, expression of a gene product, such as mRNA or protein, is at a level that is detectable in the cell.

[0425] As used herein, a “detectable” expression level, means a level that is detectable by standard techniques known to a skilled artisan, and include for example, differential display, RT (reverse transcriptase)-coupled polymerase chain reaction (PCR), Northern Blot, and/or RNase protection analyses as well as immunoaffinity-based methods for protein detection, such as flow cytometry, ELISA, or western blot. The degree of expression levels need only be large enough to be visualized or measured via standard characterization techniques.

[0426] As used herein, the term “increased expression”, “enhanced expression” or “overexpression” means any form of expression that is additional to the expression in an original or source cell that does not contain the modification for modulating a particular gene expression by a DNA-targeting system, for instance a wild-type expression level (which can be absence of expression or immeasurable expression as well). Reference herein to “increased expression,” “enhanced expression” or “overexpression” is taken to mean an increase in gene expression relative to the level in a cell that does not contain the modification, such as the original source cell prior to contacting with, or engineering to introduce, the Dna-targeting system into the a cell, such as an unmodified cell or a wild-type T cell. The increase in expression can be at least 5%, 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 100% or even more. In some cases, the increase in expression can be at least 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-food, 500- fold, 1000-fold or more.

[0427] As used herein, the term “increased transcription” refers to the level of transcription of a gene that is additional to the transcription of the gene in an original or source cell that does not contain the modification for modulating transcription by a DNA-targeting system, for instance a wild-type transcription level of a gene. Reference to increased transcription can refer to an increase in the levels of a transcribable product of a gene such as mRNA. Any of a variety of methods can be used to monitor or quantitate a level of a transcribable product such as mRNA, including but not limited to, real-time quantitative RT (reverse transcriptase)- polymerase chain reaction (qRT-PCR), Northern Blot, microarray analysis, or RNA sequencing (RNA-Seq). The increase in transcription can be at least 5%, 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 100% or even more. In some cases, the increase in transcription can be at least 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80- fold, 90-fold, 100-fold, 200-fold or more.

[0428] As used herein, an “epigenetic modification” refers to changes in the gene expression that are non-genetic modifications, i.e. not caused by changes in the DNA sequences, but are due to epigenetic changes such as events like DNA methylations or histone modifications. An epigenetic modification may result in a heritable change in gene activity and expression that occur without alteration in DNA sequence. For instance, epigenetic modifications include non- genetic modifications such as chemical modifications to the cytosine residues of DNA (DNA methylation) and histone proteins associated with DNA (histone modifications).

[0429] As used herein, the term “modification” or “modified” with reference to a T cell refers to any change or alteration in a cell that impacts gene expression in the cell. In some embodiments, the modification is an epigenetic modification that directly changes the epigenetic state of a gene or regulatory elements thereof to alter (e.g. increase) expression of a gene product. In some embodiments, a modification described herein results in increased expression of a target gene or selected polynucleotide sequence.

[0430] As used herein, a “fusion” molecule is a molecule in which two or more subunit molecules are linked, such as covalently. Examples of a fusion molecule include, but are not limited to, fusion proteins (for example, a fusion between a DNA-binding domain such as a ZFP, TALE DNA-binding domain or CRISPR-Cas protein and one or more effector domains, such as a transactivation domain). The fusion molecule also may be part of a system in which a polynucleotide component associates with a polypeptide component to form a functional system (e.g., a CRISPR/Cas system in which a single guide RNA associates with a functional domain to modulate gene expression). Fusion molecules also include fusion nucleic acids, for example, a nucleic acid encoding the fusion protein. Expression of a fusion protein in a cell can result from delivery of the fusion protein to the cell or by delivery of a polynucleotide encoding the fusion protein to a cell, where the polynucleotide is transcribed, and the transcript is translated, to generate the fusion protein.

[0431] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Among the vectors are viral vectors, such as adenoviral vectors or lentiviral vectors.

[0432] The term “expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include, but are not limited to, cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0433] The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0434] The term "polynucleotide" refers to a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomelic "nucleotides." The monomelic nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0435] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof. [0436] As used herein, “percent (%) amino acid sequence identity” and “percent identity” when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g., the subject antibody or fragment) that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various known ways, in some embodiments, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0437] In some embodiments, “operably linked” may include the association of components, such as a DNA sequence, (e.g. a heterologous nucleic acid) and a regulatory sequence(s), in such a way as to permit gene expression when the appropriate molecules (e.g. transcriptional activator proteins) are bound to the regulatory sequence. Hence, it means that the components described are in a relationship permitting them to function in their intended manner.

[0438] An amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid. The substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution. Amino acid substitutions may be introduced into a binding molecule, e.g., antibody, of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[0439] Amino acids generally can be grouped according to the following common sidechain properties:

(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

[0440] In some embodiments, conservative substitutions can involve the exchange of a member of one of these classes for another member of the same class. In some embodiments, non-conservative amino acid substitutions can involve exchanging a member of one of these classes for another class.

[0441] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.

[0442] As used herein, a “subject” or an “individual,” which are terms that are used interchangeably, is a mammal. In some embodiments, a “mammal” includes humans, nonhuman primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, monkeys, etc. In some embodiments, the subject or individual is human. In some embodiments, the subject is a patient that is known or suspected of having a disease, disorder or condition.

[0443] As used herein, the term “treating” and “treatment” includes administering to a subject an effective amount of a biological molecule, such as a therapeutic agent, so that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, for example, beneficial or desired clinical results. For instance, a biological molecule may include cells (e.g. T cells), such as cells that have been modified by a DNA-targeting system or polynucleotide(s) encoding the DNA-targeting system described herein. For purposes of this technology, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease. In some embodiments, one or more symptoms of a disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% upon treatment of the disease.

[0444] For purposes of this technology, beneficial or desired clinical results of disease treatment include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. [0445] The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes that amount of a biological molecule, such as a compound or cells, that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the biological molecule, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0446] As used herein, “adoptive cell therapy” (ACT) refers to the administration of T cells targeting a specific antigen to a subject.

[0447] As used herein, the term "autologous" is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

[0448] "Allogeneic" refers to a graft derived from a different animal of the same species.

VI. EXEMPLARY EMBODIMENTS

[0449] Among the provided embodiments are:

1. A DNA-targeting system comprising one or more DNA-targeting modules, wherein the one or more DNA-targeting modules increase transcription of one or more lymphoid cell differentiation (LCD) genes, and wherein each of the one or more DNA-targeting modules comprises a fusion protein comprising:

(a) a DNA-binding domain that binds to a target site for one of the one or more lymphoid cell differentiation genes; and

(b) at least one transcriptional activation domain that increases transcription of the one or more lymphoid cell differentiation genes.

2. The DNA-targeting system of embodiment 1, wherein the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B.

3. A DNA-targeting system comprising one or more DNA-targeting modules for increasing transcription of one or more lymphoid cell differentiation (LCD) genes, wherein each of the one or more DNA-targeting modules comprises a fusion protein comprising:

(a) a DNA-binding domain that binds to a target site for one of the one or more lymphoid cell differentiation genes, wherein the one or more lymphoid cell differentiation genes is selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B; and

4. The DNA-targeting system of any of embodiments 1-3, wherein the one or more LCD genes is selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

5. The DNA-targeting system of any of embodiments 1-4, wherein the one or more DNA targeting modules is a plurality of DNA-targeting modules, and wherein each DNA-targeting module targets one of the one or more LCD genes.

6. The DNA-targeting system of embodiment 5, wherein the plurality of DNA-targeting modules is two, three, four, five, or six DNA-targeting modules, and wherein each DNA- targeting module targets one of the one or more LCD genes.

7. The DNA-targeting system of embodiment 5 or embodiment 6, wherein the plurality of DNA-targeting modules is two DNA-targeting modules, each targeting one of the one or more LCD genes.

8. The DNA-targeting system of any of embodiments 1-7, wherein the one or more LCD genes is two genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the two genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

9. The DNA-targeting system of embodiment 5 or embodiment 6, wherein the plurality of DNA-targeting modules is three DNA-targeting modules, each targeting one of the one or more LCD genes.

10. The DNA-targeting system of any of embodiments 1-6 and 9, wherein the one or more LCD genes is three genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the three genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

11. The DNA-targeting system of embodiment 5 or embodiment 6, wherein the plurality of DNA-targeting modules is four DNA-targeting modules, each targeting one or more LCD genes.

12. The DNA-targeting system of any of embodiments 1-6 and 11, wherein the one or more LCD genes is four genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the four genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

13. The DNA-targeting system of embodiment 5 or embodiment 6, wherein the plurality of DNA-targeting modules is five DNA-targeting modules, each targeting one of the one or more LCD genes.

14. The DNA-targeting system of any of embodiments 1-6 and 13, wherein the one or more LCD genes is five genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the five genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

15. The DNA-targeting system of embodiment 5 or embodiment 6, wherein the plurality of DNA-targeting modules is six DNA-targeting modules, each targeting one of the one or more LCD genes.

16. The DNA-targeting system of any of embodiments 1-6 and 15, wherein the one or more LCD genes is six genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the six genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

17. The DNA-targeting system of any of embodiments 1-16, wherein at least one of the one or more LCD genes is RUNX3.

18. The DNA-targeting system of any of embodiments 1-16, wherein at least one of the one or more LCD genes is IL7Ra.

19. The DNA-targeting system of any of embodiments 1-18, wherein the one or more LCD genes include RUNX3 and IL7Ra.

20. The DNA-targeting system of any of embodiments 1-16, wherein at least one of the one or more LCD genes is TBX21.

21. The DNA-targeting system of any of embodiments 1-16, wherein at least one of the one or more LCD genes is CBFB.

22. The DNA-targeting system of any of embodiments 1-16, wherein at least one of the one or more LCD genes is LEF1.

23. The DNA-targeting system of any of embodiments 1-16, wherein at least one of the one or more LCD genes is MYB.

24. The DNA-targeting system of any of embodiments 1-16, wherein at least one of the one or more LCD genes is RUNX1. 25. The DNA-targeting system of any of embodiments 1-16, wherein at least one of the one or more LCD genes is SPI1.

26. The DNA-targeting system of any of embodiments 1-16, wherein at least one of the one or more LCD genes is HEY 1.

27. The DNA-targeting system of any of embodiments 1-23, wherein the one or more LCD genes are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; RUNX3, IL7Ra, and TBX21; RUNX3, CBFB, EEF1, MYB, TBX21, IE7Ra; RUNX3, CBFB, EEF1, MYB, and TBX21; RUNX3, CBFB, EEF1, and MYB; EEF1, MYB, and TBX21; EEF1 and TBX21; or EEF1 and MYB.

28. The DNA targeting system of any of embodiments 1-23 and 27, wherein the one or more DNA-targeting modules comprise:

(a) a first set of DNA-targeting modules, wherein the first set of DNA-targeting modules targets one or more ECD genes selected from the group consisting of RUNX3, IE7Ra, TBX21, and CBFB; and

(b) a second set of DNA-targeting modules, wherein the second set of DNA-targeting modules targets one or more ECD genes selected from the group consisting of RUNX3, IE7Ra, TBX21, CBFB, EEF1, and MYB.

29. The DNA targeting system of embodiment 28, wherein the one or more ECD genes targeted by the first set of DNA-targeting modules are: RUNX3 and IE7Ra; RUNX3, IE7Ra, and CBFB; or RUNX3, IE7Ra, and TBX21.

30. The DNA targeting system of embodiment 28 or embodiment 29, wherein the one or more ECD genes targeted by the second set of DNA-targeting modules are: RUNX3, CBFB, EEF1, MYB, TBX21, IE7Ra; RUNX3, CBFB, EEF1, MYB, and TBX21; RUNX3, CBFB, EEF1, and MYB; EEF1, MYB, and TBX21; EEF1 and TBX21; or EEF1 and MYB.

31. The DNA-targeting system of any of embodiments 1-3, wherein the one or more ECD genes are selected from the group consisting of TCF7, GATA3 and BCE1 IB.

32. The DNA-targeting system of any of embodiments 1-3 and 31, wherein the one or more DNA-targeting modules comprise two DNA-targeting modules that target a combination of two ECD genes selected from the group consisting of TCF7, GATA3 and BCE1 IB.

33. The DNA-targeting system of any of embodiments 1-3 and 31, wherein the one or more DNA-targeting modules comprise three DNA-targeting modules that target TCF7, GATA3 and BCE11B. 34. The DNA-targeting system of any of embodiments 1-3 and 31-33, wherein at least one of the one or more LCD genes is TCF7.

35. The DNA-targeting system of any of embodiments 1-3, and 31-34, wherein at least one of the one or more LCD genes is GATA3.

36. The DNA-targeting system of any of embodiments 1-3, and 31-35, wherein at least one of the one or more LCD genes is BCLB11.

37. The DNA-targeting system of any of embodiments 1-3, 31, and 33-36, wherein the one or more LCD genes include TCF7, GATA3, and BCLB11.

38. The DNA-targeting system of any of embodiments 1-37, wherein the target site for each of the one or more LCD genes is in the gene or a regulatory DNA element thereof.

39. The DNA-targeting system of embodiment 38, wherein the regulatory DNA element is an enhancer or a promoter of the gene.

40. The DNA-targeting system of embodiment 39, wherein the regulatory DNA element is a promoter of the gene.

41. The DNA-targeting system of any of embodiments 1-40, wherein the target site for each of the one or more LCD genes is within 1000 base pairs of the transcription start site (TSS) of the gene.

42. The DNA-targeting system of any of embodiments 1-41, wherein the target for each of the one or more LCD genes is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS of the gene.

43. The DNA-targeting system of any of embodiments 1-42, wherein the target site for each of the one or more LCD genes is within 550 base pairs of the TSS of the gene.

44. The DNA-targeting system of any of embodiments 1-17, 19, 27-30 and 38-43, wherein the target site for RUNX3 has the sequence forth in SEQ ID NO: 99 or SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

45. The DNA-targeting system of any of embodiments 1-16, 18-19, 27-30 and 38-43, wherein the target site for IL7Ra has the sequence set forth in SEQ ID NO: 103, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. 46. The DNA-targeting system of any of embodiments 1-16, 20, 27-30 and 38-43, wherein the target site for TBX21 has the sequence set forth in SEQ ID NO: 104, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

47. The DNA-targeting system of any of embodiments 1-16, 21, 27-30 and 38-43, wherein the target site for CBFB has the sequence set forth in SEQ ID NO: 106, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

48. The DNA-targeting system of any of embodiments 1-16, 22, 27-28, 30 and 38-43, wherein the target site for LEF1 has the sequence set forth in SEQ ID NO: 105, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

49. The DNA-targeting system of any of embodiments 1-16, 23, 27-28, 30 and 38-43, wherein the target site for MYB has the sequence set forth in SEQ ID NO: 93 or SEQ ID NO: 94, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

50. The DNA-targeting system of any of embodiments 1-16, 24, and 38-43, wherein the target site for RUNX1 has the sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 96, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing.

51. The DNA-targeting system of any of embodiments 1-16, 25, and 38-43, wherein the target site for SPI1 has the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing.

52. The DNA-targeting system of any of embodiments 1-16, 26, and 38-43, wherein the target site for HEY1 has the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 98, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

53. The DNA-targeting system of any of embodiments 1-3 and 31-43 , wherein:

(a) the target site for TCF7 has the sequence set forth in any one of SEQ ID NOS: 1-6, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (b) the target site for GATA3 has the sequence set forth in any one of SEQ ID NOS:7-12, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; and

(c) the target site for BCL11B has the sequence set forth in any one of SEQ ID NOS:13-18, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

54. The DNA-targeting system of any of embodiments 1-3, 31-34, 37-43, and 53, wherein the target site for TCF7 has the sequence set forth in SEQ ID NO: 1, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

55. The DNA-targeting system of any of embodiments 1-3, 31-33, 35, 37-43, and 53, wherein the target site for GATA3 has the sequence set forth in SEQ ID NO: 11, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

56. The DNA-targeting system of any of embodiments 1-3, 31-33, 36-43, and 53, wherein the target site for BCLB 11 has the sequence set forth in SEQ ID NO: 16, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing.

57. The DNA-targeting system of any of embodiments 1-3, 31, 33-43, and 53-56, wherein:

(a) the target site for TCF7 has the sequence set forth in SEQ ID NO: 1, or a complementary sequence thereof;

(b) the target site for GATA3 has the sequence set forth in SEQ ID NO: 11, or a complementary sequence thereof; and

(c) the target site for BCL1 IB has the sequence set forth in SEQ ID NO: 16, or a complementary sequence thereof.

58. The DNA-targeting system of any of embodiments 1-57, wherein the DNA-targeting system does not introduce a genetic disruption or a DNA break.

59. The DNA-targeting system of any of embodiments 1-58, wherein the fusion protein of each DNA-targeting module comprises a DNA-binding domain selected from: a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or a variant thereof; a zinc finger protein (ZFP); a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an I-Scel enzyme or a variant thereof, optionally wherein the DNA- binding domain comprises a catalytically inactive variant of any of the foregoing, wherein, when the DNA-binding domain of the fusion protein comprises a Cas protein, the DNA-targeting module further comprises one or more guide nucleic acids, for targeting the Cas protein to the target site for one of the one or more LCD genes.

60. The DNA-targeting system of any one of embodiments 1-59, wherein the DNA-binding domain is a zinc finger protein.

61. The DNA-targeting system of any one of embodiments 1-60, wherein the fusion protein of each of the plurality of DNA-targeting modules is different.

62. The DNA-targeting system of embodiment 59, wherein the DNA-targeting system comprises one fusion protein that is shared by each of the plurality of DNA-targeting modules and wherein each DNA-targeting module is characterized by comprising a different guide nucleic acid for targeting the DNA-binding domain to the target site.

63. The DNA-targeting system of any of embodiments 1-59 and 61-62, wherein the DNA- binding domain of the fusion protein is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof.

64. The DNA-targeting system of any of embodiments 1-59 and 61-63, wherein the DNA- binding domain of each of the one or more DNA-targeting modules is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof, and each of the one or more DNA-targeting modules further comprises one or more guide nucleic acids for targeting the DNA-binding domain to the target site of one of the one or more LCD gene.

65. The DNA-targeting system of any of embodiments 59 and 61-64, wherein the one or more guide nucleic acids are one or more guide RNAs (gRNAs).

66. The DNA-targeting system of any of embodiments 59 and 61-64, wherein the Cas protein or variant thereof is a deactivated (dCas) protein.

67. A DNA-targeting system comprising one or more DNA-targeting modules, wherein each of the one or more DNA-targeting modules comprises:

(a) a fusion protein comprising a DNA binding domain comprising a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and

(b) one or more gRNAs that target a target site for one or more lymphoid cell differentiation (LCD) genes, wherein the DNA-targeting system increases transcription of the one or more LCD genes. 68. The DNA-targeting system of embodiment 67, wherein the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B.

69. A DNA-targeting system comprising one or more DNA-targeting modules, wherein each of the one or more DNA-targeting modules comprises:

(b) one or more gRNAs that target a target site for one or more lymphoid cell differentiation (LCD) genes, wherein the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B.

70. The DNA-targeting system of any of embodiments 67-69, wherein the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1 and HEY1.

71. The DNA-targeting system of any of embodiments 67-70, comprising two, three, four, five, or six different guide RNAs, wherein each gRNA targets a target site of one of the one or more LCD genes.

72. The DNA-targeting system of any of embodiments 67-71, wherein the one or more LCD genes are two genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the two genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

73. The DNA-targeting system of any of embodiments 67-71, wherein the one or more LCD genes are three genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the three genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

74. The DNA-targeting system of any of embodiments 67-71, wherein the one or more LCD genes are four genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the four genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

75. The DNA-targeting system of any of embodiments 67-71, wherein the one or more LCD genes are five genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the five genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

76. The DNA-targeting system of any of embodiments 67-71, wherein the one or more LCD genes are six genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the six genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

77. The DNA-targeting system of any of embodiments 67-76, wherein at least one of the one or more LCD genes is RUNX3.

78. The DNA-targeting system of any of embodiments 67-76, wherein at least one of the one or more LCD genes is IL7Ra.

79. The DNA-targeting system of any of embodiments 67-78, wherein the one or more LCD genes include RUNX3 and IL7Ra.

80. The DNA-targeting system of any of embodiments 67-76, wherein at least one of the one or more LCD genes is TBX21.

81. The DNA-targeting system of any of embodiments 67-76, wherein at least one of the one or more LCD genes is CBFB.

82. The DNA-targeting system of any of embodiments 67-76, wherein at least one of the one or more LCD genes is LEF1.

83. The DNA-targeting system of any of embodiments 67-76, wherein at least one of the one or more LCD genes is MYB.

84. The DNA-targeting system of any of embodiments 67-76, wherein at least one of the one or more LCD genes is RUNX1.

85. The DNA-targeting system of any of embodiments 67-76, wherein at least one of the one or more LCD genes is SPI1.

86. The DNA-targeting system of any of embodiments 67-76, wherein at least one of the one or more LCD genes is HEY 1.

87. The DNA-targeting system of any of embodiments 67-83, wherein the one or more LCD genes are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; RUNX3, IL7Ra, and TBX21; RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB. 88. The DNA targeting system of any of embodiments 67-87, wherein the one or more DNA-targeting modules comprise:

(a) a first DNA-targeting module comprising a first fusion protein and a first set of gRNAs; and

(b) a second DNA-targeting module comprising a second fusion protein and a second set of gRNAs, wherein the first and second fusion proteins comprise a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and wherein the first and second set of gRNAs target a target site for one or more lymphoid cell differentiation (LCD) genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1.

89. The DNA-targeting system of embodiment 88, wherein the first set of gRNAs and the second set of gRNAs are the same.

90. The DNA-targeting system of embodiment 88 and embodiment 89, wherein the first set of gRNAs and the second set of gRNAs each target a combination of two or more of the LCD genes that are the same.

91. The DNA-targeting system of embodiment 88, wherein the first set of gRNAs and the second set of gRNAs are different.

92. The DNA-targeting system of embodiment 88 and embodiment 91, wherein the first set of gRNAs and the second set of gRNAs each target a combination of two or more of the LCD genes that are different.

93. A DNA-targeting system comprising one or more DNA-targeting modules comprising:

(1) a first DNA-targeting module comprising (a) a first fusion protein comprising a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and (b) a first set of gRNAs comprising two or more gRNAs that target a target site for two or more lymphoid cell differentiation (LCD) genes; and

(2) a second DNA-targeting module comprising (a) a second fusion protein comprising a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and (b) a second set of gRNAs comprising two or more gRNAs that target a target site for two or more lymphoid cell differentiation (LCD) genes, wherein the two or more LCD genes in the first and second set of gRNAs are independently selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1.

94. The DNA targeting system of any of embodiments 88-93, wherein the first set of gRNAs targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, and CBFB; and the second set of gRNAs targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

95. The DNA targeting system of any of embodiments 88-94, wherein the one or more LCD genes targeted by the first set of gRNAs are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; or RUNX3, IL7Ra, and TBX21.

96. The DNA targeting system of any of embodiments 88-95, wherein the one or more LCD genes targeted by the second set of gRNAs are: RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB.

97. The DNA targeting system of any of embodiments 88-96, wherein: the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1 and TBX21; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target RUNX3, CBFB, LEF1 and MYB; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1 and MYB; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1, MYB and TBX21; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target RUNX3, CBFB, LEF1, MYB and TBX21; or the first set of gRNAs target RUNX3 and IL7RA and the second set for gRNAs target RUNX3, CBFB, LEF1, MYB, TBX21 and IL7Ra.

98. The DNA targeting system of any of embodiments 88-97, wherein the dCas and the transcriptional activation domain of the first and second fusion protein are the same.

99. The DNA targeting system of any of embodiments88-99, wherein the first DNA- targeting module is present in a first lipid nanoparticle and the second DNA-targeting module is present in a second lipid nanoparticle. 100. The DNA-targeting system of any of embodiments 67-69, wherein the one or more LCD genes are selected from the group consisting of TCF7, GATA3, and BCL1 IB.

101. The DNA-targeting system of any of embodiments 66-100 , wherein the dCas protein lacks nuclease activity.

102. The DNA-targeting system of any of embodiments 66-101, wherein the dCas protein is a dCas9 protein.

103. The DNA-targeting system of embodiment 102, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

104. The DNA-targeting system of embodiment 103, wherein the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 83.

105. The DNA-targeting system of embodiment 103 or embodiment 104, wherein the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 84 or SEQ ID NO: 191, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

106. The DNA-targeting system of any of embodiments 103-105, wherein the dSaCas9 protein is set forth in SEQ ID NO: 84 or SEQ ID NO: 191.

107. The DNA-targeting system of embodiment 102, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

108. The DNA-targeting system of embodiment 107, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 63.

109. The DNA-targeting system of embodiment 107 or embodiment 108, wherein the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 64 or SEQ ID NO: 196, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

110. The DNA-targeting system of any of embodiments 107-109, wherein the dSpCas9 protein is set forth in SEQ ID NO: 64 or SEQ ID NO: 196.

111. The DNA-targeting system of any of embodiments 65-110, wherein each of the one or more gRNAs comprise a gRNA spacer that is complementary to the target site of the gene.

112. The DNA-targeting system of any of embodiments 68-77, 79, 87-99, and 101- 111, wherein the gRNA targeting a target site for RUNX3 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114, or a contiguous portion thereof of at least 14 nt.

113. The DNA-targeting system of embodiment 112, wherein the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114.

114. The DNA-targeting system of any of embodiments 68-76, 78-79, 87-99, and 101- 111, wherein the gRNA targeting a target site for IL7Ra comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 117, or a contiguous portion thereof of at least 14 nt.

115. The DNA-targeting system of embodiment 114, wherein the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO: 117.

116. The DNA-targeting system of any of embodiments 68-76, 80, 87-99, and 101- 111, wherein the gRNA targeting a target site for TBX21 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 118, or a contiguous portion thereof of at least 14 nt.

117. The DNA-targeting system of embodiment 116, wherein the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO:118.

118. The DNA-targeting system of any of embodiments 68-76, 81, 87-99, and 101- 111, wherein the gRNA targeting a target site for CBFB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 120, or a contiguous portion thereof of at least 14 nt.

119. The DNA-targeting system of embodiment 118, wherein the gRNA targeting a target site for CBFB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 120.

120. The DNA-targeting system of any of embodiments 68-76, 82, 87-94, 96-99, and 101 - 111, wherein the gRNA targeting a target site for LEF1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119, or a contiguous portion thereof of at least 14 nt.

121. The DNA-targeting system of embodiment 120, wherein the gRNA targeting a target site for LEF1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119.

122. The DNA-targeting system of any of embodiments 68-76, 83, 87-94, 96-99, and 101 - 111, wherein the gRNA targeting a target site for MYB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108, or a contiguous portion thereof of at least 14 nt.

123. The DNA-targeting system of embodiment 122, wherein the gRNA targeting a target site for MYB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108.

124. The DNA-targeting system of any of embodiments 68-76, 84, 87-93, 98-99, and 101 - 111, wherein the gRNA targeting a target site for RUNX1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110, or a contiguous portion thereof of at least 14 nt.

125. The DNA-targeting system of embodiment 124, wherein the gRNA targeting a target site for RUNX1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110.

126. The DNA-targeting system of any of embodiments 68-76, 85, 87-93, 98-99, and 101 - 111, wherein the gRNA targeting a target site for SPI1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116, or a contiguous portion thereof of at least 14 nt.

127. The DNA-targeting system of embodiment 126, wherein the gRNA targeting a target site for SPI1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116.

128. The DNA-targeting system of any of embodiments 68-76, 86-93, 98-99, and 101- 111, wherein the gRNA targeting a target site for HEY 1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112, or a contiguous portion thereof of at least 14 nt.

129. The DNA-targeting system of embodiment 128, wherein the gRNA targeting a target site for HEY 1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112.

130. The DNA-targeting system of any of embodiments68-69 and 100-111 , wherein:

(a) the gRNA targeting a target site for TCF7 comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 19-24, or a contiguous portion thereof of at least 14 nt; (b) the gRNA targeting a target site for GATA3 comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 25-30, or a contiguous portion thereof of at least 14 nt; and

(c) the gRNA targeting a target site for BCLB 11 comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 31-36, or a contiguous portion thereof of at least 14 nt.

131. The DNA-targeting system of any of embodiments 68-69, 100-111, and 120 , wherein:

(a) the gRNA targeting a target site for TCF7 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19;

(b) the gRNA targeting a target site for GATA3 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 29; and

(c) the gRNA targeting a target site for BCLB 11 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 34.132. The DNA-targeting system of any of embodiments 111-131, wherein the spacer sequence is between 14 nt and 24 nt, or between 16 nt and 22 nt in length.

133. The DNA-targeting system of any of embodiments 111-132, wherein the spacer sequence is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

134. The DNA-targeting system of any of embodiments 65-102or 107-133, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 56.

135. The DNA-targeting system of any of embodiments 65-106 or 111-133-, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 91.

136. The DNA-targeting system of any of embodiments 65-102 or 107-133, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 122.

137. The DNA-targeting system of any of embodiments 65-136, wherein the gRNA further comprises 2’ MeO modified bases and/or phosphorothiate backbone modifications.

138. The DNA-targeting system of any of 1-137, wherein the at least one transcriptional activator effector domain is selected from the group consisting of: a VP64 domain, a p65 activation domain, a p300 domain, an Rta domain, a CBP domain, a VPR domain, a VPH domain, an HSF1 domain, a TET protein domain, optionally wherein the TET protein is TET1, a SunTag domain, or a domain, portion, variant, or truncation of any of the foregoing. 139. The DNA-targeting system of any of embodiments 1-138, wherein the at least one transcriptional activator effector domain comprises at least one VP 16 domain, and/or a VP 16 tetramer (“VP64”) or a variant thereof.

140. The DNA-targeting system of any of embodiments 1-139, wherein the at least one transcriptional activator effector domain comprises a VP64 domain or a variant or portion thereof that exhibits transcriptional activation activity.

141. The DNA-targeting system of any of embodiments 1-140, wherein the at least one transcriptional activator effector domain is VP64.

142. The DNA-targeting system of embodiment 141, wherein the VP64 is positioned N- terminal and/or C-terminal to the DNA-binding domain.

143. The DNA-targeting system of any of embodiments 1-142, wherein the at least one transcriptional activator effector domain comprises two copies of VP64.

144. The DNA-targeting system of any of embodiments 1-143, wherein the at least one transcriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 60, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

145. The DNA-targeting system of any of embodiments 1-144, wherein the at least one transcriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 62, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

146. The DNA-targeting system of any of embodiments 1-145, wherein the fusion protein comprises the sequence set forth in SEQ ID NO: 58, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

147. The DNA-targeting system of any of embodiments 1-146, wherein introduction of the DNA-targeting system to a hematopoietic progenitor cell (HPC) promotes lymphoid differentiation.

148. The DNA-targeting system of embodiment 147, wherein the lymphoid differentiation is to a lymphoid progenitor cell (LPC) phenotype.

149. The DNA-targeting system of embodiment 148, wherein the LPC phenotype is an induced common lymphoid progenitor (iCLP) phenotype. 150. The DNA-targeting system of any of embodiments 147-149, wherein the lymphoid differentiation is characterized by decreased expression of CD34 relative to a HPC that was not introduced with the DNA-targeting system.

151. The DNA-targeting system of any of embodiments 147-150, wherein the lymphoid differentiation is characterized by differentiation to a CD34- cell.

152. The DNA-targeting system of any of embodiments 147-151, wherein the lymphoid differentiation is characterized by increased expression of CD45 relative to a HPC that was not introduced with the DNA-targeting system.

153. The DNA-targeting system of any of embodiments 147-152, wherein the lymphoid differentiation is characterized by differentiation to a CD45+ cell.

154. The DNA-targeting system of any of embodiments 147-153, wherein the lymphoid differentiation is characterized by increased expression of CD7 relative to a HPC that was not introduced with the DNA-targeting system.

155. The DNA-targeting system of any of embodiments 147-154, wherein the lymphoid differentiation is characterized by differentiation to a CD7+ cell.

156. The DNA-targeting system of any of embodiments 147-155, wherein the lymphoid differentiation is characterized by increased expression of both CD5 and CD7 relative to a HPC that was not introduced with the DNA-targeting system.

157. The DNA-targeting system of any of embodiments 147-155, wherein the lymphoid differentiation is characterized by differentiation to a CD5+CD7+ cell.

158. The DNA-targeting system of any of embodiments 147-157, wherein the lymphoid differentiation is characterized by increased expression of CD56 relative to a HPC that was not introduced with the DNA-targeting system.

159. The DNA-targeting system of any of embodiments 147-158, wherein the lymphoid differentiation is characterized by differentiation to a CD56+ cell.

160. The DNA-targeting system of any of embodiments 147-159, wherein the lymphoid differentiation is characterized by decreased expression of c-KIT relative to a HPC that was not introduced with the DNA-targeting system.

161. The DNA-targeting system of any of embodiments 147-160, wherein the lymphoid differentiation is characterized by differentiation to a c-KIT- cell. 162. A gRNA that targets a target site for RUNX3, wherein the target site for RUNX3 comprises the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

163. The gRNA of embodiment 162, wherein the target site for RUNX3 comprises the sequence set forth in any one of SEQ ID NO:99 or SEQ ID NO: 100.

164. The gRNA of embodiment 162 or embodiment 163 wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114, or a contiguous portion thereof of at least 14 nt.

165. The gRNA of any of embodiments 162-164, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114.

166. A gRNA targeting a target site for IL7Ra, wherein the target site for IL7Ra comprises the sequence set forth in SEQ ID NO: 103, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

167. The gRNA of embodiment 166, wherein the target site for IL7Ra comprises the sequence set forth in SEQ ID NOS: 103.

168. The gRNA of embodiment 166 or embodiment 167, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 117, or a contiguous portion thereof of at least 14 nt.

169. The gRNA of any of embodiments 166-168, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 117.

170. A gRNA targeting a target site for TBX21, wherein the target site for TBX21 comprises the sequence set forth in SEQ ID NO: 104, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

171. The gRNA of embodiment 170, wherein the target site for TBX21 comprises the sequence set forth in SEQ ID NO: 104.

172. The gRNA of embodiment 170 and 171, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 118, or a contiguous portion thereof of at least 14 nt.

173. The gRNA of any of embodiments 170-172, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119. 174. A gRNA targeting a target site for CBFB wherein the target site for CBFB comprises the sequence set forth in SEQ ID NO: 106, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

175. The gRNA of embodiment 174, wherein the target site for CBFB comprises the sequence set forth in SEQ ID NO: 106.

176. The gRNA of embodiment 174 or embodiment 175, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 120, or a contiguous portion thereof of at least 14 nt.

177. The gRNA of any of embodiments 174- 176, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 120.

178. A gRNA targeting a target site for LEF1 wherein the target site for LEF1 comprises the sequence set forth in SEQ ID NO: 105, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

179. The gRNA of embodiment 178, wherein the target site for LEF1 comprises the sequence set forth in SEQ ID NO: 105.

180. The gRNA of embodiment 178 or embodiment 179, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119, or a contiguous portion thereof of at least 14 nt.

181. The gRNA of any of embodiments 178-180, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119.

182. A gRNA targeting a target site for MYB, wherein the target site for MYB comprises the sequence set forth in SEQ ID NO: 93 or SEQ ID NO: 94, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

183. The gRNA of embodiment 182, wherein the target site for MYB comprises the sequence set forth in SEQ ID NO: 93 or SEQ ID NO: 94.

184. The gRNA of embodiment 182or embodiment 183, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108, or a contiguous portion thereof of at least 14 nt.

185. The gRNA of any of embodiments 182- 184, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108. 186. A gRNA targeting a target site for RUNX1 wherein the target site for RUNX1 comprises the sequence set forth in SEQ ID NO:95 or SEQ ID NO: 96, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

187. The gRNA of embodiment 186, wherein the target site for RUNX1 comprises the sequence set forth in SEQ ID NO:95 or SEQ ID NO: 96.

188. The gRNA of embodiment 186 or embodiment 187, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110, or a contiguous portion thereof of at least 14 nt.

189. The gRNA of any of embodiments 186- 188, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110.

190. A gRNA targeting a target site for SPI1, wherein the target site for SPI1 comprises the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

191. The gRNA of embodiment 190, wherein the target site for SPI1 comprises the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102.

192. The gRNA of embodiment 190 or embodiment 191, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116, or a contiguous portion thereof of at least 14 nt.

193. The gRNA of any of embodiments 190-192, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116.

194. A gRNA targeting a target site for HEY1, wherein the target site for HEY 1 comprises the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 98, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

195. The gRNA of embodiment 194, wherein the target site for HEY1 comprises the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 98.

196. The gRNA of embodiment 194 or embodiment 195, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112, or a contiguous portion thereof of at least 14 nt.

197. The gRNA of any of embodiments 194-196, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112. 198. A gRNA that targets a target site for TCF7, wherein the target site has the sequence set forth in any one of SEQ ID NOS: 1-6, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

199. The gRNA of embodiment 198, wherein the target site for TCF7 comprises the sequence set forth in SEQ ID NO: 1, or a complementary sequence thereof.

200. The gRNA of embodiment 198 or embodiment 199, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 19-24, or a contiguous portion thereof of at least 14 nt.

201. The gRNA of any of embodiments 198-200, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 19-24.

202. The gRNA of any of embodiments 198-201, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19.

203. A gRNA that targets a target site for GATA3, wherein the target site comprises the sequence set forth in any one of SEQ ID NOS:7-12, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

204. The gRNA of embodiment 203, wherein the target site for GATA3 comprises the sequence set forth in SEQ ID NO: 11, or a complementary sequence thereof.

205. The gRNA of embodiment 203 or embodiment 204, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 25-30, or a contiguous portion thereof of at least 14 nt.

206. The gRNA of any of embodiments 203-205, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 25-30.

207. The gRNA of any of embodiments 203-206, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 29.

208. A gRNA that targets a target site for BCLB 11, wherein the target site for BCL1 IB comprises the sequence set forth in any one of SEQ ID NOS: 13-18, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

209. The gRNA of embodiment 208, wherein the target site for BCL1 IB comprises the sequence set forth in SEQ ID NO: 16, or a complementary sequence thereof.

210. The gRNA of embodiment 208 or embodiment 209, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 31-36, or a contiguous portion thereof of at least 14 nt. 211. The gRNA of any of embodiments208-210, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 31-36.

212. The gRNA of any of embodiments208-211, wherein the gRNA targeting a target site for BCLB11 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 34.

213. The gRNA of any of embodiments 162-212, wherein the spacer sequence is between 14 nt and 24 nt, or between 16 nt and 22 nt in length.

214. The gRNA of any of embodiments 162-213, wherein the spacer sequence is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

215. The gRNA of any of embodiments 162-214, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 56.

216. The gRNA of any of embodiments 162-214, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 91.

217. The gRNA of any of embodiments 162-214, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 122.

218. The gRNA of any of embodiments 162-217, wherein the gRNA further comprises 2’ MeO modified bases and/or phosphorothiate backbone modifications.

219. A combination comprising two or more gRNAs of any of embodiments 162-218.

220. The combination of embodiment 219, comprising two, three, four, five, or six different guide RNAs, wherein each gRNA targets a target site of a different LCD genes.

221. A combination of gRNAs comprising two or more gRNAs, wherein the combination of gRNAs targets a target site for one or more lymphoid cell differentiation (LCD) genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1.

222. The combination of gRNAs of embodiment 221, wherein the target site for each of the one or more LCD genes is in the gene or a regulatory DNA element thereof.

223. The combination of gRNAs of embodiment 222, wherein the regulatory DNA element is an enhancer or a promoter.

224 The combination of gRNAs of embodiment 222 or embodiment 223, wherein the regulatory DNA element is a promoter of the gene. 225. The combination of gRNAs of any of embodiments 221-224, wherein the target site for each of the one or more LCD genes is independently within 1000 base pairs of the transcription start site (TSS).

226. The combination of gRNAs of any of embodiments 221-225, wherein the target site for each of the one or more LCD genes is independently within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS of the gene.

227. The combination of gRNAs of any of embodiments 221-225, wherein the target site for each of the one or more LCD genes is within 550 base pairs of the TSS of the gene.

228. The combination of gRNAs of any of embodiments 221-227 comprising two, three, four, five, or six different guide RNAs, wherein each gRNA targets a target site of one o the one or more LCD genes.

229. The combination of gRNAs of any of embodiments 221-228, wherein the one or more LCD genes are two genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the two genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

230. The combination of gRNAs of any of embodiments 221-228, wherein the one or more LCD genes are three genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the three genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

231. The combination of gRNAs of any of embodiments 221-228, wherein the one or more LCD genes are four genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the four genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

232. The combination of gRNAs of any of embodiments 221-228, wherein the one or more LCD genes are five genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPIl,and HEY1; optionally where the five genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

233. The combination of gRNAs of any of embodiments 221-228, wherein the one or more LCD genes are six genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the six genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. 234. The combination of any of embodiments 221-233, wherein at least one of the one or more LCD genes is RUNX3.

235. The combination of any of embodiments 221-233, wherein at least one of the one or more LCD genes is IL7Ra.

236. The combination of any of embodiments 221-235, wherein the one or more LCD genes include RUNX3 and IL7Ra.

237. The combination of any of embodiments 221-233, wherein at least one of the one or more LCD genes is TBX21.

238. The combination of any of embodiments 221-233, wherein at least one of the one or more LCD genes is CBFB.

239. The combination of any of embodiments 221-233, wherein at least one of the one or more LCD genes is LEF1.

240. The combination of any of embodiments 221-233, wherein at least one of the one or more LCD genes is MYB.

241. The combination of any of embodiments 221-233, wherein at least one of the one or more LCD genes is RUNX1.

242. The combination of any of embodiments 221-233, wherein at least one of the one or more LCD genes is SPI1.

243. The combination of any of embodiments 221-233, wherein at least one of the one or more LCD genes is HEY 1.

244. The combination of any of embodiments 221-243, wherein the one or more LCD genes are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; RUNX3, IL7Ra, and TBX21; RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB.

245. The combination of gRNAs of any of embodiments 221-240 and 244, wherein the two or more gRNAs comprise:

(a) a first set of gRNAs, wherein the first set of gRNAs target one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, and CBFB; and

(b) a second set of gRNAs, wherein the second set gRNAs target one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB. 246. The combination of gRNAs of embodiment 245, wherein the one or more LCD genes targeted by the first set of gRNAs are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; or RUNX3, IL7Ra, and TBX21.

247. The combination of gRNAs of embodiment 245 or embodiment 246, wherein the one or more LCD genes targeted by the second set of gRNAs are: RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB.

248. The combination of gRNAs of any of embodiments 245-247, wherein: the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1 and TBX21; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target RUNX3, CBFB, LEF1 and MYB; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1 and MYB; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1, MYB and TBX21; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target RUNX3, CBFB, LEF1, MYB and TBX21; or the first set of gRNAs target RUNX3 and IL7RA and the second set for gRNAs target RUNX3, CBFB, LEF1, MYB, TBX21 and IL7Ra.

249. The combination of gRNAs of any of embodiments 221-234, 236, and 244-248, wherein the target site for RUNX3 has the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100 , a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

250. The combination of gRNAs of any of embodiments 221-234, 236, and 244-249, wherein the gRNA targeting a target site for RUNX3 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114, or a contiguous portion thereof of at least 14 nt.

251. The combination of gRNAs of any of embodiments 221-233, 235-236, and 244-248, wherein the target site for IL7Ra has the sequence set forth in SEQ ID NO: 103, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. 252. The combination of gRNAs of any of embodiments 221-233, 235-236, 244-248, and 251, wherein the gRNA targeting a target site for IL7Ra comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 117, or a contiguous portion thereof of at least 14 nt.

253. The combination of gRNAs of any of embodiments 221-233, 237, and 244-248, wherein the target site for TBX21 has the sequence set forth in SEQ ID NO: 104, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing.

254. The combination of gRNAs of any of embodiments 221-233, 237, 244-248, and 253, wherein the gRNA targeting a target site for TBX21 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 118, or a contiguous portion thereof of at least 14 nt.

255. The combination of gRNAs of any of embodiments 221-233, 238, and 244-248, wherein the target site for CBFB has the sequence set forth in SEQ ID NO: 106, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

256. The combination of gRNAs of any of embodiments 221-233, 238, 244-248, and 255, wherein the gRNA targeting a target site for CBFB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 120, or a contiguous portion thereof of at least 14 nt.

257. The combination of gRNAs of any of embodiments 221-233, 239, 244-245, and 247- 248, wherein the target site for LEF1 has the sequence set forth in SEQ ID NO: 105, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

258. The combination of gRNAs of any of embodiments 221-233, 239, 244-245, 247-248 and 257, wherein the gRNA targeting a target site for LEF1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119, or a contiguous portion thereof of at least 14 nt.

259. The combination of gRNAs of any of embodiments 221-233, 240, 244-245, and 247- 248, wherein the target site for MYB has the sequence set forth in SEQ ID NO: 93 or SEQ ID NO: 94, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing. 260. The combination of gRNAs of any of embodiments 221-233, 240, 244-245, 247-248, and 259, wherein the gRNA targeting a target site for MYB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108, or a contiguous portion thereof of at least 14 nt.

261. The combination of gRNAs of any of embodiments 221-233 and 241, wherein the target site for RUNX1 has the sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 96, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing.

262. The combination of gRNAs of any of embodiments 221-233, 241, and 261, wherein the gRNA targeting a target site for RUNX1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110, or a contiguous portion thereof of at least 14 nt.

263. The combination of gRNAs of any of embodiments 221-233 and 242, wherein the target site for SPI1 has the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing.

264. The combination of gRNAs of any of embodiments 221-233, 242, and 263, wherein the gRNA targeting a target site for SPI1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116, or a contiguous portion thereof of at least 14 nt.

265. The combination of gRNAs of any of embodiments 221-233, and 243, wherein the target site for HEY1 has the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 98, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing.

266. The combination of gRNAs of any of embodiments 221-233, 243, and 265, wherein the gRNA targeting a target site for HEY 1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112, or a contiguous portion thereof of at least 14 nt.

267. The combination of gRNAs of any of embodiments 221-266, wherein the two or more guide RNAs independently comprise a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length. 268. The combination of gRNAs of any of embodiments 221-267, wherein the two or more guide RNAs independently comprise a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

269. The combination of gRNAs of any of embodiments 221-268, wherein the two or more guide RNAs each further comprise a scaffold sequence set forth in SEQ ID NO: 56.

270. The combination of gRNAs of any of embodiments221-268, wherein the two or more guide RNAs each further comprise a scaffold sequence set forth in SEQ ID NO: 91.

271. The combination of gRNAs of any of embodiments 221-268, wherein the two or more guide RNAs each further comprise a scaffold sequence set forth in SEQ ID NO: 122.

272. The combination of gRNAs of any of embodiments 221-271, wherein the two or more gRNAs independently further comprise 2’ MeO modified bases and/or phosphorothiate backbone modifications.

273. A Cas-guide RNA (gRNA) combination comprising:

(a) a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof fused to a transcriptional activation domain; and

(b) the combination of gRNAs of any of embodiments 219, 220, and 221-272.

274. A Cas-guide RNA (gRNA) combination comprising:

(b) one or more guide RNAs, each selected from the gRNA of any of embodiments 162-218.

275. The Cas-gRNA combination of embodiment 273 or embodiment 274, wherein the Cas protein or variant thereof is a deactivated (dCas) protein.

276. The Cas-gRNA combination of embodiment 275, wherein the dCas protein lacks nuclease activity.

277. The Cas-gRNA combination of embodiment 275 or embodiment 276, wherein the dCas protein is a dCas9 protein.

278. The Cas-gRNA combination of embodiment 277, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

279. The Cas-gRNA combination of embodiment 278, wherein the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 83. 280. The Cas-gRNA combination of embodiment 278 or embodiment 279, wherein the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 84 or SEQ ID NO: 191, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

281. The Cas-gRNA combination of any of embodiments 278-280, wherein the dSaCas9 protein is set forth in SEQ ID NO: 84 or SEQ ID NO: 191.

282. The Cas-gRNA combination of embodiment 277, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

283. The Cas-gRNA combination of embodiment 282, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 63.

284. The Cas-gRNA combination any of embodiment 282 or embodiment 283, wherein the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 64 or SEQ ID NO: 196, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

285. The Cas-gRNA combination of any of embodiments 282-284, wherein the dSpCas9 protein is set forth in SEQ ID NO: 64 or SEQ ID NO: 196.

286. A polynucleotide encoding the DNA-targeting system of any of embodiments 1-161.

287. A polynucleotide encoding at least one DNA-targeting module of the DNA-targeting system of any of embodiments 1-161.

288. A polynucleotide encoding the gRNA of any of embodiments 162-218.

289. A polynucleotide encoding the combination of gRNAs of any of embodiments 219, 220, and 221-272.

290. A polynucleotide encoding the Cas-gRNA combination of any of embodiments 273- 285.

291. A vector comprising the polynucleotide of any of embodiments 286-290.

292. The vector of embodiment 291 that is a viral vector.

293. The vector of embodiment 291 that is a lipid nanoparticle.

294. A pharmaceutical composition comprising the DNA-targeting system of any of embodiments 1-161, the Cas-gRNA combination of any of embodiments 273-285, the polynucleotide of any of embodiments 286-290, or the vector of embodiment 292 or embodiment 293. 295. The pharmaceutical composition of embodiment 294 comprising a pharmaceutically acceptable excipient.

296. A method of differentiating a population of hematopoietic progenitor cells (HPCs) to a differentiated population of cells, the method comprising introducing the DNA-targeting system of any one of embodiments 1-161, the Cas-gRNA combination of any of embodiments 273-285, the polynucleotide of any of embodiments 286-290, the vector of embodiment 292 or embodiment 293, or a combination thereof, into a population of HPCs, and culturing the HPCs under conditions for their differentiation.

297. A method of differentiating a population of hematopoietic progenitor cells (HPCs) to a differentiated population of cells, the method comprising introducing the pharmaceutical composition of embodiment 294 or embodiment 295 into a population of HPCs, and culturing the HPCs under conditions for their differentiation.

298. The method of any of embodiment 296 or embodiment 297, wherein the population of HPCs are induced hematopoietic progenitor cells (iHPCs).

299. The method of any of embodiment 296 or embodiment 297, wherein the population of stem cells are primary hematopoietic progenitor cells.

300. The method of any of embodiments 296-298, wherein the HPCs comprise cells engineered with a recombinant receptor, optionally a chimeric antigen receptor.

301. The method of embodiment 300, wherein the differentiated cells comprise cells that express a recombinant receptor, optionally a chimeric antigen receptor.

302. The method of any of embodiments 296-301, wherein cells of the differentiated population of cells are lymphoid progenitor cells.

303. The method of embodiment 302, wherein the lymphoid progenitor cells are induced common lymphoid progenitor cells (iCLPs).

304. The method of any of embodiments 296-303, wherein the introducing decreases expression of CD34 in the differentiated population of cells relative to the population of HPCs.

305. The method of any of embodiments 296-304, wherein cells of the differentiated population of cells are CD34- cells.

306. The method of any of embodiments 296-305, wherein the introducing increases expression of CD45 in the differentiated population of cells relative to the population of HPCs.

307. The method of any of embodiments 296-306, wherein cells of the differentiated population of cells are CD45+ cells. 308. The method of any of embodiments 296-307, wherein the introducing increases expression of CD7 in the differentiated population of cells relative to the population of HPCs.

309. The method of any of embodiments 296-308, wherein cells of the differentiated population of cells are CD7+ cells.

310. The method of any of embodiments 296-309, wherein the introducing increases expression of both CD5 and CD7 in the differentiated population of cells relative to the population of HPCs.

311. The method of any of embodiments 296-310, wherein cells of the differentiated population of cells are CD5+CD7+ cells.

312. The method of any of embodiments 296-311, wherein the introducing increases expression of CD56 in the differentiated population of cells relative to the population of HPCs.

313. The method any of embodiments 296-312, wherein cells of the differentiated population of cell are CD56+ cells.

314. The method of any of embodiments 296-313, wherein the introducing decreases expression of c-KIT in the differentiated population of cells relative in the population of HPCs.

315. The method any of embodiments 296-314, wherein cells of the differentiated population of cells are c-KIT" cells.

316. The method of any of embodiments 296-315, wherein the introducing is by transient delivery into the population of stem HPCs.

317. The method of embodiment 316, wherein the transient delivery comprises electroporation, transfection, or transduction.

318. The method of embodiment 317, wherein the transient delivery comprises transfection using lipid nanoparticles (LNPs).

319. The method of embodiment 317 or embodiment 318, wherein the transient delivery is repeated at least once.

320. The method of any of embodiments 296-319, wherein the DNA-targeting system of any one of embodiments 1-161, the Cas-gRNA combination of any of embodiments 273-285, the polynucleotide of any of embodiments286-290, the vector of embodiment 292 or embodiment 293, or a combination thereof, is transiently expressed and/or transiently present in the population of HPCs.

321. The method of any of any of embodiments 296-320, wherein the introducing increases transcription of one or more lymphoid cell differentiation (LCD) genes, selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL1 IB, in the population of HPCs.

322. The method of any of embodiments 296-321, wherein the introduced DNA-targeting system comprises at least two DNA-targeting modules and wherein each of the at least two modules are introduced at different times.

323. The method of embodiment 322, wherein that at least two DNA-targeting modules is a first set and second set of DNA-targeting modules that are introduced at different times.

324. The method of embodiment 323, wherein:

(a) the first set of DNA-targeting modules targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, and CBFB; and

(b) a second set of DNA-targeting modules, wherein the second set of DNA-targeting modules targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

325. The method of embodiment 324, wherein the one or more LCD genes targeted by the first set of DNA-targeting modules are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; or RUNX3, IL7Ra, and TBX21.

326. The method of embodiment 324 or embodiment 326, wherein the one or more LCD genes targeted by the second set of DNA-targeting modules are: RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB.

327. The method of any of embodiments 302-326, wherein the lymphoid progenitor cells express a recombinant receptor, optionally a chimeric antigen receptor.

328. A population of lymphoid progenitor cells produced by the method of any of embodiments 302-327.

329. The method of any of embodiment 296-301 and 304-328, wherein cells of the differentiated population of cells are lymphoid cells (LCs).

330. A method of generating lymphoid cells (LCs), the method comprising culturing the population of lymphoid progenitor cells produced by the method of any of embodiments 302- 327 or the population of lymphoid progenitor cells of embodiment 328 under conditions to differentiate cells of the population to lymphoid cells (LCs) to produce a population comprising LCs. 331. The method of embodiment 329 or embodiments 330, wherein the LCs are induced T

(iT) cells, induced B (iB) cells or induced natural killer (iNK) cells.

332. The method of any of embodiments 329-331, wherein the LCs are induced Natural Killer (iNK) cells.

333. The method of embodiment 331 or embodiment 332, wherein the iNK cells are CD56+CD3- cells.

334. The method of any of embodiments 331-333, wherein the iNK cells are further characterized by one or more of the following: DNAM1+, NKG2D+, NKP30+ and/or CD16+.

335. The method of any of embodiments 329, wherein the LCs express a recombinant receptor, optionally a chimeric antigen receptor.

336. The method of any of embodiments 296-335, that is carried out in vitro or ex vivo.

337. The method of any of embodiments 296-335, wherein the HPCs are human HPCs.

338. A population of differentiated cells produced by the method of any of embodiments 296-329

339. A population of lymphoid cells (LCs) produced by the method of embodiment 329 or embodiments 330-337.

340. A method of treating a disease or condition in a subject, the method comprising administering to the subject the population of differentiated cells of embodiment 338, the population of lymphoid progenitor cells of embodiment 328, or the population of lymphoid cells of embodiment 339.

VII. EXAMPLES

[0450] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0451] The current Examples illustrate the use of DNA-targeting systems for activation of genes to facilitate differentiation. In some aspects, the DNA-targeting systems target genes that are activated by Notch signaling, thereby bypassing the need for an extrinsic Notch signal to facilitate stem cell differentiation. Such DNA-targeting systems may provide a useful alternative to protocols that rely on the provision of extrinsic signals, such as Notch ligands. The DNA- targeting systems provide several potential advantages, including reduced cost, reduced hands- on time for cell-culture, and greater ability to scale up differentiation protocols to larger numbers of cells. Example 1 : Activation of lymphoid cell differentiation genes using CRISPR/Cas-based DNA- targeting systems

[0452] Hematopoietic Progenitor Cells (HPCs) were electroporated with CRISPR/Cas- based DNA-targeting systems for transcriptional activation of lymphoid cell differentiation (LCD) genes, and assessed for targeted gene activation.

[0453] HPCs were electroporated on Day 0 with DNA-targeting systems for transcriptional activation of LCD genes. Each DNA-targeting system included the fusion protein dSpCas9- 2xVP64 (SEQ ID NO:58) and a gRNA for targeting a target site for an LCD gene selected from TCF7, GATA3, and BCL11B (as shown in Table El). dSpCas9-2xVP64 is an exemplary nuclease-inactive Cas (dCas) fusion protein for transcriptional activation of gRNA-targeted genes, comprising dSpCas9 fused to 2 copies of the transcriptional activator effector domain VP64. The dSpCas9 is positioned in between an N-terminal VP64 sequence (SEQ ID NO:60) and a C-terminal VP64 sequence (SEQ ID NO: 62). The DNA target sites for the gRNAs are set forth in SEQ ID NOs: 1-18. The corresponding gRNA spacer sequences are set forth in SEQ ID NOs: 19-54. The full-length gRNA sequences, which include a spacer sequence and a SpCas9 gRNA scaffold sequence (SEQ ID NO: 56), are set forth in SEQ ID NOs: 37-54. In these experiments, the gRNAs included 2’ MeO base and phosphorothiate backbone modifications to enhance stability and increase potency. The sequences for the modified full-length gRNAs are set forth in SEQ ID NOs: 65-82.

Table El. gRNAs for targeting TCF7, GATA3, and BCL11B.

[0454] HPCs were generated from induced pluripotent stem cells (iPSCs) using the STEMdiff Hematopoietic kit. DNA-targeting systems were delivered by electroporation to generated HPCs for transient expression. The DNA-targeting systems were delivered as mRNA encoding the dSpCas9-2xVP64 fusion protein and pre-transcribed gRNA at a mRNA:gRNA molar ratio of approximately 1:100. Negative control cells (“Mock” in figures) were electroporated with only the dSpCas9-2xVP64 fusion protein (i.e., without a guide RNA). Positive control cells underwent a standard protocol for stem cell differentiation (“StemDiff” in figures) including culture in wells coated with Lymphoid Coating Material (LCM) and were not electroporated.

[0455] HPCs electroporated with the DNA-targeting systems were cultured in flat bottom tissue culture plates and assessed for expression of the targeted genes at various time points post-electroporation. Gene expression was assessed by RT-qPCR, with expression measured as fold change in comparison to expression levels at Day 1, and normalized to a control gene, GAPDH. As shown in FIG. 1 and Table E2, cells delivered with DNA-targeting systems for activation of target genes exhibited transcriptional upregulation of BCL1 IB, GATA3, or TCF7. Notably, the DNA-targeting systems were capable of increasing expression of the target genes to biologically relevant levels. As shown in FIG. 1, target gene expression was increased to a degree comparable to cells undergoing a standard protocol for stem cell differentiation (StemDiff), and expression was increased for up to 1 week post-electroporation. In contrast, negative control cells (Mock) did not exhibit increased expression of any of the target genes.

[0456] The cells delivered with the DNA-targeting systems were also assessed for viability, growth, and transduction efficiency. Table E2 summarizes the results at Day 2 postelectroporation for RNA expression, viability, cell growth, and transduction efficiency, for DNA-targeting systems with each gRNA. Viability was measured as a percentage of viable cells using the Cellaca MX automated cell counter with an AO/PI stain for LIVE/DEAD cells. Cell growth was measured as fold change in cell numbers after electroporation. Fold change was established by dividing number of cells at harvest time by the number of cells plated postelectroporation. In these experiments, the dSpCas9-2xVP64 fusion protein included a P2A selfcleaving peptide (SEQ ID NO:87) and a mCherry fluorescent tag (SEQ ID NO: 88) as a marker for successful transduction of the fusion protein. Transduction efficiency was measured as the percentage of cells expressing mCherry. As shown in Table E2, cells were successfully transduced with the DNA-targeting systems and remained viable following electroporation. As shown in FIG. 2, cells delivered with the DNA-targeting systems remained viable following electroporation, exhibiting viability similar to positive (StemDiff) and negative (Mock) control cells for up to one week.

Table E2. Day 2 post-electroporation results for DNA-targeting systems with indicated gRNAs

[0457] The results showed that DNA-targeting systems comprising a dCas-effector fusion protein for transcriptional activation and gene-targeting gRNAs can facilitate transcriptional activation of LCD genes to biologically relevant levels. The results support the utility of DNA- targeting systems for transcriptional activation to facilitate lymphoid cell differentiation, such as differentiation of common lymphoid progenitors (CLP) and related cell lineages.

Example 2 : Simultaneous activation of a plurality of LCD genes using a CRISPR/Cas-based multiplexed DNA-targeting system

[0458] Hematopoietic Progenitor Cells (HPCs) were electroporated with a multiplexed DNA-targeting system for simultaneous transcriptional activation of TCF7, GATA3, and BCL1 IB, and assessed for targeted gene activation.

[0459] gRNAs were selected based on results from Example 1 for inclusion in a DNA- targeting system for multiplexed activation of TCF7, GATA3, and BCL1 IB. The TCF7/GATA3/BCL11B multiplexed DNA-targeting system included dSaCas9-2xVP64, and the gRNAs TCF7_A (targeting SEQ ID NO:1), GATA3_E (targeting SEQ ID NO: 11), and BCL11B_D (targeting SEQ ID NO: 16). As shown in FIG. 3, gRNAs TCF7_A, GATA3_E and BCL11B_D all resulted in strong activation of their respective target genes in comparison to the mock treated control cells.

[0460] Hematopoietic Progenitor Cells (HPCs)were electroporated with the TCF7/GATA3/BCL1 IB multiplexed DNA-targeting system on Day 0, or on both Day 0 and Day 3, for transient expression as in Example 1. Negative control cells (“Mock” in figures) were electroporated but not delivered with a gRNA. Positive control cells underwent a standard protocol for stem cell differentiation (“StemDiff” in figures) including culture in LCM coated wells and were not electroporated.

[0461] Cells were assessed for expression of targeted genes TCF7, GATA3, and BCL1 IB on Day 0 (pre-electroporation), Day 3, Day 6, and Day 9. As shown in FIGS. 4A-4C, delivery of the multiplexed DNA-targeting system led to increased expression of all three of the targeted genes. HPCs electroporated with the multiplexed DNA-targeting system on Day 0 and Day 3 exhibited prolonged increased expression of TCF7, GATA3, and BCE1 IB at least until Day 9, indicating a synergistic effect of multiple deliveries, and showing that the degree and timing of gene activation using the DNA-targeting systems is tunable. The results show that multiplexed DNA-targeting systems comprising a dCas-effector fusion protein for transcriptional activation and gene-targeting gRNAs can facilitate simultaneous transcriptional activation of a plurality of lymphoid cell differentiation genes. The results support the utility of multiplexed DNA-targeting systems for transcriptional activation to facilitate differentiation, including for lymphoid lineages such as common lymphoid progenitors (CEPs) and downstream lineages.

Example 3 : Induction of lymphoid cell differentiation using a multiplexed DNA-targeting system

[0462] Hematopoietic Progenitor Cells (HPCs)were electroporated with a multiplexed DNA-targeting system for simultaneous transcriptional activation of TCF7, GATA3, and BCE1 IB, and assessed for markers of lymphoid differentiation.

[0463] HPCs electroporated with the TCF7/GATA3/BCE1 IB multiplexed DNA-targeting system as in Example 2 were assessed by flow cytometry for cell surface markers indicative of lymphoid differentiation.

[0464] Hematopoietic progenitor cells undergoing differentiation typically exhibit decreased expression of CD34. As shown in FIG. 5, HPCs electroporated with the multiplexed DNA- targeting system exhibited decreased CD34 expression at Day 6 in comparison to mock negative controls, as assessed by mean fluorescence intensity (MFI) of CD34 expression measured by flow cytometry. CD34 expression was decreased in cells electroporated at Day 0, and also in cells electroporated at Day 0 and Day 3. [0465] Differentiation of hematopoietic progenitor cells into lymphoid lineages such as natural killer (NK) cells can exhibit increased expression of CD56. As shown in FIGS. 6A-6C, HPCs electroporated with the multiplexed DNA-targeting system exhibited increased CD56 expression at Day 6 (FIG. 6A) and Day 9 (FIG. 6B and FIG. 6C) in comparison to mock negative controls, as assessed by percentage of cells positive for CD56 expression. CD56 expression was increased in cells electroporated at Day 0, and also in cells electroporated at Day 0 and Day 3.

[0466] The results show that multiplexed DNA-targeting systems comprising a dCas- effector fusion protein for transcriptional activation and gene-targeting gRNAs can facilitate changes in cell surface markers associated with lymphoid differentiation. The results support the utility of DNA-targeting systems, including multiplexed DNA-targeting systems, for transcriptional activation of lymphoid cell differentiation genes to facilitate stem cell differentiation into lymphoid cells, such as common lymphoid progenitors (CLPs), without the need for extrinsic signaling ligands.

Example 4 : Screen for Notch target genes that promote induced common lymphoid progenitor (iCLP) differentiation

[0467] A library of gRNAs targeting genes activated by Notch signaling was screened in a pooled format in Hematopoietic Progenitor Cells (HPCs) expressing an exemplary dCas9- transcriptional activator fusion protein to identify lymphoid cell differentiation genes. Cells were assessed for expression of cell surface markers associated with induced common lymphoid progenitor (iCLP) differentiation. FIG. 7A shows the screen workflow, which is further described below.

[0468] Briefly, a library of approximately 250 gRNAs was generated. The library was composed of gRNAs targeted to regions from 550 to 25 base pairs upstream of the transcriptional start site (TSS) of 50 human genes, with approximately 5 gRNAs per gene. On day 0, HPCs generated from iPSCs (also termed iHPCs) as described in Example 1 were thawed and transduced with lentiviral constructs encoding the gRNA library. On day 1, the cells were transfected with LNPs encapsulating mRNA encoding the dSpCas9-2xVP64 fusion protein (SEQ ID NO: 58).

[0469] On day 4, the cells were transfected a second time with LNPs encapsulating mRNA encoding the dSpCas9-2xVP64 fusion protein. Cells were analyzed by flow cytometry for expression of cell surface markers associated with CLP differentiation including: CD4+CD8+, CD5+CD7+, CD7+, CD45highCD34-, and CD45high. On day 7, cells were transferred to fresh media and analyzed by flow cytometry for cell surface markers of CLP differentiation. On day 8, cells were transfected a third time with LNPs encapsulating mRNA encoding the dSpCas9- 2xVP64 fusion protein and screened using flow cytometry to analyze cell surface markers of CLP differentiation. On day 11, cells were harvested and sorted based on increased expression of CD45 (CD45 high) compared to control cells that were not transduced with the gRNA library. The CD45high phenotype was chosen for sorting as it exhibited the clearest phenotypic change in the presence of the gRNA library. An exemplary flow cytometry expression plot and sorted populations are shown in FIG. 7B.

[0470] Genes enriched in the CD45high population were identified based on sequencing analysis. Exemplary results depicted in FIG. 7C show hits for target genes that were upregulated in the CD45high population. Table E3 summarizes gene targets whose activation increased the percentage of cells with CD45high expression in iHPCs.

[0471] Table E3: Summary of Activation Screen Gene Hits

[0472] Table E4 shows exemplary enriched gRNAs for the gene hits described above. The DNA target sites for the gRNAs are set forth in SEQ ID NOs: 93-106. The corresponding gRNA spacer sequences are set forth in SEQ ID NOs: 107-120. . In these experiments, each gRNA further comprised a scaffold sequence for SpCas9, comprising the sequence:GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 56). The gRNAs included 2’ MeO base and phosphorothiate backbone modifications to enhance stability and increase potency.

Table E4. Enriched gRNA Sequences from Activation Screen

Example 5 : Singleplex and multiplex activation of lymphoid cell differentiation genes using LNP delivery of CRISPR/Cas-based DNA-targeting systems

[0473] iHPCs generated as described in Example 1 were transfected with an exemplary dCas9-transcriptional activator fusion protein and one or more gRNAs for singleplex or multiplex targeting of lymphoid cell differentiation (LCD) gene(s) identified in Example 4 (summarized in Table E3). Transfected cells were assessed for targeted gene activation by monitoring gene expression.

[0474] Specifically, iHPCs generated as described in Example 1 were transfected with LNPs for delivery of DNA-targeting systems for transcriptional activation of the one or more LCD genes. The LNPs encapsulated a DNA-targeting system comprising mRNA encoding the dSpCas9-2xVP64 fusion protein (SEQ ID NO:58) and one or more gRNAs for targeting a target site for an LCD gene identified in Example 4 (as shown in Table E4) or a non-targeting (NT) gRNA control. The DNA target site for the NT gRNA is set forth in SEQ ID NO: 92. In these experiments, each gRNA further comprised a scaffold sequence for SpCas9, comprising the sequence: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA AAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 122. The gRNAs included 2’ MeO base and phosphorothiate backbone modifications to enhance stability and increase potency. [0475] Gene expression was assessed by RT-qPCR on Day 2 post-transfection, with expression measured as fold change in comparison to the NT control gRNA. Gene expression of MYB, RUNX1, HEY1, RUNX3, and SPI were assessed after delivery of a singleplex or multiplex DNA-targeting system (exemplified using two different combinations of gRNAs to target sites in these genes). As shown in FIG. 8A activation of MYB, RUNX1, HEY1, and RUNX3 gene expression was much higher when cells were delivered with a multiplex DNA- targeting system targeting MYB, RUNX1 and HEY1 or RUNX3, SPI and IL7Ra than when delivered with a singleplex DNA-targeting system targeting each respective gene. SPI activation was slightly higher when cells were delivered with a singleplex DNA-targeting system compared to a multiplex DNA-targeting system. As shown in FIG. 8B, cells delivered with a singleplex DNA-targeting system targeting IL7Ra, TBX21, LEF1, or CBFB all showed increased expression of the target genes compared to the NT control.

[0476] The results showed that LNP-based delivery of singleplex or multiplex DNA- targeting systems comprising a dCas-effector fusion protein for transcriptional activation and gene-targeting gRNAs can facilitate transcriptional activation of LCD genes. The results support the utility of LNP delivery of DNA-targeting systems for transcriptional activation to facilitate differentiation of HPCs into common lymphoid progenitors (CLPs) and downstream lymphoid cell lineages.

Example 6 : Assessment of LCD gene activation for iCLP differentiation using extrinsic Notch ligands and singleplex CRISPR/Cas-based DNA-targeting systems

[0477] To assess how the LCD genes identified in Example 4 influence the progression of induced common lymphoid progenitor (iCLP) differentiation, HPCs were provided with an extrinsic Notch ligand and transfected with LNPs encapsulating a DNA-targeting system comprising an exemplary dCas9-transcriptional activator fusion protein and a single gRNA targeting an LCD gene. Cells were assessed for expression of cell surface markers associated with iCLP differentiation at multiple time points after LNP transfection.

[0478] iHPCs, generated as described in Example 1, were cultured in wells coated with Lymphoid Coating Material (LCM) comprising membrane-bound DLL4. On Day 0, HPCs were transfected with LNPs encapsulating DNA-targeting systems for transcriptional activation of LCD genes. Each DNA-targeting system included an mRNA encoding the dSpCas9-2xVP64 fusion protein (SEQ ID NO:58) and a gRNA for targeting a target site for an LCD gene selected from HEY1_B (SEQ ID NO: 98), TBX21 (SEQ ID NO: 104), SPI1_A (SEQ ID NO: 101), RUNX1_B (SEQ ID NO: 96), LEF1 (SEQ ID NO: 105) and MYB_B (SEQ ID NO: 94) or a non-targeting (NT) gRNA.

[0479] To assess early stage iCLP differentiation, HPCs were delivered with DNA-targeting systems for transcriptional activation of HEY1, TBX21, SPI1 or RUNX1 on Day 0 and assessed for cell surface marker expression on Day 4 using flow cytometry. HPCs undergoing differentiation typically exhibit increased expression of CD45RA and decreased expression of CD34 (CD45RA+CD34-). As shown in FIG. 9A, only HPCs delivered with the TBX21 DNA- targeting system exhibited an increase in cells expressing CD45RA+CD34- markers compared to the NT control. Another set of markers associated with differentiation of HPCs into CLPs is increased expression of CD7 and negative expression of CD 14. As shown in FIG. 9B, HPCs delivered with the HEY1, TBX21, or RUNX1 DNA-targeting system exhibited increased CD7+ cells compared to the NT control and minimal CD 14 expression.

[0480] To assess late stage iCLP differentiation, HPCs were delivered with DNA-targeting systems for transcriptional activation of LEF1 or MYB on Day 0 and again on Day 4 and assessed for cell surface marker expression on Day 7 using flow cytometry. HPCs undergoing late-stage differentiation typically express both CD7 and CD5 (CD7+CD5+). As shown in FIG. 9C, HPCs delivered with either the LEF1 or MYB DNA-targeting system exhibited increased CD7+CD5+ cells compared to the NT control.

Example 7 : Identification of LCD gene combinations that induce iCLP differentiation using multiplex CRISPR/Cas-based DNA-targeting systems

[0481] To assess if multiplex targeted activation of a combination of LCD genes could support iCLP differentiation from HPCs without providing extrinsic Notch ligands, combinations of gRNAs targeting LCD genes identified in the screen described in Example 4 were tested.

[0482] iHPCs, generated as described in Example 1, were transfected with LNPs encapsulating a multiplex DNA-targeting system comprising an mRNA encoding the dSpCas9- 2xVP64 fusion protein (SEQ ID NO:58) and gRNAs for targeting 3 LCD genes simultaneously. 56 unique combinations of 3 gRNAs targeting a target site for an LCD gene selected from the group consisting of MYB_B (SEQ ID NO: 94), RUNX3_A (SEQ ID NO: 99), RUNX1_B (SEQ ID NO: 96), SPI1_A (SEQ ID NO: 101), HEY1_B (SEQ ID NO: 98), IL7Ra (SEQ ID NO: 103), TBX21 (SEQ ID NO: 104), and LEF1 (SEQ ID NO: 105) were tested.

[0483] Negative control cells were transfected with LNPs encapsulating the dSpCas9- 2xVP64 fusion protein and the NT guide RNA. Positive control cells underwent a standard protocol for stem cell differentiation comprising culture in wells coated with Lymphoid Coating Material (LCM) and were not transfected.

[0484] HPCs were transfected with LNPs for delivery of a multiplex DNA-targeting system on Day 0 and Day 4. Cells were assessed for expression of cell surface markers associated with iCLP differentiation on Day 7 using flow cytometry. Exemplary flow plots are shown in FIGS. 10A-10J. As shown in FIG. 10A, positive control cells cultured with LCM to induce CLP differentiation exhibit (from left to right) a majority of CD34-CD45RA+ cells (68.8%), few to no cells expressing IRL3Ra or CD 14, a majority of CD7+ cells (60.6%) with some CD7+CD5+ cells (24.9%), and a majority of ckit mid+ cells (77.2%) with some ckit mid+CD56+ cells (10.3%). As shown in FIG. 10B, the negative control cells exhibit only 36.6% CD34-CD45RA+ cells, few to no cells expressing IRL3Ra or CD14, a majority of CD7-CD5- cells (59.8%), and almost all (97.5%) cells were CD56-ckit high.

[0485] As shown in FIGS. 10C - 10F, some guide RNA combinations did not result in changes in marker expression indicative of CLP differentiation. For example, multiplex targeting of RUNX3, SPI1, and IL7Ra (FIG. 10C), RUNX1, HEY1, and TBX21 (FIG. 10 D), HEY1, IL7Ra, and TBX21 (FIG. 10E) and MYB, IL7Ra, and LEF1 (FIG. 10F) resulted in more CD7-CD5- cells than the negative control cells.

[0486] Notably, targeting of RUNX3 and IL7a in combination with MYB (FIG. 10G), RUNX1 (FIG. 10H), TBX21 (FIG. 101), or LEF1 (FIG. 10J) each resulted in increased in CD7+ and ckit mid+ cells. Thus, certain combinations of LCD genes can promote cell surface maker expression indicative of CLP differentiation without providing an extrinsic Notch ligand.

[0487] The results show that multiplexed DNA-targeting systems comprising a dCas- effector fusion protein for transcriptional activation and gRNAs targeting lymphoid cell differentiation genes promote the expression of cell surface markers associated with common lymphoid progenitors (CLPs). The results further support the utility of DNA-targeting systems, including multiplexed DNA-targeting systems, for transcriptional activation of lymphoid cell differentiation genes to facilitate stem cell differentiation into lymphoid cells, such as common lymphoid progenitors (CLPs), without the need for extrinsic signaling ligands. Example 8 : Functional assessment of iCLP cells generated using multiplex CRISPR/Cas- based DNA-targeting systems

[0488] To assess if transcriptional activation of LCD genes using CRISPR/Cas based DNA- targeting systems produced functional induced common lymphoid progenitors (iCLPs), HPCs were transfected with LNPs encapsulating a DNA-targeting system comprising an exemplary dCas9-transcriptional activator fusion protein and various combinations of two or more gRNAs targeting different LCD genes. Cells were subsequently grown in Natural Killer (NK) cell induction media, assessed for NK cell surface markers, and cell functionality was assessed in a tumor cell killing assay.

[0489] iHPCs containing an endogenous HER2 CAR transgenewere transfected with LNPs encapsulating a multiplex DNA-targeting system comprising an mRNA encoding the dSpCas9- 2xVP64 fusion protein (SEQ ID NO:58) and two or more LCD gene targeting gRNAs at Day 0. Cells were transfected again at Day 4 with the dCas9 effector and a second set of LCD targeting gRNAs. 3 groups of target genes were tested for transfection at Day 0 in combination with 6 groups of target genes for transfection at Day 4 for a total of 18 target gene combinations. The combinations of target genes tested are summarized in Table E5 below.

Table E5. LCD target gene combinations for iCLP differentiation

[0490] HPCs were grown for eight days after the initial LNP transfection in SFEMII media (Stem Cell Technologies, Catalog No. 09605) with Lymphoid Progenitor Expansion Supplement (Stem Cell Technologies, Catalog No. 09915) At Day 8, cells were subjected to a NK differentiation protocol comprising cell culture in wells coated with DLL4 ligand and RetroNectin.

[0491] At Day 14, cells were assayed using flow cytometry for cell surface markers associated with an induced Natural Killer (iNK) phenotype including CD45+EGFRt+, CD56+, CD16+, DNAM-1, and NKG2D+. Cells were sequentially gated as follows: live cells, CD45+EGFRt+, CD56+, and CD16+, DNAM-1+ or NKG2D+. FIGS. 11A-11C shows the percentage of cells from the parent gate as assessed by flow cytometry . All the tested gRNA combinations resulted in a similar iNK phenotype.

[0492] Next, a cytotoxicity co-culture assay was performed to assess the capacity of the induced Natural Killer (iNK) cells to kill HER2-positive tumor cells through the endogenously integrated HER2 CAR transgene . iNK cells were cocultured with the HER2 antigen-expressing NSCLC cell line H1975 target cells at an approximately 1:1, 1:2, or 1:5 effector to target cell ratio for up to 112 hours. The Hl 975 tumor cell line was cultured alone (target alone) as a negative control. iNKs derived from LCM cultured cells were co-cultured with the Hl 975 targets cells as a positive control (POS CTRL) Target cell killing was assessed via Incucyte live cell imaging analysis. [0493] Exemplary data for the 3-1 through 3-6 LCD gene combinations are shown in FIGS. 12A-12B. As shown in FIG. 12A, all the combinations except for the 3-5 combination (Day 0: RUNX3, IL7Ra + Day 4: LEF1, TBX21) exhibited robust tumor killing capacity similar to the positive control cells. FIG. 12B shows that the 3-1 (Day 0: RUNX3, IL7Ra + Day 4: RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra), 3-2 (Day 0: RUNX3, IL7Ra + Day 4: RUNX3, CBFB, LEF1, MYB, TBX21), and 3-4 (Day 0: RUNX3, IL7Ra + Day 4: LEF1, MYB, TBX21) LCD gene combinations most closely mirrored the tumor killing capacity of the positive control cells.

[0494] The results show that multiplexed DNA-targeting systems comprising a dCas- effector fusion protein and gRNAs targeting lymphoid cell differentiation genes can produce induced common lymphoid progenitors (iCLPs) that can be further differentiated into functionally mature lymphoid cells, such as iNK cells. The results further support the utility of DNA-targeting systems for transcriptional activation of lymphoid cell differentiation genes to facilitate stem cell differentiation into lymphoid cells, such as common lymphoid progenitors (CLPs), without the need for extrinsic signaling ligands.

[0495] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

SEQUENCES

Claims

2. The DNA-targeting system of claim 1, wherein the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B.

4. The DNA-targeting system of any of claims 1-3, wherein the one or more LCD genes is selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

5. The DNA-targeting system of any of claims 1-4, wherein the one or more DNA targeting modules is a plurality of DNA-targeting modules, and wherein each DNA-targeting module targets one of the one or more LCD genes.

6. The DNA-targeting system of claim 5, wherein the plurality of DNA-targeting modules is two, three, four, five, or six DNA-targeting modules, and wherein each DNA-targeting module targets one of the one or more LCD genes.

7. The DNA-targeting system of claim 5 or claim 6, wherein the plurality of DNA-targeting modules is two DNA-targeting modules, each targeting one of the one or more LCD genes.

8. The DNA-targeting system of any of claims 1-7, wherein the one or more LCD genes is two genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the two genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

9. The DNA-targeting system of claim 5 or claim 6, wherein the plurality of DNA-targeting modules is three DNA-targeting modules, each targeting one of the one or more LCD genes.

10. The DNA-targeting system of any of claims 1-6 and 9, wherein the one or more LCD genes is three genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the three genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

11. The DNA-targeting system of claim 5 or claim 6, wherein the plurality of DNA-targeting modules is four DNA-targeting modules, each targeting one or more LCD genes.

12. The DNA-targeting system of any of claims 1-6 and 11, wherein the one or more LCD genes is four genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the four genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

13. The DNA-targeting system of claim 5 or claim 6, wherein the plurality of DNA-targeting modules is five DNA-targeting modules, each targeting one of the one or more LCD genes.

14. The DNA-targeting system of any of claims 1-6 and 13, wherein the one or more LCD genes is five genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the five genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

15. The DNA-targeting system of claim 5 or claim 6, wherein the plurality of DNA-targeting modules is six DNA-targeting modules, each targeting one of the one or more LCD genes.

16. The DNA-targeting system of any of claims 1-6 and 15, wherein the one or more LCD genes is six genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the six genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

17. The DNA-targeting system of any of claims 1-16, wherein at least one of the one or more LCD genes is RUNX3.

18. The DNA-targeting system of any of claims 1-16, wherein at least one of the one or more LCD genes is IL7Ra.

19. The DNA-targeting system of any of claims 1-18, wherein the one or more LCD genes include RUNX3 and IL7Ra.

20. The DNA-targeting system of any of claims 1-16, wherein at least one of the one or more LCD genes is TBX21.

21. The DNA-targeting system of any of claims 1-16, wherein at least one of the one or more LCD genes is CBFB.

22. The DNA-targeting system of any of claims 1-16, wherein at least one of the one or more LCD genes is LEF1.

23. The DNA-targeting system of any of claims 1-16, wherein at least one of the one or more LCD genes is MYB.

24. The DNA-targeting system of any of claims 1-16, wherein at least one of the one or more LCD genes is RUNX1.

25. The DNA-targeting system of any of claims 1-16, wherein at least one of the one or more LCD genes is SPI1.

26. The DNA-targeting system of any of claims 1-16, wherein at least one of the one or more LCD genes is HEY 1.

27. The DNA-targeting system of any of claims 1-23, wherein the one or more LCD genes are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; RUNX3, IL7Ra, and TBX21; RUNX3, CBFB, EEF1, MYB, TBX21, and IE7Ra; RUNX3, CBFB, EEF1, MYB, and TBX21; RUNX3, CBFB, EEF1, and MYB; EEF1, MYB, and TBX21; EEF1 and TBX21; or EEF1 and MYB.

28. The DNA targeting system of any of claims 1-23 and 27, wherein the one or more DNA- targeting modules comprise:

29. The DNA targeting system of claim 28, wherein the one or more ECD genes targeted by the first set of DNA-targeting modules are: RUNX3 and IE7Ra; RUNX3, IE7Ra, and CBFB; or RUNX3, IE7Ra, and TBX21.

30. The DNA targeting system of claim 28 or claim 29, wherein the one or more ECD genes targeted by the second set of DNA-targeting modules are: RUNX3, CBFB, EEF1, MYB, TBX21, and IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB.

3E The DNA-targeting system of any of claims 1-3, wherein the one or more LCD genes are selected from the group consisting of TCF7, GATA3 and BCL1 IB.

32. The DNA-targeting system of any of claims 1-3 and 31, wherein the one or more DNA- targeting modules comprise two DNA-targeting modules that target a combination of two LCD genes selected from the group consisting of TCF7, GATA3 and BCL1 IB.

33. The DNA-targeting system of any of claims 1-3 and 31, wherein the one or more DNA- targeting modules comprise three DNA-targeting modules that target TCF7, GATA3 and BCL11B.

34. The DNA-targeting system of any of claims 1-3 and 31-33, wherein at least one of the one or more LCD genes is TCF7.

35. The DNA-targeting system of any of claims 1-3, and 31-34, wherein at least one of the one or more LCD genes is GATA3.

36. The DNA-targeting system of any of claims 1-3, and 31-35, wherein at least one of the one or more LCD genes is BCLB 11.

37. The DNA-targeting system of any of claims 1-3, 31, and 33-36, wherein the one or more LCD genes include TCF7, GATA3, and BCLB 11.

38. The DNA-targeting system of any of claims 1-37, wherein the target site for each of the one or more LCD genes is in the gene or a regulatory DNA element thereof.

39. The DNA-targeting system of claim 38, wherein the regulatory DNA element is an enhancer or a promoter of the gene.

40. The DNA-targeting system of claim 39, wherein the regulatory DNA element is a promoter of the gene.

41. The DNA-targeting system of any of claims 1-40, wherein the target site for each of the one or more LCD genes is within 1000 base pairs of the transcription start site (TSS) of the gene.

42. The DNA-targeting system of any of claims 1-41, wherein the target for each of the one or more LCD genes is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS of the gene.

43. The DNA-targeting system of any of claims 1-42, wherein the target site for each of the one or more LCD genes is within 550 base pairs of the TSS of the gene.

44. The DNA-targeting system of any of claims 1-17, 19, 27-30 and 38-43, wherein the target site for RUNX3 has the sequence forth in SEQ ID NO: 99 or SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

45. The DNA-targeting system of any of claims 1-16, 18-19, 27-30 and 38-43, wherein the target site for IL7Ra has the sequence set forth in SEQ ID NO: 103, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

46. The DNA-targeting system of any of claims 1-16, 20, 27-30 and 38-43, wherein the target site for TBX21 has the sequence set forth in SEQ ID NO: 104, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

47. The DNA-targeting system of any of claims 1-16, 21, 27-30 and 38-43, wherein the target site for CBFB has the sequence set forth in SEQ ID NO: 106, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

48. The DNA-targeting system of any of claims 1-16, 22, 27-28, 30 and 38-43, wherein the target site for LEF1 has the sequence set forth in SEQ ID NO: 105, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

49. The DNA-targeting system of any of claims 1-16, 23, 27-28, 30 and 38-43, wherein the target site for MYB has the sequence set forth in SEQ ID NO: 93 or SEQ ID NO: 94, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

50. The DNA-targeting system of any of claims 1-16, 24, and 38-43, wherein the target site for RUNX1 has the sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 96, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

51. The DNA-targeting system of any of claims 1-16, 25, and 38-43, wherein the target site for SPI1 has the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

52. The DNA-targeting system of any of claims 1-16, 26, and 38-43, wherein the target site for HEY1 has the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 98, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

53. The DNA-targeting system of any of claims 1-3 and 31-43 , wherein:

(a) the target site for TCF7 has the sequence set forth in any one of SEQ ID NOS: 1-6, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;

(b) the target site for GATA3 has the sequence set forth in any one of SEQ ID NOS:7-12, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; and (c) the target site for BCL11B has the sequence set forth in any one of SEQ ID NOS:13-18, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

54. The DNA-targeting system of any of claims 1-3, 31-34, 37-43, and 53, wherein the target site for TCF7 has the sequence set forth in SEQ ID NO: 1, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

55. The DNA-targeting system of any of claims 1-3, 31-33, 35, 37-43, and 53, wherein the target site for GATA3 has the sequence set forth in SEQ ID NO: 11, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

56. The DNA-targeting system of any of claims 1-3, 31-33, 36-43, and 53, wherein the target site for BCLB11 has the sequence set forth in SEQ ID NO: 16, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

57. The DNA-targeting system of any of claims 1-3, 31, 33-43, and 53-56, wherein:

58. The DNA-targeting system of any of claims 1-57, wherein the DNA-targeting system does not introduce a genetic disruption or a DNA break.

59. The DNA-targeting system of any of claims 1-58, wherein the fusion protein of each DNA-targeting module comprises a DNA-binding domain selected from: a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or a variant thereof; a zinc finger protein (ZFP); a transcription activator- like effector (TALE); a meganuclease; a homing endonuclease; or an I-Scel enzyme or a variant thereof, optionally wherein the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing, wherein, when the DNA-binding domain of the fusion protein comprises a Cas protein, the DNA-targeting module further comprises one or more guide nucleic acids, for targeting the Cas protein to the target site for one of the one or more LCD genes.

60. The DNA-targeting system of any one of claims 1-59, wherein the DNA-binding domain is a zinc finger protein.

61. The DNA-targeting system of any one of claims 5-60, wherein the fusion protein of each of the plurality of DNA-targeting modules is different.

62. The DNA-targeting system of claim 59, wherein the DNA-targeting system comprises one fusion protein that is shared by each of the plurality of DNA-targeting modules and wherein each DNA-targeting module is characterized by comprising a different guide nucleic acid for targeting the DNA-binding domain to the target site.

63. The DNA-targeting system of any of claims 1-59 and 61-62, wherein the DNA-binding domain of the fusion protein is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof.

64. The DNA-targeting system of any of claims 1-59 and 61-63, wherein the DNA-binding domain of each of the one or more DNA-targeting modules is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof, and each of the one or more DNA-targeting modules further comprises one or more guide nucleic acids for targeting the DNA-binding domain to the target site of one of the one or more LCD gene.

65. The DNA-targeting system of any of claims 59 and 61-64, wherein the one or more guide nucleic acids are one or more guide RNAs (gRNAs).

66. The DNA-targeting system of any of claims 59 and 61-65, wherein the Cas protein or variant thereof is a deactivated (dCas) protein.

(b) one or more gRNAs that target a target site for one or more lymphoid cell differentiation (LCD) genes, wherein the DNA-targeting system increases transcription of the one or more LCD genes.

68. The DNA-targeting system of claim 67, wherein the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL11B.

70. The DNA-targeting system of any of claims 67-69, wherein the one or more LCD genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1 and HEY1.

71. The DNA-targeting system of any of claims 67-70, comprising two, three, four, five, or six different guide RNAs, wherein each gRNA targets a target site of one of the one or more LCD genes.

72. The DNA-targeting system of any of claims 67-71, wherein the one or more LCD genes are two genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the two genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

73. The DNA-targeting system of any of claims 67-71, wherein the one or more LCD genes are three genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the three genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

74. The DNA-targeting system of any of claims 67-71, wherein the one or more LCD genes are four genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the four genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

75. The DNA-targeting system of any of claims 67-71, wherein the one or more LCD genes are five genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the five genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

76. The DNA-targeting system of any of claims 67-71, wherein the one or more LCD genes are six genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the six genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

77. The DNA-targeting system of any of claims 67-76, wherein at least one of the one or more LCD genes is RUNX3.

78. The DNA-targeting system of any of claims 67-76, wherein at least one of the one or more LCD genes is IL7Ra.

79. The DNA-targeting system of any of claims 67-78, wherein the one or more LCD genes include RUNX3 and IL7Ra.

80. The DNA-targeting system of any of claims 67-76, wherein at least one of the one or more LCD genes is TBX21.

81. The DNA-targeting system of any of claims 67-76, wherein at least one of the one or more LCD genes is CBFB.

82. The DNA-targeting system of any of claims 67-76, wherein at least one of the one or more LCD genes is LEF1.

83. The DNA-targeting system of any of claims 67-76, wherein at least one of the one or more LCD genes is MYB.

84. The DNA-targeting system of any of claims 67-76, wherein at least one of the one or more LCD genes is RUNX1.

85. The DNA-targeting system of any of claims 67-76, wherein at least one of the one or more LCD genes is SPI1.

86. The DNA-targeting system of any of claims 67-76, wherein at least one of the one or more LCD genes is HEY 1.

87. The DNA-targeting system of any of claims 67-83, wherein the one or more LCD genes are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; RUNX3, IL7Ra, and TBX21; RUNX3, CBFB, LEF1, MYB, TBX21, and IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB.

88. The DNA targeting system of any of claims 67-87, wherein the one or more DNA- targeting modules comprise:

(b) a second DNA-targeting module comprising a second fusion protein and a second set of gRNAs, wherein the first and second fusion proteins comprise a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and wherein the first and second set of gRNAs target a target site for one or more lymphoid cell differentiation (LCD) genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1.

89. The DNA-targeting system of claim 88, wherein the first set of gRNAs and the second set of gRNAs are the same.

90. The DNA-targeting system of claim 88 and claim 89, wherein the first set of gRNAs and the second set of gRNAs each target a combination of two or more LCD genes that are the same.

91. The DNA-targeting system of claim 88, wherein the first set of gRNAs and the second set of gRNAs are different.

92. The DNA-targeting system of claim 88 and claim 91, wherein the first set of gRNAs and the second set of gRNAs each target a combination of two or more LCD genes that are different.

(2) a second DNA-targeting module comprising (a) a second fusion protein comprising a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and (b) a second set of gRNAs comprising two or more gRNAs that target a target site for two or more lymphoid cell differentiation (LCD) genes, wherein the two or more LCD genes targeted by the first and second set of gRNAs are independently selected from the group consisting of RUNX3, IL7Ra, TBX21, CBEB, LEE1, MYB, RUNX1, SPI1, and HEY1.

94. The DNA targeting system of claim 88 or claim 93, wherein: (a) the first set of gRNAs targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, and CBFB; and

(b) the second set of gRNAs targets one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

95. The DNA targeting system of any of claims 88 and 93-94, wherein the one or more LCD genes targeted by the first set of gRNAs are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; or RUNX3, IL7Ra, and TBX21.

96. The DNA targeting system of any of claims 88 and 93-95, wherein the one or more LCD genes targeted by the second set of gRNAs are: RUNX3, CBFB, LEF1, MYB, TBX21, IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB.

97. The DNA targeting system of any of claims 88 and 93-96, wherein: the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1 and TBX21; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target RUNX3, CBFB, LEF1 and MYB; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1 and MYB; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1, MYB and TBX21; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target RUNX3, CBFB, LEF1, MYB and TBX21; or the first set of gRNAs target RUNX3 and IL7RA and the second set for gRNAs target RUNX3, CBFB, LEF1, MYB, TBX21 and IL7Ra.

98. The DNA targeting system of any of claims 88-97, wherein the dCas and the transcriptional activation domain of the first and second fusion protein are the same.

99. The DNA targeting system of any of claims 88-99, wherein the first DNA-targeting module is present in a first lipid nanoparticle and the second DNA-targeting module is present in a second lipid nanoparticle.

100. The DNA-targeting system of any of claims 67-69, wherein the one or more LCD genes are selected from the group consisting of TCF7, GATA3, and BCL1 IB.

101. The DNA-targeting system of any of claims 66-100, wherein the dCas protein lacks nuclease activity.

102. The DNA-targeting system of any of claims 66-101, wherein the dCas protein is a dCas9 protein.

103. The DNA-targeting system of claim 102, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

104. The DNA-targeting system of claim 103, wherein the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 83.

105. The DNA-targeting system of claim 103 or claim 104, wherein the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 84 or SEQ ID NO: 191, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

106. The DNA-targeting system of any of claims 103-105, wherein the dSaCas9 protein is set forth in SEQ ID NO: 84 or SEQ ID NO: 191.

107. The DNA-targeting system of claim 102, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

108. The DNA-targeting system of claim 107, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 63.

109. The DNA-targeting system of claim 107 or claim 108, wherein the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 64 or SEQ ID NO: 196, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

110. The DNA-targeting system of any of claims 107-109, wherein the dSpCas9 protein is set forth in SEQ ID NO: 64 or SEQ ID NO: 196.

111. The DNA-targeting system of any of claims 65-110, wherein each of the one or more gRNAs comprise a gRNA spacer that is complementary to the target site of the gene.

112. The DNA-targeting system of any of claims 68-77, 79, 87-99, and 101- 111, wherein the gRNA targeting a target site for RUNX3 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114, or a contiguous portion thereof of at least 14 nt.

113. The DNA-targeting system of claim 112, wherein the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114.

114. The DNA-targeting system of any of claims 68-76, 78-79, 87-99, and 101- 111, wherein the gRNA targeting a target site for IL7Ra comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 117, or a contiguous portion thereof of at least 14 nt.

115. The DNA-targeting system of claim 114, wherein the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO: 117.

116. The DNA-targeting system of any of claims 68-76, 80, 87-99, and 101- 111, wherein the gRNA targeting a target site for TBX21 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 118, or a contiguous portion thereof of at least 14 nt.

117. The DNA-targeting system of claim 116, wherein the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO:118.

118. The DNA-targeting system of any of claims 68-76, 81, 87-99, and 101- 111, wherein the gRNA targeting a target site for CBFB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 120, or a contiguous portion thereof of at least 14 nt.

119. The DNA-targeting system of claim 118, wherein the gRNA targeting a target site for CBFB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO:

120.

120. The DNA-targeting system of any of claims 68-76, 82, 87-94, 96-99, and 101- 111, wherein the gRNA targeting a target site for LEF1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119, or a contiguous portion thereof of at least 14 nt.

121. The DNA-targeting system of claim 120, wherein the gRNA targeting a target site for LEF1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119.

122. The DNA-targeting system of any of claims 68-76, 83, 87-94, 96-99, and 101- 111, wherein the gRNA targeting a target site for MYB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108, or a contiguous portion thereof of at least 14 nt.

123. The DNA-targeting system of claim 122, wherein the gRNA targeting a target site for MYB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108.

124. The DNA-targeting system of any of claims 68-76, 84, 87-93, 98-99, and 101- 111, wherein the gRNA targeting a target site for RUNX1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110, or a contiguous portion thereof of at least 14 nt.

125. The DNA-targeting system of claim 124, wherein the gRNA targeting a target site for RUNX1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110.

126. The DNA-targeting system of any of claims 68-76, 85, 87-93, 98-99, and 101- 111, wherein the gRNA targeting a target site for SPI1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116, or a contiguous portion thereof of at least 14 nt.

127. The DNA-targeting system of claim 126, wherein the gRNA targeting a target site for SPI1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116.

128. The DNA-targeting system of any of claims 68-76, 86-93, 98-99, and 101- 111, wherein the gRNA targeting a target site for HEY 1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112, or a contiguous portion thereof of at least 14 nt.

129. The DNA-targeting system of claim 128, wherein the gRNA targeting a target site for HEY1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112.

130. The DNA-targeting system of any of claims 68-69 and 100-111 , wherein:

131. The DNA-targeting system of any of claims 68-69, 100-111, and 130, wherein:

(c) the gRNA targeting a target site for BCLB 11 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 34.

132. The DNA-targeting system of any of claims 111-131, wherein the spacer sequence is between 14 nt and 24 nt, or between 16 nt and 22 nt in length.

133. The DNA-targeting system of any of claims 111-132, wherein the spacer sequence is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

134. The DNA-targeting system of any of claims 65-102 and 107-133, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 56.

135. The DNA-targeting system of any of claims 65-106 and 111 - 133-, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 91.

136. The DNA-targeting system of any of claims 65-102 and 107-133, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 122.

137. The DNA-targeting system of any of claims 65-136, wherein the gRNA further comprises 2’ MeO modified bases and/or phosphorothiate backbone modifications.

138. The DNA-targeting system of any of 1-137, wherein the at least one transcriptional activator effector domain is selected from the group consisting of: a VP64 domain, a p65 activation domain, a p300 domain, an Rta domain, a CBP domain, a VPR domain, a VPH domain, an HSF1 domain, a TET protein domain, optionally wherein the TET protein is TET1, a SunTag domain, and a domain, portion, variant, or truncation of any of the foregoing.

139. The DNA-targeting system of any of claims 1-138, wherein the at least one transcriptional activator effector domain comprises at least one VP 16 domain, and/or a VP 16 tetramer (“VP64”) or a variant thereof.

140. The DNA-targeting system of any of claims 1-139, wherein the at least one transcriptional activator effector domain comprises a VP64 domain or a variant or portion thereof that exhibits transcriptional activation activity.

141. The DNA-targeting system of any of claims 1-140, wherein the at least one transcriptional activator effector domain is VP64.

142. The DNA-targeting system of claim 141, wherein the VP64 is positioned N-terminal and/or C-terminal to the DNA-binding domain.

143. The DNA-targeting system of any of claims 1-142, wherein the at least one transcriptional activator effector domain comprises two copies of VP64.

144. The DNA-targeting system of any of claims 1-143, wherein the at least one transcriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 60, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

145. The DNA-targeting system of any of claims 1-143, wherein the at least one transcriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 62, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

146. The DNA-targeting system of any of claims 1-145, wherein the fusion protein comprises the sequence set forth in SEQ ID NO: 58, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

147. The DNA-targeting system of any of claims 1-146, wherein introduction of the DNA- targeting system to a hematopoietic progenitor cell (HPC) promotes lymphoid differentiation.

148. The DNA-targeting system of claim 147, wherein the lymphoid differentiation is to a lymphoid progenitor cell (LPC) phenotype.

149. The DNA-targeting system of claim 148, wherein the LPC phenotype is an induced common lymphoid progenitor (iCLP) phenotype.

150. The DNA-targeting system of any of claims 147-149, wherein the lymphoid differentiation is characterized by decreased expression of CD34 relative to a HPC that was not introduced with the DNA-targeting system.

151. The DNA-targeting system of any of claims 147-150, wherein the lymphoid differentiation is characterized by differentiation to a CD34- cell.

152. The DNA-targeting system of any of claims 147-151, wherein the lymphoid differentiation is characterized by increased expression of CD45 relative to a HPC that was not introduced with the DNA-targeting system.

153. The DNA-targeting system of any of claims 147-152, wherein the lymphoid differentiation is characterized by differentiation to a CD45+ cell.

154. The DNA-targeting system of any of claims 147-153, wherein the lymphoid differentiation is characterized by increased expression of CD7 relative to a HPC that was not introduced with the DNA-targeting system.

155. The DNA-targeting system of any of claims 147-154, wherein the lymphoid differentiation is characterized by differentiation to a CD7+ cell.

156. The DNA-targeting system of any of claims 147-155, wherein the lymphoid differentiation is characterized by increased expression of both CD5 and CD7 relative to a HPC that was not introduced with the DNA-targeting system.

157. The DNA-targeting system of any of claims 147-155, wherein the lymphoid differentiation is characterized by differentiation to a CD5+CD7+ cell.

158. The DNA-targeting system of any of claims 147-157, wherein the lymphoid differentiation is characterized by increased expression of CD56 relative to a HPC that was not introduced with the DNA-targeting system.

159. The DNA-targeting system of any of claims 147-158, wherein the lymphoid differentiation is characterized by differentiation to a CD56+ cell.

160. The DNA-targeting system of any of claims 147-159, wherein the lymphoid differentiation is characterized by decreased expression of c-KIT relative to a HPC that was not introduced with the DNA-targeting system.

161. The DNA-targeting system of any of claims 147-160, wherein the lymphoid differentiation is characterized by differentiation to a c-KIT- cell.

162. A gRNA that targets a target site for RUNX3, wherein the target site for RUNX3 comprises the sequence set forth in SEQ ID NO:99 or SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

163. The gRNA of claim 162, wherein the target site for RUNX3 comprises the sequence set forth in any one of SEQ ID NO:99 or SEQ ID NO: 100.

164. The gRNA of claim 162 or claim 163 wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114, or a contiguous portion thereof of at least 14 nt.

165. The gRNA of any of claims 162-164, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114.

167. The gRNA of claim 166, wherein the target site for IL7Ra comprises the sequence set forth in SEQ ID NO: 103.

168. The gRNA of claim 166 or claim 167, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 117, or a contiguous portion thereof of at least 14 nt.

169. The gRNA of any of claims 166-168, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 117.

171. The gRNA of claim 170, wherein the target site for TBX21 comprises the sequence set forth in SEQ ID NO: 104.

172. The gRNA of claim 170 and 171, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 118, or a contiguous portion thereof of at least 14 nt.

173. The gRNA of any of claims 170-172, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 118.

174. A gRNA targeting a target site for CBFB wherein the target site for CBFB comprises the sequence set forth in SEQ ID NO: 106, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

175. The gRNA of claim 174, wherein the target site for CBFB comprises the sequence set forth in SEQ ID NO: 106.

176. The gRNA of claim 174 or claim 175, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 120, or a contiguous portion thereof of at least 14 nt.

177. The gRNA of any of claimsl74-176, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 120.

179. The gRNA of claim 178, wherein the target site for LEF1 comprises the sequence set forth in SEQ ID NO: 105.

180. The gRNA of claim 178 or claim 179, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119, or a contiguous portion thereof of at least 14 nt.

181. The gRNA of any of claims 178-180, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119.

183. The gRNA of claim 182, wherein the target site for MYB comprises the sequence set forth in SEQ ID NO: 93 or SEQ ID NO: 94.

184. The gRNA of claim 182 or claim 183, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108, or a contiguous portion thereof of at least 14 nt.

185. The gRNA of any of claims 182-184, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108.

186. A gRNA targeting a target site for RUNX1 wherein the target site for RUNX1 comprises the sequence set forth in SEQ ID NO:95 or SEQ ID NO: 96, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

187. The gRNA of claim 186, wherein the target site for RUNX1 comprises the sequence set forth in SEQ ID NO:95 or SEQ ID NO: 96.

188. The gRNA of claim 186 or claim 187, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110, or a contiguous portion thereof of at least 14 nt.

189. The gRNA of any of claims 186-188, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110.

191. The gRNA of claim 190, wherein the target site for SPI1 comprises the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102.

192. The gRNA of claim 190 or claim 191, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116, or a contiguous portion thereof of at least 14 nt.

193. The gRNA of any of claims 190-192, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116.

195. The gRNA of claim 194, wherein the target site for HEY1 comprises the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 98.

196. The gRNA of claim 194 or claim 195, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112, or a contiguous portion thereof of at least 14 nt.

197. The gRNA of any of claims 194-196, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112.

198. A gRNA that targets a target site for TCF7, wherein the target site has the sequence set forth in any one of SEQ ID NOS: 1-6, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

199. The gRNA of claim 198, wherein the target site for TCF7 comprises the sequence set forth in SEQ ID NO: 1, or a complementary sequence thereof.

200. The gRNA of claim 198 or claim 199, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 19-24, or a contiguous portion thereof of at least 14 nt.

201. The gRNA of any of claims 198-200, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 19-24.

202. The gRNA of any of claims 198-201, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19.

204. The gRNA of claim 203, wherein the target site for GATA3 comprises the sequence set forth in SEQ ID NO: 11, or a complementary sequence thereof.

205. The gRNA of claim 203 or claim 204, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 25-30, or a contiguous portion thereof of at least 14 nt.

206. The gRNA of any of claims 203-205, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 25-30.

207. The gRNA of any of claims 203-206, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 29

209. The gRNA of claim 208, wherein the target site for BCL1 IB comprises the sequence set forth in SEQ ID NO: 16, or a complementary sequence thereof.

210. The gRNA of claim 208 or claim 209, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 31-36, or a contiguous portion thereof of at least 14 nt.

211. The gRNA of any of claims 208-210, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 31-36.

212. The gRNA of any of claims 208-211, wherein the gRNA targeting a target site for BCLB 11 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 34.

213. The gRNA of any of claims 162-212, wherein the spacer sequence is between 14 nt and 24 nt, or between 16 nt and 22 nt in length.

214. The gRNA of any of claims 162-213, wherein the spacer sequence is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

215. The gRNA of any of claims 162-214, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 56.

216. The gRNA of any of claims 162-214, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 91.

217. The gRNA of any of claims 162-214, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 122

218. The gRNA of any of claims 162-217, wherein the gRNA further comprises 2’ MeO modified bases and/or phosphorothiate backbone modifications.

219. A combination comprising two or more gRNAs of any of claims 162-218.

220. The combination of claim 219, comprising two, three, four, five, or six different guide RNAs, wherein each gRNA targets a target site of a different LCD genes.

222. The combination of gRNAs of claim 221, wherein the target site for each of the one or more LCD genes is in the gene or a regulatory DNA element thereof.

223. The combination of gRNAs of claim 222, wherein the regulatory DNA element is an enhancer or a promoter.

224. The combination of gRNAs of claim 222 or claim 223, wherein the regulatory DNA element is a promoter of the gene.

225. The combination of gRNAs of any of claims 221-224, wherein the target site for each of the one or more LCD genes is independently within 1000 base pairs of the transcription start site (TSS).

226. The combination of gRNAs of any of claims 221-225, wherein the target site for each of the one or more LCD genes is independently within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS of the gene.

227. The combination of gRNAs of any of claims 221-225, wherein the target site for each of the one or more LCD genes is within 550 base pairs of the TSS of the gene.

228. The combination of gRNAs of any of claims 221-227 comprising two, three, four, five, or six different guide RNAs, wherein each gRNA targets a target site of one of the one or more LCD genes.

229. The combination of gRNAs of any of claims 221-228, wherein the one or more LCD genes are two genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the two genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

230. The combination of gRNAs of any of claims 221-228, wherein the one or more LCD genes are three genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the three genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

231. The combination of gRNAs of any of claims 221-228, wherein the one or more LCD genes are four genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the four genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

232. The combination of gRNAs of any of claims 221-228, wherein the one or more LCD genes are five genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the five genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

233. The combination of gRNAs of any of claims 221-228, wherein the one or more LCD genes are six genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, and HEY1; optionally where the six genes are selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

234. The combination of any of claims 221-233, wherein at least one of the one or more LCD genes is RUNX3.

235. The combination of any of claims 221-233, wherein at least one of the one or more LCD genes is IL7Ra.

236. The combination of any of claims 221-235, wherein the one or more LCD genes include RUNX3 and IL7Ra.

237. The combination of any of claims 221-233, wherein at least one of the one or more LCD genes is TBX21.

238. The combination of any of claims 221-233, wherein at least one of the one or more LCD genes is CBFB.

239. The combination of any of claims 221-233, wherein at least one of the one or more LCD genes is LEF1.

240. The combination of any of claims 221-233, wherein at least one of the one or more LCD genes is MYB.

241. The combination of any of claims 221-233, wherein at least one of the one or more LCD genes is RUNX1.

242. The combination of any of claims 221-233, wherein at least one of the one or more LCD genes is SPI1.

243. The combination of any of claims 221-233, wherein at least one of the one or more LCD genes is HEY 1.

244. The combination of any of claims 221-243, wherein the one or more LCD genes are:

RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; RUNX3, IL7Ra, and TBX21; RUNX3, CBFB, LEF1, MYB, TBX21, and IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB,

LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB.

245. The combination of gRNAs of any of claims 221-240 and 244, wherein the two or more gRNAs comprise:

(b) a second set of gRNAs, wherein the second set gRNAs target one or more LCD genes selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, and MYB.

246. The combination of gRNAs of claim 245, wherein the one or more LCD genes targeted by the first set of gRNAs are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; or RUNX3, IL7Ra, and TBX21.

247. The combination of gRNAs of claim 245 or claim 246, wherein the one or more LCD genes targeted by the second set of gRNAs are: RUNX3, CBFB, LEF1, MYB, TBX21, and IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB.

248. The combination of gRNAs of any of claims 245-247, wherein: the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1 and TBX21; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target RUNX3, CBFB, LEF1 and MYB; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1 and MYB; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target LEF1, MYB and TBX21; the first set of gRNAs target RUNX3 and IL7RA and the second set of gRNAs target RUNX3, CBFB, LEF1, MYB and TBX21; or the first set of gRNAs target RUNX3 and IL7RA and the second set for gRNAs target RUNX3, CBFB, LEF1, MYB, TBX21 and IL7Ra.

249. The combination of gRNAs of any of claims 221-234, 236, and 244-248, wherein the target site for RUNX3 has the sequence set forth in SEQ ID NO: 99 or SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

250. The combination of gRNAs of any of claims 221-234, 236, and 244-249, wherein the gRNA targeting a target site for RUNX3 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 113 or SEQ ID NO: 114, or a contiguous portion thereof of at least 14 nt.

251. The combination of gRNAs of any of claims 221-233, 235-236, and 244-248, wherein the target site for IL7Ra has the sequence set forth in SEQ ID NO: 103, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

252. The combination of gRNAs of any of claims 221-233, 235-236, 244-248, and 251, wherein the gRNA targeting a target site for IL7Ra comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 117, or a contiguous portion thereof of at least 14 nt.

253. The combination of gRNAs of any of claims 221-233, 237, and 244-248, wherein the target site for TBX21 has the sequence set forth in SEQ ID NO: 104, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

254. The combination of gRNAs of any of claims 221-233, 237, 244-248, and 253, wherein the gRNA targeting a target site for TBX21 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 118, or a contiguous portion thereof of at least 14 nt.

255. The combination of gRNAs of any of claims 221-233, 238, and 244-248, wherein the target site for CBFB has the sequence set forth in SEQ ID NO: 106, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

256. The combination of gRNAs of any of claims 221-233, 238, 244-248, and 255, wherein the gRNA targeting a target site for CBFB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 120, or a contiguous portion thereof of at least 14 nt.

257. The combination of gRNAs of any of claims 221-233, 239, 244-245, and 247-248, wherein the target site for LEF1 has the sequence set forth in SEQ ID NO: 105, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

258. The combination of gRNAs of any of claims 221-233, 239, 244-245, 247-248 and 257, wherein the gRNA targeting a target site for LEF1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 119, or a contiguous portion thereof of at least 14 nt.

259. The combination of gRNAs of any of claims 221-233, 240, 244-245, and 247-248, wherein the target site for MYB has the sequence set forth in SEQ ID NO: 93 or SEQ ID NO: 94, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

260. The combination of gRNAs of any of claims 221-233, 240, 244-245, 247-248, and 259, wherein the gRNA targeting a target site for MYB comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108, or a contiguous portion thereof of at least 14 nt.

261. The combination of gRNAs of any of claims 221-233 and 241, wherein the target site for RUNX1 has the sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 96, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

262. The combination of gRNAs of any of claims 221-233, 241, and 261, wherein the gRNA targeting a target site for RUNX1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 109 or SEQ ID NO: 110, or a contiguous portion thereof of at least 14 nt.

263. The combination of gRNAs of any of claims 221-233 and 242, wherein the target site for SPI1 has the sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing.

264. The combination of gRNAs of any of claims 221-233, 242, and 263, wherein the gRNA targeting a target site for SPI1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116, or a contiguous portion thereof of at least 14 nt.

265. The combination of gRNAs of any of claims 221-233, and 243, wherein the target site for HEY1 has the sequence set forth in SEQ ID NO: 97 or SEQ ID NO: 98, a contiguous portion thereof of at least 14 nucleotides (nt) , or a complementary sequence of any of the foregoing.

266. The combination of gRNAs of any of claims 221-233, 243, and 265, wherein the gRNA targeting a target site for HEY 1 comprises a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112, or a contiguous portion thereof of at least 14 nt.

267. The combination of gRNAs of any of claims 221-266, wherein the two or more guide RNAs independently comprise a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length.

268. The combination of gRNAs of any of claims 221-267, wherein the two or more guide RNAs independently comprise a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

269. The combination of gRNAs of any of claims 221-268, wherein the two or more guide RNAs each further comprise a scaffold sequence set forth in SEQ ID NO: 56.

270. The combination of gRNAs of any of claims 221-268, wherein the two or more guide RNAs each further comprise a scaffold sequence set forth in SEQ ID NO: 91.

271. The combination of gRNAs of any of claims 221-268, wherein the two or more guide RNAs each further comprise a scaffold sequence set forth in SEQ ID NO: 122

272. The combination of gRNAs of any of claims 221-271, wherein the two or more gRNAs independently further comprise 2’ MeO modified bases and/or phosphorothiate backbone modifications.

273. A Cas-guide RNA (gRNA) combination comprising:

(b) the combination of gRNAs of any of claims 219, 220, and 221-272.

274. A Cas-guide RNA (gRNA) combination comprising:

(b) one or more guide RNAs, each selected from the gRNA of any of claims 162-218

275. The Cas-gRNA combination of claim 273 or claim 274, wherein the Cas protein or variant thereof is a deactivated (dCas) protein.

276. The Cas-gRNA combination of claim 275, wherein the dCas protein lacks nuclease activity.

277. The Cas-gRNA combination of claim 275 or claim 276, wherein the dCas protein is a dCas9 protein.

278. The Cas-gRNA combination of claim 277, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

279. The Cas-gRNA combination of claim 278, wherein the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 83.

280. The Cas-gRNA combination of claim 278 or claim 279, wherein the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 84 or SEQ ID NO: 191, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

281. The Cas-gRNA combination of any of claims 278-280, wherein the dSaCas9 protein is set forth in SEQ ID NO: 84 or SEQ ID NO: 191.

282. The Cas-gRNA combination of claim 277, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

283. The Cas-gRNA combination of claim 282, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 63.

284. The Cas-gRNA combination any of claim 282 or claim 283, wherein the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 64 or SEQ ID NO: 196, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

285. The Cas-gRNA combination of any of claims 282-284, wherein the dSpCas9 protein is set forth in SEQ ID NO: 64 or SEQ ID NO: 196.

286. A polynucleotide encoding the DNA-targeting system of any of claims 1-161.

287. A polynucleotide encoding at least one DNA-targeting module of the DNA-targeting system of any of claims 1-161.

288. A polynucleotide encoding the gRNA of any of claims 162-218.

289. A polynucleotide encoding the combination of gRNAs of any of claims 219-272.

290. A polynucleotide encoding the Cas-gRNA combination of any of claims 273-285.

291. A vector comprising the polynucleotide of any of claims 286-290.

292. The vector of claim 291 that is a viral vector.

293. The vector of claim 291 that is a lipid nanoparticle.

294. A pharmaceutical composition comprising the DNA-targeting system of any of claims 1-161, the Cas-gRNA combination of any of claims 273-285, the polynucleotide of any of claims 286-290, or the vector of claim 292 or claim 293.

295. The pharmaceutical composition of claim 294 comprising a pharmaceutically acceptable excipient.

296. A method of differentiating a population of hematopoietic progenitor cells (HPCs) to a differentiated population of cells, the method comprising introducing the DNA-targeting system of any one of claims 1-161, the Cas-gRNA combination of any of claims 273-285, the polynucleotide of any of claims 286-290, the vector of claim 292 or claim 293, or a combination thereof, into a population of HPCs, and culturing the HPCs under conditions for their differentiation.

297. A method of differentiating a population of hematopoietic progenitor cells (HPCs) to a differentiated population of cells, the method comprising introducing the pharmaceutical composition of claim 294 or claim 295 into a population of HPCs, and culturing the HPCs under conditions for their differentiation.

298. The method of any of claim 296 or claim 297, wherein the population of HPCs are induced hematopoietic progenitor cells (iHPCs).

299. The method of any of claim 296 or claim 297, wherein the population of stem cells are primary hematopoietic progenitor cells.

300. The method of any of claims 296-298, wherein the HPCs comprise cells engineered with a recombinant receptor, optionally a chimeric antigen receptor.

301. The method of claim 300, wherein the differentiated cells comprise cells that express a recombinant receptor, optionally a chimeric antigen receptor.

302. The method of any of claims 296-301, wherein cells of the differentiated population of cells are lymphoid progenitor cells.

303. The method of claim 302, wherein the lymphoid progenitor cells are induced common lymphoid progenitor cells (iCLPs).

304. The method of any of claims 296-303, wherein the introducing decreases expression of CD34 in the differentiated population of cells relative to the population of HPCs.

305. The method of any of claims 296-304, wherein cells of the differentiated population of cells are CD34- cells.

306. The method of any of claims 296-305, wherein the introducing increases expression of CD45 in the differentiated population of cells relative to the population of HPCs.

307. The method of any of claims 296-306, wherein cells of the differentiated population of cells are CD45+ cells.

308. The method of any of claims 296-307, wherein the introducing increases expression of CD7 in the differentiated population of cells relative to the population of HPCs.

309. The method of any of claims 296-308, wherein cells of the differentiated population of cells are CD7+ cells.

310. The method of any of claims 296-309, wherein the introducing increases expression of both CD5 and CD7 in the differentiated population of cells relative to the population of HPCs.

311. The method of any of claims 296-310, wherein cells of the differentiated population of cells are CD5+CD7+ cells.

312. The method of any of claims 296-311, wherein the introducing increases expression of CD56 in the differentiated population of cells relative to the population of HPCs.

313. The method any of claims 296-312, wherein cells of the differentiated population of cell are CD56+ cells.

314. The method of any of claims 296-313, wherein the introducing decreases expression of c-KIT in the differentiated population of cells relative in the population of HPCs.

315. The method any of claims 296-314, wherein cells of the differentiated population of cells are c-KIT" cells.

316. The method of any of claims 296-315, wherein the introducing is by transient delivery into the population of stem HPCs.

317. The method of claim 316, wherein the transient delivery comprises electroporation, transfection, or transduction.

318. The method of claim 317, wherein the transient delivery comprises transfection using lipid nanoparticles (LNPs).

319. The method of claim 317 or claim 318, wherein the transient delivery is repeated at least once.

320. The method of any of claims 296-319, wherein the DNA-targeting system of any one of claims 1-161, the Cas-gRNA combination of any of claims 273-285, the polynucleotide of any of claims286-290, the vector of claim 292 or claim 293, or a combination thereof, is transiently expressed and/or transiently present in the population of HPCs.

321. The method of any of any of claims 296-320, wherein the introducing increases transcription of one or more lymphoid cell differentiation (LCD) genes, selected from the group consisting of RUNX3, IL7Ra, TBX21, CBFB, LEF1, MYB, RUNX1, SPI1, HEY1, TCF7, GATA3, and BCL1 IB, in the population of HPCs.

322. The method of any of claims 296-321, wherein the introduced DNA-targeting system comprises at least two DNA-targeting modules and wherein each of the at least two modules are introduced at different times.

323. The method of claim 322, wherein the at least two DNA-targeting modules is a first set and second set of DNA-targeting modules that are introduced at different times.

324. The method of claim 323, wherein:

325. The method of claim 324, wherein the one or more LCD genes targeted by the first set of DNA-targeting modules are: RUNX3 and IL7Ra; RUNX3, IL7Ra, and CBFB; or RUNX3, IL7Ra, and TBX21.

326. The method of claim 324 or claim 326, wherein the one or more LCD genes targeted by the second set of DNA-targeting modules are: RUNX3, CBFB, LEF1, MYB, TBX21, and IL7Ra; RUNX3, CBFB, LEF1, MYB, and TBX21; RUNX3, CBFB, LEF1, and MYB; LEF1, MYB, and TBX21; LEF1 and TBX21; or LEF1 and MYB.

327. The method of any of claims 302-326, wherein the lymphoid progenitor cells express a recombinant receptor, optionally a chimeric antigen receptor.

328. A population of lymphoid progenitor cells produced by the method of any of claims 302-327.

329. The method of any of claim 296-301 and 304-328, wherein cells of the differentiated population of cells are lymphoid cells (LCs).

330. A method of generating lymphoid cells (LCs), the method comprising culturing the population of lymphoid progenitor cells produced by the method of any of claims 302-327 or the population of lymphoid progenitor cells of claim 328 under conditions to differentiate cells of the population to lymphoid cells (LCs) to produce a population comprising LCs.

331. The method of claim 329 or claims 330, wherein the LCs are induced T (iT) cells, induced B (iB) cells or induced natural killer (iNK) cells.

332. The method of any of claims 329-331, wherein the LCs are induced Natural Killer (iNK) cells.

333. The method of claim 331 or claim 332, wherein the iNK cells are CD56+CD3- cells.

334. The method of any of claims 331-333, wherein the iNK cells are further characterized by one or more of the following: DNAM1+, NKG2D+, NKP30+ and/or CD16+.

335. The method of any of claims 329, wherein the LCs express a recombinant receptor, optionally a chimeric antigen receptor.

336. The method of any of claims 296-335, that is carried out in vitro or ex vivo.

337. The method of any of claims 296-335, wherein the HPCs are human HPCs.

338. A population of differentiated cells produced by the method of any of claims 296-329

339. A population of lymphoid cells (LCs) produced by the method of claim 329 or claims 330-337.

340. A method of treating a disease or condition in a subject, the method comprising administering to the subject the population of differentiated cells of claim 338, the population of lymphoid progenitor cells of claim 328, or the population of lymphoid cells of claim 339.