CN114874983A

CN114874983A - Method for identifying T cell regulatory genes

Info

Publication number: CN114874983A
Application number: CN202111644258.1A
Authority: CN
Inventors: 袁鹏飞; 金鸣; 张永建; 杨晓梅; 杨玲; 苏美华; 宗会明
Original assignee: Edigene Inc
Current assignee: Beijing Jiyin Medical Technology Co ltd
Priority date: 2020-12-29
Filing date: 2021-12-29
Publication date: 2022-08-09
Also published as: WO2022143783A1; TW202242139A

Abstract

The present application provides methods of identifying genes that modulate the sensitivity or resistance of T cells (e.g., allogeneic T cells or CAR-T cells (e.g., allogeneic CAR-T cells)) to NK cell killing. Also provided are modified T cells (e.g., allogeneic T cells or CAR-T cells (e.g., allogeneic CAR-T cells)) that are resistant to NK cell killing, as well as methods and kits for producing the same.

Description

Method for identifying T cell regulatory genes

Cross Reference to Related Applications

This application claims priority to international patent application PCT/CN2020/140860, filed on 29/12/2020, 2020 and the contents of which are incorporated herein by reference in their entirety.

Technical Field

The present application relates to methods of identifying genes that modulate the sensitivity or resistance of T cells (e.g., allogeneic T cells or T cells expressing chimeric antigen receptors (CAR-T cells) like allogeneic CAR-T cells) to NK cell killing. Also provided are modified T cells (e.g., allogeneic T cells or CAR-T cells (e.g., allogeneic CAR-T cells)) that are resistant to NK cell killing, as well as methods and kits for producing the same.

Background

Immunotherapeutic approaches, including adoptive T cell therapy (e.g., CAR-T), play an increasingly important role in the treatment of cancer, viral infections, and other pathophysiological autoimmune diseases. Allogeneic T cell therapy has become a more attractive approach than autologous T cell therapy, which typically requires a lengthy and expensive custom-made manufacturing process and is not suitable for all patients, where T cells are from healthy donors and can provide off-the-shelf products suitable for many patients, not just one person. One of the major challenges of allogeneic approaches is host rejection, where the patient's immune system (e.g., host T cells, NK cells) will recognize injected non-HLA-matched T cells as foreign and reject them. To overcome this problem, researchers have used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) (CRISPR/Cas9) systems to knock-out the β -2 microglobulin (B2M) required for Human Leukocyte Antigen (HLA) class I expression in CAR-T cells to prevent host TCR α β cells from recognizing donor CAR-T cells as foreign by HLA class I (Ren et al, Clin.cancer Res.2017; 23: 2255-. However, cells with reduced HLAI-like expression are also the target of NK cells, which represent a barrier to prevent allogeneic T cell rejection (Liu et al. curr. Res. Transl. Med.2018; 66: 39-42).

The activity of NK cells is regulated by a complex interaction of various cell surface inhibitory and activating receptors. Inhibitory receptors include the killer immunoglobulin-like receptor (KIR) and CD94/NKG2A, recognize Major Histocompatibility Complex (MHC) or HLAI-like molecules, allow NK cells to recognize autologous cells and prevent them from attacking host tissues. In the absence of matching MHC class I molecules, the inhibitory effect of NK cytotoxicity is released, and this balance is shifted to NK cell activation by activating receptor engagement. During viral infection or malignant transformation, transformed cells reduce the expression of MHC class I antigens on the cell surface to avoid recognition by T cells. NK cells can recognize such transformed cells as "altered self," with abnormal levels of MHC class I expression leading to reduced involvement of KIRs and increased stimulatory receptor expression to provide effector responses and cytotoxic killing of the transformed cells (Nayyar et al.

The CRISPR/Cas9 system is capable of editing at a target genomic locus in an efficient and specific manner. One of its broad applications is the identification of the function of coding genes, non-coding RNAs and regulatory elements by high throughput convergent screening in combination with next generation sequencing ("NGS") analysis. By introducing a pooled single-stranded guide RNA ("sgRNA") or paired guide RNA ("pgRNA") library into cells expressing Cas9 or catalytically inactive Cas9(dCas9) fused to an effector domain, researchers can conduct multiple gene screens by making different mutations, large genome deletions, transcriptional activation, or transcriptional repression.

To generate a high quality gRNA cell library for any given pooled CRISPR screen, a low multiplicity of infection ("MOI") must be used in the cell library construction process to ensure that each cell contains on average less than one sgRNA or pgRNA to minimize False Discovery Rate (FDR) for the screen. To further reduce FDR and improve data reproducibility, deep coverage and multiple biological replications of grnas are often required to obtain highly statistically significant hits, resulting in increased workload. Other difficulties may arise when large whole genome screens are performed, when the cellular material used to construct the library is limited, or when more challenging screens (i.e., in vivo screens) are performed that make it difficult to obtain experimental replicates or control MOI. The "internal barcode (" iBAR ") approach previously developed by the applicant (see WO2020125762, the contents of which are incorporated herein by reference in their entirety) provides a reliable and efficient screening strategy for large scale target identification in eukaryotic cells, where the false positive and false negative rates are much lower and allows the generation of cell libraries using high MOI. For example, the iBAR method can reduce the starting cell number by more than 20 fold (e.g., MOI of 3) to more than 70 fold (e.g., MOI of 10) compared to traditional CRISPR/Cas screens with a low MOI of 0.3, while maintaining high efficiency and accuracy. The iBAR system is particularly useful for cell-based screens with limited cell numbers, or for in vivo screens where viral infection of specific cells or tissues is difficult to control at low MOI.

The disclosures of all publications, patents, patent applications, and published patent applications cited herein are hereby incorporated by reference in their entirety.

Summary of The Invention

In one aspect, the invention provides a method of identifying a target gene in a T cell (e.g., an allogeneic T cell or CAR-T cell (e.g., an allogeneic CAR-T cell)) that modulates T cell activity, comprising: a) providing a T cell library comprising a plurality of T cells, wherein each of the plurality of T cells has a mutation (e.g., an inactivating mutation) at a hit gene in the genome ("hit gene mutation"), wherein the hit genes at least two of the plurality of T cells are different from each other; b) treating the T cell library with NK cells; c) obtaining T cells from a T cell library that are sensitive or resistant to NK cell killing; and d) identifying the hit gene in the T cell obtained in step c), thereby identifying a target gene in the T cell that modulates T cell activity. In some embodiments, the T cell library is generated by genome-wide gene editing of an initial population of T cells. In some embodiments, the T cells in the initial T cell population express a CAR.

In some embodiments according to any of the above methods, the library of T cells is generated by contacting a population of naive T cells (e.g., allogeneic T cells or CAR-T cells (e.g., allogeneic CAR-T cells)) with: i) a library of single-stranded guide RNAs ("sgrnas") comprising a plurality of sgRNA constructs, wherein each sgRNA construct comprises or encodes a sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary (e.g., any of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site of a hit gene in a genome; and optionally, ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein under conditions that allow introduction of the sgRNA construct and optionally the Cas component into the initial population of T cells. In some embodiments, the Cas protein is Cas 9. In some embodiments, each sgRNA comprises a guide sequence fused to a second sequence, wherein the second sequence comprises a repeat-trans-repeat stem loop that interacts with Cas 9. In some embodiments, the second sequence of each sgRNA further comprises stem loop 1, stem loop 2, and/or stem loop 3. In some embodiments, each sgRNA further comprises an Internal Barcode (iBAR) sequence ("sgRNA) ^iBAR "), wherein each sgRNA ^iBAR Can be operated with a Cas protein (e.g., Cas9) to modify the hit (e.g., cleave the hit, or modulate the hit expression). In some embodiments, the Cas protein is Cas9, and each sgRNA ^iBAR The iBAR sequence of (a) is inserted into the loop region of the repeat-trans-repeat stem loop. In some embodiments, each sgRNA ^iBAR The iBAR sequence of (a) is inserted into the loop region of the repeat-trans-repeat stem loop. In some embodiments, each sgRNA ^iBAR Comprising in the 5 'to 3' direction a first stem sequence and a second stem sequence, wherein the first stem sequence hybridizes to the second stem sequence to form a double-stranded RNA (dsRNA) region that interacts with a Cas protein, and the iBAR sequence is located atBetween the 3 'end of the first stem sequence and the 5' end of the second stem sequence. In some embodiments, each guide sequence comprises from about 17 to about 23 nucleotides. In some embodiments, each iBAR sequence comprises from about 1 to about 50 nucleotides (e.g., about 6 nucleotides). In some embodiments, comprising a plurality of sgrnas ^iBAR sgRNA library of constructs ("sgRNA) ^iBAR Library ") comprises multiple sets of sgrnas ^iBAR Construct, wherein each group of sgRNAs ^iBAR The constructs comprise three or more (e.g., 3, 4, 5, 6, or more) sgrnas ^iBAR Constructs, each comprising or encoding a sgRNA ^iBAR Three or more (e.g., 3, 4, 5, 6, or more) sgrnas therein ^iBAR The guide sequences of the constructs are identical, with three or more (e.g., 3, 4, 5, 6, or more) sgrnas ^iBAR The iBAR sequence of each of the constructs is different from each other, and wherein each group of sgrnas ^iBAR The guide sequence of the construct is complementary to a different target site in the genome (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary). In some embodiments, each set of sgrnas ^iBAR The construct comprises 4 sgrnas ^iBAR Constructs, and 4 sgrnas ^iBAR The iBAR sequences of each of the constructs differ from each other. In some embodiments, the sgRNA ^iBAR The library comprises at least about 100 groups (e.g., at least about any one of 1,000, 10,000, 50,000, or more) of sgrnas ^iBAR Constructs. In some embodiments, different sets of sgrnas ^iBAR At least two sgRNAs in a construct ^iBAR The iBAR sequences of the constructs are identical (e.g., first and second sets of sgrnas ^iBAR Two sets of sgrnas of the construct ^iBAR With at least 1, 2, 3, 4, or more iBAR sequences shared between constructs). In some embodiments, at least two sets of sgrnas ^iBAR The iBAR sequences of the constructs were identical. In some embodiments, a sgRNA library comprising a plurality of sgRNA constructs comprises or encodes a gene having complementarity to a target site of each annotated gene in a genome (e.g., at least about 50%, 60%, 70%, 80%, 90%, 95%, 96% >),Any one of 97%, 98%, 99%, or 100% complement). In some embodiments, a plurality of sgrnas are included ^iBAR sgRNA of constructs ^iBAR The library comprises or encodes sgrnas having guide sequences that are complementary (e.g., any of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to the target site of each annotated gene in the genome ^iBAR . In some embodiments, a sgRNA library (or sgrnas) ^iBAR Library) of at least about 95% (e.g., any of at least about 96%, 97%, 98%, 99%, or 100%) of sgrnas ^iBAR Construct) into the initial T cell population. In some embodiments, the T cell library is for each sgRNA ^iBAR Having an average coverage of at least about 100-fold (e.g., at least about any of 200-, 500-, 1,000-, 5,000-, or more-fold). In some embodiments, the library of T cells has an average coverage of at least about 400-fold (e.g., at least any one of about 600-, 800-, 1,000-, 2,000-, 8,000-, 12,000-fold, or more) per sgRNA. In some embodiments, the sgRNA library (or sgRNA) ^iBAR Library) comprises at least about 400 (e.g., at least about any of 600, 1000, 5000, 10,000, 50,000, 100,000, 300,000, 600,000, or more) sgRNA constructs (or sgrnas) ^iBAR Construct). In some embodiments, the sgRNA library (or sgrnas) ^iBAR Library) comprises at least about 150,000 (e.g., at least any one of about 300,000, 600,000, or more) sgrnas construct(s) (or sgrnas) ^iBAR Construct). In some embodiments, the initial T cell population expresses a Cas (e.g., Cas9) protein. In some embodiments, the method further comprises contacting the initial population of T cells or the T cell library with: i) a sgRNA construct comprising or encoding a sgRNA that comprises a guide sequence that is complementary (e.g., at least any one of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site in a B2M gene ("B2M sgRNA"); and optionally, ii) allowing the B2M sgRNA construct and optionallyUnder conditions in which the Cas component of (a) is introduced into the initial population of T cells or T cell library, a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein. In some embodiments, the T cells in the initial T cell population comprise a B2M mutation (e.g., an inactivated B2M mutation). In some embodiments, each sgRNA construct (or sgRNA) in the sgRNA library ^iBAR Each sgRNA in the library ^iBAR Construct) and/or the B2M sgRNA construct is RNA. In some embodiments, each sgRNA construct (or sgRNA) in the sgRNA library ^iBAR Each sgRNA in the library ^iBAR Construct) and/or the B2M sgRNA construct is a plasmid. In some embodiments, each sgRNA construct (or sgRNA) in the sgRNA library ^iBAR Each sgRNA in the library ^iBAR Construct) and/or the B2M sgRNA construct is a viral vector, such as a lentiviral vector. In some embodiments, each sgRNA construct (or sgRNA) in the sgRNA library ^iBAR Each sgRNA in the library ^iBAR Construct) and/or the B2M sgRNA construct is a virus, such as a lentivirus. In some embodiments, the sgRNA library (or sgrnas) ^iBAR Library) and/or B2M sgRNA construct is contacted with the initial population of T cells at a multiplicity of infection (MOI) of at least about 2, such as 3.

In some embodiments according to any of the above methods, the treating with NK cells comprises: i) an initial processing step comprising contacting a library of T cells with NK cells; ii) an optional first enrichment step comprising sorting the mixture of treated cells to obtain a first subpopulation of T cells that are sensitive or resistant to NK cell killing; iii) an optional first recovery step comprising culturing a first subpopulation of T cells; and iv) optionally a second treatment step comprising contacting the first subpopulation of T cells with NK cells. In some embodiments, the initial processing step comprises contacting the T cell library with NK cells for at least about 48 hours, such as any of about 48 hours, 72 hours, 5 days, or 10 days. In some embodiments, the method comprises a first enrichment step. In some embodiments, the first enrichment step comprises sorting a mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a first subpopulation of T cells that are resistant to NK cell killing ("first viable enrichment"). In some embodiments, the first enrichment step comprises sorting a mixture of B2M negative (or defective) and dead treated cells, thereby obtaining a first subpopulation of T cells that are sensitive to NK cell killing ("first death enrichment"). In some embodiments, the method further comprises staining the mixture of treated cells with an anti-B2M antibody prior to sorting. In some embodiments, the method further comprises staining the mixture of treated cells with Propidium Iodide (PI) prior to sorting, wherein PI staining indicates cell death. In some embodiments, the method comprises a first recovery step. In some embodiments, the first recovery step comprises culturing the first subpopulation of T cells for at least about 24 hours, such as about 48 hours. In some embodiments, the method comprises a second treatment step. In some embodiments, the second treatment step comprises contacting the first T cell subpopulation with NK cells for at least about 48 hours, e.g., 96 hours. In some embodiments, the ratio of NK cells to T cells in the T cell library in the initial processing step is about 0.1:1 to about 20:1 (e.g., about 0.3:1 to about 1:1, or about 0.5:1 to about 20:1), such as about 0.5:1 or about 1: 1. In some embodiments, the ratio of NK cells to T cells in the first T cell subpopulation in the second treatment step is from about 0.1:1 to about 20:1 (e.g., from about 0.3:1 to about 1:1, or from about 1:1 to about 10:1), such as about 0.3: 1.

In some embodiments according to any of the above methods, obtaining T cells from the library of T cells that are sensitive or resistant to NK cell killing comprises: i) a sorting step comprising sorting the cells obtained from step b) to obtain a second subpopulation of T cells sensitive or resistant to NK cell killing; and ii) optionally a second recovery step comprising culturing a second subpopulation of T cells prior to harvesting the cells. In some embodiments, the sorting step comprises sorting B2M negative (or defective) and viable cells obtained from step B), thereby obtaining a second T cell subpopulation that is resistant to NK cell killing ("harvest live sorting"). In some embodiments, the sorting step comprises sorting B2M negative (or defective) and dead cells obtained from step B), thereby obtaining a second subpopulation of T cells that are sensitive to NK cell killing ("harvest-dead sorting"). In some embodiments, the method further comprises staining the cells obtained from step B) with an anti-B2M antibody prior to sorting. In some embodiments, the method further comprises staining the cells obtained from step b) with PI prior to sorting, wherein PI staining indicates cell death. In some embodiments, the method includes a second recovery step. In some embodiments, the second recovery step comprises culturing the second subpopulation of T cells for at least about 24 hours, such as about 48 hours.

In some embodiments of any of the methods above, steps b) and c) comprise: i) an initial treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 0.5:1 for about 72 hours; ii) an enrichment step comprising sorting a mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a first T cell subpopulation that is resistant to NK cell killing; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; iv) a second treatment step comprising contacting the restored first T cell subpopulation with NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3: 1; and v) a sorting step comprising sorting the final mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a second T cell subpopulation that is resistant to NK cell killing.

In some embodiments of any of the methods above, steps b) and c) comprise: i) a treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 0.5:1 for about 10 days; and ii) a sorting step comprising sorting a mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing.

In some embodiments of any of the methods above, steps b) and c) comprise: i) a treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 1:1 for about 48 hours; ii) a sorting step comprising sorting a mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing; and iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours prior to harvesting the cells.

In some embodiments of any of the methods above, steps b) and c) comprise: i) a treatment step comprising contacting a T cell library with NK cells for about 48 hours at a ratio of NK cells to T cells of about 1: 1; ii) an enrichment step comprising sorting a mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a first T cell subpopulation that is resistant to NK cell killing; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and iv) a sorting step comprising sorting the recovered first subpopulation of T cells that are B2M negative (or defective) and survive, thereby obtaining a second subpopulation of T cells that are resistant to NK cell killing.

In some embodiments according to any of the methods above, identifying hits in the T cells obtained from step c) comprises: i) identifying a sequence comprising a hit gene mutation (e.g., an inactivating mutation) in the T cell obtained from step c); and ii) identifying a hit corresponding to a sequence comprising a hit mutation (e.g., an inactivating mutation). In some embodiments, identifying hits in the T cells obtained from step c) comprises: i) identifying sgRNA sequences in the T cells obtained from step c); and ii) identifying a hit gene corresponding to the guide sequence of the sgRNA. In some embodiments, hit gene mutations (e.g., inactivating mutations) or sgRNA sequences are identified by DNA sequencing or RNA sequencing. In some embodiments, hit gene mutations (e.g., inactivating mutations) or sgRNA sequences are identified by Next Generation Sequencing (NGS). In some embodiments, identifying the target gene comprises: i) obtaining a sequence comprising a hit gene mutation (e.g., an inactivating mutation) in the final T cell subpopulation obtained from step c); ii) ordering sequences comprising hit gene mutations (e.g., inactivating mutations) based on sequence counts; and iii) identifying a hit gene corresponding to a sequence comprising a hit gene mutation (e.g., an inactivating mutation) that is ranked above a predetermined threshold level. In some embodiments, identifying the target gene comprises: i) obtaining sgRNA sequences in the final T cell sub-population obtained from step c); ii) based on the order Column counting the corresponding guide sequences of the sgRNA sequences; and iii) identifying the hit genes corresponding to the leader sequences ranked above a predetermined threshold level. In some embodiments, the sgRNA is sgRNA ^iBAR And identifying the target gene comprises: i) obtaining sgRNAs in the final T cell subpopulation obtained from step c) ^iBAR A sequence; ii) sequence count based on sgRNA ^iBAR Ordering respective ones of the sequences, wherein the ordering comprises based on sgRNAs corresponding to the guide sequences ^iBAR Data consistency between iBAR sequences in the sequence adjusts the ordering of each pilot sequence; and iii) identifying the hit genes corresponding to the leader sequences ranked above a predetermined threshold level. In some embodiments, the method is a positive screen. In some embodiments, the method is a negative screen. In some embodiments, the sequence counts are normalized for median ratio and then modeled for mean-square difference. In some embodiments, the sgRNA library is a sgRNA ^iBAR Library, and based on sgRNAs corresponding to the leader sequences ^iBAR Data consistency between iBAR sequences in the sequence, the variance of each guide sequence is adjusted. In some embodiments, the sequence counts obtained from the final T cell subpopulation obtained in step c) are compared to corresponding sequence counts obtained from a control T cell subpopulation to provide a fold change (e.g., an actual fold change, or a derivative of a fold change, such as a log2 or log10 fold change). In some embodiments, the control T cell subpopulation is obtained from the same T cell library cultured under the same conditions and not subjected to NK cell treatment, and optionally subjected to the same acquisition method in step c). In some embodiments, the sgrnas corresponding to each guide sequence are determined based on the direction of fold change of each iBAR sequence ^iBAR Data consistency between iBAR sequences in a sequence, wherein the variance of the pilot sequence increases if the fold changes of the iBAR sequences are in different directions relative to each other (e.g., increasing versus decreasing, increasing versus invariant, or decreasing versus invariant). In some embodiments, the method further comprises culturing the same T cell library under the same conditions without undergoing NK cell processingAnd optionally subjected to the same acquisition method in step c) to obtain a subpopulation of control T cells, wherein the presence of a hit corresponding to a sequence from the subpopulation of control T cells comprising a hit mutation (e.g., inactivating mutation) or a guide sequence of the sgRNA is identified, while the absence in the T cells obtained from step c) from the T cell library treated with NK cells identifies the hit as a target gene.

In some embodiments according to any of the above methods, the method comprises subjecting the library of T cells from step a) to at least two of 4 separate assays prior to step d): (I) test I: i) an initial processing step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 0.5:1 for about 72 hours; ii) an enrichment step comprising sorting a mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a first T cell subpopulation that is resistant to NK cell killing; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; iv) a second treatment step comprising contacting the restored first T cell subpopulation with NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3: 1; and v) a sorting step comprising sorting the final mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a second T cell subpopulation that is resistant to NK cell killing; (II) run II: i) a treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 0.5:1 for about 10 days; and ii) a sorting step comprising sorting a mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing; (III) run III: i) a treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 1:1 for about 48 hours; ii) a sorting step comprising sorting a mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing; and iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours prior to harvesting the cells; and (IV) test IV: i) a treatment step comprising contacting a T cell library with NK cells for about 48 hours at a ratio of NK cells to T cells of about 1: 1; ii) an enrichment step comprising sorting a mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a first T cell subpopulation that is resistant to NK cell killing; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and iv) a sorting step comprising sorting the recovered first subpopulation of T cells that are B2M negative (or defective) and survive, thereby obtaining a second subpopulation of T cells that are resistant to NK cell killing. In some embodiments, identifying the target gene comprises identifying hits from at least two of 4 separate trials, wherein: i) hits identified as depleted (and/or having at least about 2-fold depletion, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more depletion) from the final T cell subpopulation (viable) in at least one assay of FDR ≦ 0.01, or in at least two assays of FDR ≦ 0.05, are identified as target genes whose mutation (e.g., inactivation) renders the T cells susceptible to NK cell killing; and/or ii) hits identified as enriched from the final T cell subpopulation (and/or having at least about a 2-fold enrichment, such as any of at least about a 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more enrichment) in at least one assay at FDR ≦ 0.05, or in at least two assays at FDR ≦ 0.15, are identified as target genes whose mutation (e.g., inactivation) renders T cells resistant to NK cell killing.

In some embodiments according to any of the above methods, the method comprises performing at least two separate different treatments with NK cells in step b) on the T cell library from step a), and obtaining T cells sensitive or resistant to NK cell killing from each of the treatments of step c). In some embodiments, identifying the target gene comprises identifying a hit gene in a T cell obtained from at least two separate distinct treatments with NK cells, wherein: i) hits identified as having been depleted (and/or having at least about 2-fold depletion, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more depletion) from a final T cell subpopulation that is resistant to NK cell killing in at least one NK cell treatment at FDR ≦ 0.01, or in at least two separate different NK cell treatments at FDR ≦ 0.05, are identified as target genes whose mutation (e.g., inactivation) sensitizes T cells to NK cell killing; ii) hits identified as enriched from (and/or having at least about 2-fold enrichment, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more enrichment) a final T cell subpopulation that is resistant to NK cell killing in at least one NK cell treatment at FDR ≦ 0.05, or in at least two separate different NK cell treatments at FDR ≦ 0.15, are identified as target genes whose mutation (e.g., inactivation) renders the T cells resistant to NK cell killing; iii) hits identified as depleted (and/or having at least about 2-fold depletion, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more depletion) from a final T cell subpopulation that is sensitive to NK cell killing in at least one NK cell treatment with FDR ≦ 0.05, or in at least two separate different NK cell treatments with FDR ≦ 0.15, are identified as target genes whose mutation (e.g., inactivation) renders T cells resistant to NK cell killing; and/or iv) hits identified as enriched from (and/or having at least about 2-fold enrichment, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more enrichment) in at least one NK cell treatment with FDR ≦ 0.01, or in at least two separate different NK cell treatments with FDR ≦ 0.15, are identified as target genes whose mutation (e.g., inactivation) renders T cells susceptible to NK cell killing.

In some embodiments according to any of the methods above, the method further comprises validating the target gene by: a) modifying a T cell by making a mutation (e.g., an inactivating mutation) in a target gene of the T cell; and b) determining the sensitivity or resistance of the modified T cell to NK cell killing. In some embodiments, the method further comprises generating a mutation (e.g., an inactivating mutation) in B2M of the T cell.

In another aspect, there is also provided a method of producing a modified T cell (e.g., an allogeneic T cell or CAR-T cell (e.g., an allogeneic CAR-T cell)) comprising inactivation of a target gene identified in a host T cell by any of the methods described above. In some embodiments, the host T cell further comprises a mutation (e.g., an inactivating mutation) in B2M. In some embodiments, the host T cell expresses a CAR. In some embodiments, the method further comprises introducing a nucleic acid encoding a CAR into the host T cell or modified T cell. In some embodiments, the host T cell is allogeneic.

Also provided are modified T cells (e.g., allogeneic T cells or CAR-T cells (e.g., allogeneic CAR-T cells)) comprising a mutation (e.g., an inactivating mutation) in a target gene, wherein the target gene is selected from the group consisting of: TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34 and PACS 2. In some embodiments, the modified T cell further comprises a mutation (e.g., an inactivating mutation) in B2M. In some embodiments, the target gene is PSCS 2. In some embodiments, the modified T cell further expresses a CAR. In some embodiments, the modified T cell is allogeneic.

Also provided are compositions comprising one or more sgrnas (or sgrnas) ^iBAR ) sgRNA (or sgRNA) of a construct ^iBAR ) Library, wherein each sgRNA (or sgRNA) ^iBAR ) The construct comprises or encodes a sgRNA (or sgRNA) ^iBAR ) And wherein each sgRNA (or sgRNA) ^iBAR ) Comprising a guide sequence that is complementary (e.g., at least any one of about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site in a target gene selected from the group consisting of: TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34 and PACS 2. In some embodiments, the sgRNA (or sgRNA) ^iBAR ) The library also includes sgRNA constructs that comprise or encode sgrnas whose guide sequences are complementary (e.g., at least any one of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to the target site in B2MRNA。

Also provided are kits and articles of manufacture useful in the methods described herein, e.g., kits for generating modified T cells (e.g., allogeneic T cells or CAR-T cells (such as allogeneic CAR-T cells)) that are resistant to NK cell killing.

Specifically, the present application relates to the following technical solutions:

1. a method of identifying a target gene that modulates the activity of a T cell in said T cell comprising:

a) providing a T cell library comprising a plurality of T cells, wherein each of the plurality of T cells has a mutation at a hit in the genome ("hit mutation"), wherein the hits in at least two of the plurality of T cells are different from each other;

b) treating the T cell library with NK cells;

c) obtaining from the library of T cells that are sensitive or resistant to NK cell killing; and

d) identifying the hit gene in the T-cell obtained in step c), thereby identifying the target gene that modulates the activity of said T-cell in the T-cell.

2. The method of item 1, wherein the T cell library is generated by genome-wide gene editing of an initial population of T cells.

3. The method of

item

1 or 2, wherein the T cell library is generated by contacting an initial population of T cells with: i) a single-stranded guide RNA ("sgRNA") library comprising a plurality of sgRNA constructs, wherein each sgRNA construct comprises or encodes a sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary to a target site of a hit gene in a genome; and optionally, ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein under conditions that allow introduction of the sgRNA construct and optionally the Cas component into the initial population of T cells.

4. The method of item 3, wherein the Cas protein is Cas 9.

5. The method of item 4, wherein each sgRNA comprises a guide sequence fused to a second sequence, wherein the second sequence comprises a repeat-trans-repeat stem loop that interacts with Cas 9.

6. The method of clause 5, wherein the second sequence of each sgRNA further comprises stem loop 1, stem loop 2, and/or stem loop 3.

7. The method of any one of items 3-6, wherein each sgRNA further comprises an Internal Barcode (iBAR) sequence ("sgRNA ^iBAR "), wherein each sgRNA ^iBAR Can be operated with the Cas protein to modify the hit gene.

8. The method of item 7, wherein each sgRNA ^iBAR Is inserted into the loop region of the repeat-trans-repeat stem loop.

9. The method of item 7, wherein each sgRNA ^iBAR Comprising in the 5 'to 3' direction a first stem sequence and a second stem sequence, wherein the first stem sequence hybridizes to the second stem sequence to form a double stranded rna (dsrna) region that interacts with the Cas protein, and wherein the iBAR sequence is located between the 3 'end of the first stem sequence and the 5' end of the second stem sequence.

10. The method of any one of claims 3-9, wherein each guide sequence comprises about 17 to about 23 nucleotides.

11. The method of any one of items 7-10, wherein each iBAR sequence comprises from about 1 to about 50 nucleotides.

12. The method of any one of claims 7-11, wherein a plurality of sgrnas are included ^iBAR sgRNA library of constructs ("sgRNA) ^iBAR Library ") comprises multiple sets of sgrnas ^iBAR Construct, wherein each group of sgRNAs ^iBAR The construct comprises three or more sgrnas ^iBAR Constructs, each comprising or encoding a sgRNA ^iBAR Three or more sgRNAs ^iBAR The guide sequences of the constructs are identical, wherein three or more sgRNAs ^iBAR The iBAR sequence of each of the constructs is different from each other, and wherein each group of sgrnas ^iBAR The leader sequence of the construct is complementary to a different target site in the genome.

13. The method of item 12, wherein each group of sgrnas ^iBAR The construct comprises 4 sgrnas ^iBAR Construct, and wherein 4 sgrnas ^iBAR The iBAR sequences of each of the constructs differ from each other.

14. The method of item 12 or 13, wherein the sgRNA ^iBAR The library comprises at least about 100 sgRNAs ^iBAR Constructs.

15. The method of any one of claims 12-14, wherein at least two groups of sgrnas ^iBAR The iBAR sequences of the constructs were identical.

16. The method of any one of items 3-15, wherein a sgRNA library comprising a plurality of sgRNA constructs comprises or encodes sgrnas having a guide sequence complementary to a target site of each annotated gene in a genome.

17. The method of any one of claims 3-16, wherein at least about 95% of sgRNA constructs in the sgRNA library are introduced into the initial population of T cells.

18. The method of any one of claims 12-17, wherein the T cell library is for each sgRNA ^iBAR Has a coverage of at least about 100 times.

19. The method of any one of items 3-18, wherein the library of T cells has at least about 400-fold coverage per sgRNA.

20. The method of any one of claims 3-16, wherein the sgRNA library comprises at least about 150000 sgRNA constructs.

21. The method of any one of items 2-20, wherein the initial population of T cells expresses a Cas protein.

22. The method of any one of claims 3-21, further comprising contacting the initial population of T cells or the T cell library with: i) a sgRNA construct comprising or encoding a sgRNA that comprises a guide sequence complementary to a target site in a B2M gene ("B2M sgRNA"); and optionally, ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein under conditions that allow introduction of the B2M sgRNA construct and optionally the Cas component into the initial population of T cells or the T cell library.

23. The method of any one of items 2-21, wherein the T cells in the initial T cell population comprise the B2M mutation.

24. The method of any one of items 3-23, wherein each sgRNA construct in the sgRNA library and/or the B2M sgRNA construct is an RNA.

25. The method of any one of claims 3-23, wherein each sgRNA construct in the sgRNA library and/or the B2M sgRNA construct is a plasmid.

26. The method of any one of claims 3-23, wherein each sgRNA construct in the sgRNA library and/or the B2M sgRNA construct is a viral vector.

27. The method of clause 26, wherein the viral vector is a lentiviral vector.

28. The method of any one of claims 3-23, wherein each sgRNA construct in the sgRNA library and/or the B2M sgRNA construct is a virus.

29. The method of clause 28, wherein the virus is a lentivirus.

30. The method of any one of claims 26-29, wherein the sgRNA library and/or the B2M sgRNA construct is contacted with the initial population of T cells at a multiplicity of infection (MOI) of at least about 2.

31. The method of any one of claims 1-30, wherein treating with NK cells comprises:

i) An initial processing step comprising contacting the library of T cells with the NK cells;

ii) an optional first enrichment step comprising sorting the mixture of treated cells to obtain a first subpopulation of T cells sensitive or resistant to NK cell killing;

iii) an optional first recovery step comprising culturing a first subpopulation of T cells; and

iv) an optional second treatment step comprising contacting a first subpopulation of T cells with said NK cells.

32. The method of clause 31, wherein the initial treatment step comprises contacting the library of T cells with the NK cells for at least about 48 hours.

33. The method of clause 31 or 32, wherein the initial treatment step comprises contacting the T cell library with NK cells for at least about 5 days.

34. The method of any one of items 31-33, wherein the method comprises a first enrichment step.

35. The method of clause 34, wherein the first enrichment step comprises sorting a mixture of B2M negative or defective and viable treated cells, thereby obtaining a first subpopulation of T cells that are resistant to NK cell killing ("first viable enrichment").

36. The method of clause 34, wherein the first enrichment step comprises sorting a mixture of B2M negative or defective and dead treated cells, thereby obtaining a first subpopulation of T cells that are sensitive to NK cell killing ("first death enrichment").

37. The method of clause 35 or 36, further comprising staining the mixture of treated cells with an anti-B2M antibody prior to sorting.

38. The method of any one of items 35-37, further comprising staining the mixture of treated cells with Propidium Iodide (PI) prior to sorting, wherein PI staining indicates cell death.

39. The method of any of clauses 31-35, 37 and 38, wherein the method comprises a first recovery step.

40. The method of clause 39, wherein the first recovery step comprises culturing the first subpopulation of T cells for at least about 24 hours.

41. The method of any of items 31-35 and 37-40, wherein the method comprises a second processing step.

42. The method of clause 41, wherein the second treatment step comprises contacting a first subpopulation of T cells with the NK cells for at least about 48 hours.

43. The method of any one of items 1-42, wherein in the initial processing step, the ratio of NK cells to T cells in the T cell library is about 0.1:1 to about 20: 1.

44. The method of any one of items 1-43, wherein in the initial processing step, the ratio of NK cells to T cells in the T cell library is about 0.5: 1.

45. The method of any one of items 1-43, wherein in the initial processing step, the ratio of NK cells to T cells in the T cell library is about 1: 1.

46. The method of any one of items 1-35 and 37-45, wherein in the second treatment step, the ratio of NK cells to T cells in the first T cell subpopulation is from about 0.1:1 to about 20: 1.

47. The method of any one of items 1-35 and 37-46, wherein in the second processing step, the ratio of NK cells to T cells in the first T cell subpopulation is about 0.3: 1.

48. The method of any one of claims 1-47, wherein obtaining T cells from the T cell library that are sensitive or resistant to NK cell killing comprises:

i) a sorting step comprising sorting the cells obtained from step b) to obtain a second subpopulation of T cells sensitive or resistant to NK cell killing; and

ii) an optional second recovery step comprising culturing a second subpopulation of T cells prior to harvesting the cells.

49. The method of clause 48, wherein the sorting step comprises sorting the B2M negative or defective and viable cells obtained from step B), thereby obtaining a second T cell subpopulation that is resistant to NK cell killing ("harvest live sorting").

50. The method of clause 48, wherein the sorting step comprises sorting the B2M negative or defective and dead cells obtained from step B), thereby obtaining a second subpopulation of T cells that are sensitive to NK cell killing ("harvest-dead sorting").

51. The method of clauses 49 or 50, further comprising staining the cells obtained from step B) with an anti-B2M antibody prior to sorting.

52. The method of any one of items 49-51, further comprising staining the cells obtained from step b) with PI prior to sorting, wherein PI staining indicates cell death.

53. The method of any of clauses 48-49 and 51-52, wherein the method comprises a second recovery step.

54. The method of clause 53, wherein the second recovery step comprises culturing the second subpopulation of T cells for at least about 24 hours.

55. The method of any one of items 1-32, wherein steps b) and c) comprise:

i) an initial processing step comprising contacting the library of T cells with the NK cells for about 72 hours at a ratio of NK cells to T cells of about 0.5: 1;

ii) an enrichment step comprising sorting a mixture of B2M negative or defective and viable treated cells, thereby obtaining a first T cell subpopulation that is resistant to NK cell killing;

iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours;

iv) a second treatment step comprising contacting the restored first T cell subpopulation with said NK cells at a ratio of NK cells to T cells of about 0.3:1 for about 96 hours; and

v) a sorting step comprising sorting the final mixture of B2M negative or defective and viable treated cells, thereby obtaining a second T cell subpopulation that is resistant to NK cell killing.

56. The method of any one of items 1-32, wherein steps b) and c) comprise:

i) a treatment step comprising contacting the T cell library with the NK cells at a ratio of NK cells to T cells of about 0.5:1 for about 10 days; and

ii) a sorting step comprising sorting a mixture of B2M negative or defective and viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing.

57. The method of any one of items 1-32, wherein steps b) and c) comprise:

i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1: 1;

ii) a sorting step comprising sorting a mixture of B2M negative or defective and viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing; and

iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours prior to harvesting the cells.

58. The method of any one of items 1-32, wherein steps b) and c) comprise:

iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and

iv) a sorting step comprising sorting the recovered first subpopulation of T cells that are B2M negative or defective and viable, thereby obtaining a second subpopulation of T cells that are resistant to NK cell killing.

59. The method of any one of items 1-58, wherein identifying hits in the T cells obtained from step c) comprises:

i) identifying sequences comprising the hit gene mutation in the T cells obtained from step c); and

ii) identifying a hit gene corresponding to the sequence comprising the hit gene mutation.

60. The method of any one of items 3-58, wherein identifying the hit genes in the T cells obtained from step c) comprises:

i) Identifying sgRNA sequences in the T cells obtained from step c); and

ii) identifying a hit gene corresponding to the guide sequence of the sgRNA.

61. The method of clauses 59 or 60, wherein the hit gene mutation or the sgRNA sequence is identified by DNA sequencing or RNA sequencing.

62. The method of any one of items 59-61, wherein the hit gene mutation or the sgRNA sequence is identified by Next Generation Sequencing (NGS).

63. The method of any one of items 59 and 61-62, wherein identifying the target gene comprises:

i) obtaining a sequence comprising the hit gene mutation in the final T cell subpopulation obtained from step c);

ii) ordering sequences comprising the hit gene mutations based on sequence counts; and

iii) identifying a hit gene corresponding to a sequence comprising a hit gene mutation ranked above a predetermined threshold level.

64. The method of any one of items 60-62, wherein identifying the target gene comprises:

i) obtaining sgRNA sequences in the final T cell subpopulation obtained from step c);

ii) ordering the respective guide sequences of the sgRNA sequences based on sequence count; and

iii) identifying the hit genes corresponding to the leader sequences ranked above a predetermined threshold level.

65. The method of any one of items 60-62 and 64, wherein the sgRNA is a sgRNA ^iBAR And wherein identifying the target gene comprises:

i) obtaining sgRNAs in the final T cell subpopulation obtained from step c) ^iBAR A sequence;

ii) sequence count based on sgRNA ^iBAR Ordering respective ones of the sequences, wherein the ordering comprises based on sgRNAs corresponding to the guide sequences ^iBAR Data consistency between iBAR sequences in the sequence adjusts the ordering of each pilot sequence; and

66. The method of any one of items 63-65, which is a positive screen.

67. The method of any one of items 63-65, which is a negative screen.

68. The method of any one of items 63-67, wherein the sequence counts are subjected to median ratio normalization followed by mean-variance modeling.

69. The method of clause 68, wherein the method is based on sgRNA corresponding to the guide sequence ^iBAR Data consistency between iBAR sequences in the sequence adjusts the variance of each pilot sequence.

70. The method of any one of clauses 63-69, wherein the sequence counts obtained from the final T cell subpopulation obtained in step c) are compared to the corresponding sequence counts obtained from the control T cell subpopulation to provide a fold-change.

71. The method of clause 70, wherein the control T cell subpopulation is obtained from the same T cell library cultured under the same conditions and not subjected to NK cell treatment, and optionally subjected to the same obtaining method in step c).

72. The method of clause 70 or 71, wherein the sgrnas corresponding to each guide sequence are determined based on the direction of fold change of each iBAR sequence ^iBAR Data consistency between iBAR sequences in a sequence, wherein the variance of the leader sequence increases if the fold changes of the iBAR sequences are in different directions relative to each other.

73. The method of any one of items 59-62, further comprising culturing the same T cell library under the same conditions without undergoing NK cell treatment, and optionally undergoing the same acquisition method in step c) to obtain a control T cell subpopulation, wherein identifying the presence of a hit gene corresponding to a sequence comprising a hit gene mutation or a guide sequence of a sgRNA from the control T cell subpopulation, but not in the T cells obtained from step c) from the NK cell treated T cell library, identifies the hit gene as a target gene.

74. The method of any one of items 1-32 and 61-72, wherein the method comprises prior to step d), subjecting the T cell library from step a) to at least two of 4 separate assays:

(I) Test I:

v) a sorting step comprising sorting the final mixture of B2M negative or defective and viable treated cells, thereby obtaining a second T cell subpopulation that is resistant to NK cell killing;

(II) test II:

i) a treatment step comprising contacting the library of T cells with the NK cells for about 10 days at a ratio of NK cells to T cells of about 0.5: 1; and

ii) a sorting step comprising sorting a mixture of B2M negative or defective and viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing;

(III) run III:

iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours prior to harvesting the cells; and

(IV) test IV:

75. The method of clause 74, wherein identifying the target gene comprises identifying hits from at least two of the 4 separate trials, wherein:

i) in at least one experiment with FDR ≦ 0.01, hits identified as depleted from the final T cell subpopulation in at least two experiments with FDR ≦ 0.05 were identified as target genes whose mutations sensitize T cells to NK cell killing; and/or

ii) hits identified as enriched from the final T cell subpopulation in at least one trial with FDR ≦ 0.05 or at least two trials with FDR ≦ 0.15 are identified as target genes whose mutation renders the T cells resistant to NK cell killing.

76. The method of any one of items 1-32 and 61-72, wherein the method comprises at least two separate distinct treatments with NK cells of the T cell library from step a) in step b), and obtaining T cells sensitive or resistant to NK cell killing from each treatment of step c).

77. The method of clause 76, wherein identifying the target gene comprises identifying a hit gene in a T cell obtained from at least two separate and distinct treatments with NK cells, wherein:

i) hits identified as depleted from a final T-cell subpopulation that is resistant to NK cell killing in at least one NK cell treatment with FDR ≦ 0.01, or in at least two separate different NK cell treatments with FDR ≦ 0.05, are identified as target genes whose mutations sensitize said T-cells to NK cell killing;

ii) hits identified as enriched from a final T cell subpopulation that is resistant to NK cell killing in at least one NK cell treatment with FDR ≦ 0.05, or in at least two separate different NK cell treatments with FDR ≦ 0.15 are identified as target genes whose mutations render said T cells resistant to NK cell killing;

iii) hits identified as depleted from a final T cell subpopulation that is sensitive to NK cell killing in at least one NK cell treatment with FDR ≦ 0.05, or in at least two separate different NK cell treatments with FDR ≦ 0.15, are identified as target genes whose mutation renders said T cells resistant to NK cell killing; and/or

iv) hits identified as enriched from a final T cell subpopulation that is sensitive to NK cell killing in at least one NK cell treatment with FDR ≦ 0.01, or in at least two separate different NK cell treatments with FDR ≦ 0.05 are identified as target genes whose mutations render said T cells sensitive to NK cell killing.

78. The method of any one of items 1-77, further comprising validating the target gene by:

a) modifying the T cell by making a mutation in a target gene of the T cell; and

b) determining the sensitivity or resistance of the modified T cell to NK cell killing.

79. The method of clause 78, further comprising making a mutation in B2M of the T cell.

80. The method of any one of claims 2-79, wherein T cells in the initial T cell population express a Chimeric Antigen Receptor (CAR).

81. A method of producing a modified T cell, comprising inactivating a target gene identified by the method of any one of items 1-80 in a host T cell.

82. The method of clause 81, wherein the host T cell further comprises a mutation in B2M.

83. The method of clauses 81 or 82, wherein the host T cell expresses a CAR.

84. The method of clauses 81 or 82, further comprising introducing a nucleic acid encoding a CAR into the host T cell.

85. The method of any one of claims 81-84, wherein the host T cell is an allogeneic T cell.

86. A modified T cell comprising a mutation in a target gene, wherein the target gene is selected from the group consisting of: TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34 and PACS 2.

87. The modified T cell of clause 86, wherein the modified T cell further comprises a mutation in B2M.

88. The modified T cell of clauses 86 or 87, wherein the target gene is PSCS 2.

89. The modified T cell of any one of claims 86-88, wherein the modified T cell further expresses a CAR.

90. The modified T cell of any one of claims 86-88, wherein the modified T cell is allogeneic.

91. A sgRNA library comprising one or more sgRNA constructs, wherein each sgRNA construct comprises or encodes a sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary to a target site in a target gene selected from the group consisting of: TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34 and PACS 2.

92. The sgRNA library of item 91, wherein the sgRNA library further comprises a sgRNA construct comprising or encoding a sgRNA whose guide sequence is complementary to a target site in B2M, as described below.

93. A kit for generating modified T cells resistant to NK cell killing, comprising the sgRNA library of item 91 or 92.

94. The kit of item 93, further comprising a Cas protein or a nucleic acid encoding the Cas protein.

95. The kit of clauses 93 or 94, further comprising an isolated nucleic acid encoding a CAR.

In addition, the application also relates to the following technical scheme:

b) treating the T cell library with NK cells;

d) identifying the hit gene in the T cell obtained in step c), thereby identifying a target gene that modulates the activity of said T cell in the T cell.

3. The method of

item

4. The method of item 3, wherein the Cas protein is Cas 9.

9. The method of item 7, wherein each sgRNA ^iBAR Comprising in the 5 'to 3' direction a first stem sequence and a second stem sequence, wherein the first stem sequence hybridizes to the second stem sequence to form a double-stranded RNA (dsRNA) region that interacts with the Cas protein,and wherein the iBAR sequence is located between the 3 'end of the first stem sequence and the 5' end of the second stem sequence.

18. The method of any one of items 12-17, whereinThe T cell library for each sgRNA ^iBAR Has a coverage of at least about 100 times.

27. The method of clause 26, wherein the viral vector is a lentiviral vector.

29. The method of clause 28, wherein the virus is a lentivirus.

39. The method of any one of items 31-35, 37 and 38, wherein the method comprises a first recovery step.

45. The method of any one of claims 1-43, wherein in the initial processing step, the ratio of NK cells to T cells in the T cell library is about 1: 1.

55. The method of any one of items 1-32, wherein steps b) and c) comprise:

56. The method of any one of items 1-32, wherein steps b) and c) comprise:

57. The method of any one of items 1-32, wherein steps b) and c) comprise:

58. The method of any one of items 1-32, wherein steps b) and c) comprise:

iii) a recovery step comprising culturing the first subpopulation of T cells for about 48 hours; and

i) Identifying sgRNA sequences in the T cells obtained from step c); and

ii) identifying a hit gene corresponding to the guide sequence of the sgRNA.

i) at a temperature obtained from step c)Obtaining sgRNA from the final T cell subset ^iBAR A sequence;

66. The method of any one of items 63-65, which is a positive screen.

67. The method of any one of items 63-65, which is a negative screen.

(I) Test I:

(II) run II:

(III) run III:

(IV) test IV:

83. The method of clauses 81 or 82, wherein the host T cell expresses a CAR.

86. A modified T cell comprising a mutation in a target gene, wherein the target gene is selected from the group consisting of: TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FACNB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34, and PACS 2.

87. The modified T cell of item 86, wherein the target gene encoding a protein is inactivated by mutation.

88. The modified T cell of clause 86, wherein the modified T cell further comprises a mutation in B2M.

89. The modified T cell of claim 88, wherein the B2M-encoding protein is inactivated by mutation.

90. The modified T cell of any one of claims 86-89, wherein the target gene is PSCS 2.

91. The modified T cell of clause 90, wherein the target gene PSCS 2-encoding protein is mutated to inactivate.

92. The modified T cell of any one of claims 86-91, wherein the modified T cell further expresses a CAR.

93. The modified T cell of any one of claims 86-92, wherein the modified T cell is allogeneic.

94. A sgRNA library comprising one or more sgRNA constructs, wherein each sgRNA construct comprises or encodes a sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary to a target site in a target gene selected from the group consisting of: TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34 and PACS 2.

95. The sgRNA library of item 94, wherein the sgRNA library further comprises a sgRNA construct comprising or encoding a sgRNA whose guide sequence is complementary to a target site in B2M.

96. A composition or kit for generating modified T cells resistant to NK cell killing, comprising the sgRNA library of item 94 or 95.

97. The composition or kit of clause 96, further comprising a Cas protein or a nucleic acid encoding the Cas protein.

98. The composition or kit of clauses 96 or 97, further comprising an isolated nucleic acid encoding a CAR.

99. A modified T cell that is resistant to NK cell killing, wherein a protein encoded by one or more target genes selected from the group consisting of: TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34 and PACS 2.

100. The modified T cell of item 99, which is a CAR-expressing cell.

Drawings

Figure 1 shows an exemplary procedure for screening genes associated with NK cell killing of T cells.

FIG. 2 shows Cas9 ⁺ B2M ^- sgRNA ^iBAR Exemplary screening methods for T cell libraries.

FIGS. 3A-3D show the results from the screens of experiments 3-6 and top ranked candidates, identifying genes conferring NK cell killing resistance phenotype (positive side) or sensitive phenotype (negative side) after T cell gene knock-out. The top-ranked genes with FDR < 0.15 are dark grey above the dotted line.

FIG. 4 shows the Venn plot (FDR ≦ 0.15) for top ranked candidates in various screening assays.

FIG. 5 shows Cas9 ⁺ sgRNA ^iBAR An exemplary target gene identification workflow for a T cell library.

Detailed Description

The present application provides methods of identifying target genes in T cells (e.g., allogeneic T cells or CAR-T cells (e.g., allogeneic CAR-T cells)) that modulate T cell activity (e.g., in response to NK cell treatment). The method comprises the following steps: a) providing a T cell library comprising a plurality of T cells, wherein each of the plurality of T cells has a mutation (e.g., an inactivating mutation) at a hit in the genome ("hit mutation"), wherein hits in at least two of the plurality of T cells are different from each other; b) treating the T cell library with NK cells; c) obtaining T cells from a T cell library that are sensitive or resistant to NK cell killing; and d) identifying the hit gene in the T cell, thereby identifying a target gene in the T cell that modulates T cell activity. In some embodiments, the one or more mutations (e.g., inactivating mutations) at the one or more hits are by or a construct (e.g., a vector such as a viral vector, or a virus such as a lentivirus) encoding a CRISPR/Cas guide RNA (e.g., a single stranded guide RNA) such as comprising an iBAR sequence (sgRNA) as described herein ^iBAR ) Produced by the sgRNA of (a). Use of sgRNAs described herein ^iBAR Screening assays for molecules, constructs, panels, or libraries provide reliable and efficient screening strategies for large-scale target identification in eukaryotic cells (e.g., T cells), with much lower rates of false positives and false negatives, and allow for the generation of cell libraries using high MOI. The target genes identified herein, particularly those whose mutation (e.g., inactivation) renders T cells more resistant to NK cell killing, are particularly useful in adoptive T cell therapies (e.g., CAR-T). For example, allogeneic T cells (e.g., allogeneic CAR-T cells) can be modified to inactivate one or more target genes identified herein, thereby avoiding rejection from host NK cells.

Therefore, the present inventionIn one aspect, a method is provided for identifying a target gene in a T cell (e.g., an allogeneic T cell or CAR-T cell (e.g., an allogeneic CAR-T cell)) that modulates T cell activity, comprising: a) providing sgRNA libraries or sgrnas comprising one or more hits in a targeted genome (e.g., the human whole genome) ^iBAR A library of T cells of the library; b) treating the T cell library with NK cells; c) Obtaining T cells from a T cell library that are sensitive or resistant to NK cell killing; and d) identifying the hit gene in the T cell, thereby identifying a target gene in the T cell that modulates T cell activity. In some embodiments, the sgRNA library comprises one or more sgRNA constructs, wherein each sgRNA construct comprises or encodes a sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site of a hit gene in the genome. In some embodiments, the sgRNA ^iBAR The library comprises multiple sets of sgRNAs ^iBAR Construct, wherein each group of sgRNAs ^iBAR The construct comprises three or more (e.g., 4) sgrnas ^iBAR Constructs, each construct comprising or encoding a sgRNA ^iBAR Wherein each sgRNA ^iBAR Comprising a leader sequence and an iBAR sequence, three or more (e.g., 4) sgRNAs ^iBAR The guide sequences of the constructs are identical and complementary to the same target site in the genome (e.g., any one of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary), three or more (e.g., 4) sgrnas thereof ^iBAR The iBAR sequences of each of the constructs are different from each other, with each set of sgrnas ^iBAR The guide sequences of the constructs are complementary to different target sites in the genome (e.g., different genes or different sites within the same gene), and wherein each sgRNA is complementary to a different target site in the genome ^iBAR Can be manipulated with a Cas protein (e.g., Cas9) to modify (e.g., cut or modulate expression) the target site. In some embodiments, the sgRNA library or sgRNA ^iBAR The library is a whole genome library, i.e., each annotated gene in the genome is targeted. In thatIn some embodiments, more than one (e.g., 2, 3, 4, or more, such as 2) leader sequence is designed for each hit.

Also provided are sgrnas or sgrnas for performing the screening methods described herein ^iBAR A molecule, construct, panel or library. Also provided are compositions comprising sgrnas or sgrnas ^iBAR Modified T cells (e.g., allogeneic T cells or CAR-T cells (e.g., allogeneic CAR-T cells)), of molecules, constructs, sets, or libraries, and methods of producing the same. Also provided are target genes whose mutation (e.g., inactivation, such as knock-out) renders the T cell more sensitive or more resistant to NK cell killing. Also provided are sgRNA molecules, constructs or libraries against target genes whose mutation (e.g., inactivation) renders T cells more resistant to NK cell killing, modified T cells comprising the same (e.g., allogeneic T cells or CAR-T cells (e.g., allogeneic CAR-T cells)), pharmaceutical compositions and kits thereof.

I. Definition of

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto. Any reference signs in the claims shall not be construed as limiting the scope. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

As used herein, "internal barcode" or "iBAR" refers to an index inserted or attached to a molecule that can be used to track the identity and properties of the molecule. For example, ibars can be short nucleotide sequences of guide RNAs inserted or attached to CRISPR/Cas systems, as exemplified herein. Multiple ibars can be used to track the performance of single stranded guide RNA sequences in one experiment, providing duplicate data for statistical analysis without the need for repeated experiments.

The "CRISPR system" or "CRISPR/Cas system" is collectively referred to as the transcript and other elements involved in CRISPR-associated ("Cas") gene expression and/or directing its activity. For example, the CRISPR/Cas system can include a sequence encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active portion of tracrRNA), a tracr-mate sequence (e.g., including "direct repeats" and portions of the direct repeats processed by tracrRNA in endogenous CRISPR systems), a guide sequence (also referred to as a "spacer" in endogenous CRISPR systems), and other sequences and transcripts derived from CRISPR loci.

In the context of CRISPR complex formation, "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence promotes formation of the CRISPR complex. Complete complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. The CRISPR complex can comprise a guide sequence that hybridizes to a target sequence and complexes with one or more Cas proteins.

The term "guide sequence" refers to a contiguous sequence of nucleotides in a guide RNA that has partial or complete complementarity to a target sequence in a target polynucleotide and can hybridize to the target sequence through base pairing facilitated by the Cas protein. In the CRISPR/Cas9 system, the target sequence is adjacent to the PAM site. The PAM sequence and its complementary sequence on the other strand together form a PAM site.

The terms "single-stranded guide RNA," "synthetic guide RNA," and "sgRNA" are used interchangeably to refer to polynucleotide sequences that comprise a guide sequence and sgRNA functions and/or any other sequences required for the sgrnas to interact with one or more Cas proteins to form a CRISPR complex. In some embodiments, the sgRNA comprises a second sequence that is complementary to a second sequence comprising a tracr sequence derived from a tracr rna and a tracr mate sequence derived from a crRNAA fused leader sequence. the tracr sequence may contain all or part of the sequence of a tracrRNA from a naturally occurring CRISPR/Cas system. The term "guide sequence" refers to a nucleotide sequence within a guide RNA that specifies a target site, and may be used interchangeably with the terms "guide" or "spacer". The term "tracr mate sequence" may also be used interchangeably with the term "direct repeat sequence". As used herein, "sgRNA ^iBAR "refers to a single stranded guide RNA having an iBAR sequence.

The term "operable with a Cas protein" means that the guide RNA can interact with the Cas protein to form a CRISPR complex.

As used herein, the term "wild-type" is a term of art understood by a skilled artisan and means a typical form of an organism, strain, gene, or characteristic, as it occurs in nature, as distinguished from a mutant or variant form.

As used herein, the term "variant" is to be understood as an expression having a characteristic that deviates from a naturally occurring pattern.

"complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence through traditional Watson-Crick base pairing or other unconventional types. Percent complementarity refers to the percentage of residues (e.g., 5, 6, 7, 8, 9, 10 out of 10, i.e., 50%, 60%, 70%, 80%, 90%, and 100% complementary) in a nucleic acid molecule that can form hydrogen bonds (e.g., watson-crick base pairing) with a second nucleic acid sequence. By "fully complementary" is meant that all consecutive residues of one nucleic acid sequence will form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or to two nucleic acids that hybridize under stringent conditions.

As used herein, "stringent conditions" for hybridization means that a nucleic acid having complementarity to a target sequence hybridizes significantly to the target sequence under such conditions and does not substantially hybridize to non-target sequences. Stringent conditions generally depend on the sequence and vary according to a number of factors. Generally, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described In detail In Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology With Nucleic Acid Probes Part 1, Second Chapter "Overview of principles of Hybridization And the strategy of Nucleic Acid probe assay", Elsevier, N.Y..

"hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding may occur by watson-crick base pairing, Hoogstein binding, or any other sequence specific means. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. The hybridization reaction may constitute a step in a broader process, such as the initiation of PCR, or enzymatic cleavage of a polynucleotide. Sequences that are capable of hybridizing to a given sequence are referred to as "complements" of the given sequence.

As used herein, "construct" refers to a nucleic acid molecule (e.g., DNA or RNA), or a vector capable of delivering such a nucleic acid molecule. For example, when used in the context of a sgRNA, a construct refers to a sgRNA molecule, a nucleic acid molecule (e.g., an isolated DNA or viral vector) encoding the sgRNA, or a vector capable of delivering a nucleic acid molecule encoding the sgRNA, such as a lentivirus carrying a nucleic acid molecule encoding the sgRNA. When used in the context of a protein, a construct refers to a nucleic acid molecule comprising a nucleotide sequence that can be transcribed into RNA or expressed as a protein. The construct may comprise the necessary regulatory elements operably linked to the nucleotide sequence which allow transcription or expression of the nucleotide sequence when the construct is present in a host cell.

As used herein, "operably linked" refers to the expression of a gene under the control of a regulatory element (e.g., a promoter) to which it is spatially linked. The regulatory element may be located 5 '(upstream) or 3' (downstream) of the gene under its control. The distance between a regulatory element (e.g., a promoter) and a gene can be about the same as the distance between the regulatory element (e.g., a promoter) and the gene it naturally controls and from which the regulatory element is derived. As is known in the art, changes in distance may be incorporated without loss of function of the regulatory element (e.g., promoter).

The term "vector" is used to describe a nucleic acid molecule that can be engineered to contain one or more cloned polynucleotides that can be propagated in a host cell. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, no free ends (e.g., circular); a nucleic acid molecule comprising DNA, RNA, or both; and other kinds of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". A recombinant expression vector may comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector comprises one or more regulatory elements, which may be selected for expression on the basis of the host cell, i.e. operably linked to the nucleic acid sequence to be expressed.

"host cell" refers to a cell that may or has been the recipient of a vector or isolated polynucleotide. The host cell may be a prokaryotic cell or a eukaryotic cell. In some embodiments, the host cell is a eukaryotic cell, which can be cultured in vitro and modified using the methods described herein. The term "cell" includes primary subject cells and their progeny.

"multiplicity of infection" or "MOI" are used interchangeably herein to refer to the ratio of a pathogen (e.g., phage, virus, or bacterium) to its target of infection (e.g., cell or organism). For example, when referring to a group of cells inoculated with a viral particle, the multiplicity of infection or MOI is the ratio between the number of viral particles (e.g., viral particles comprising a sgRNA library) and the number of target cells present in the mixture during viral transduction.

As used herein, a "phenotype" of a cell refers to an observable feature or trait of the cell, such as its morphology, development (e.g., growth, proliferation, differentiation, or death), biochemical or physiological properties, phenology, or behavior. The phenotype may result from the expression of a gene in the cell, the influence of environmental factors, or an interaction between the two. In some embodiments, the phenotype is resistance or sensitivity to killing (e.g., by NK cells). In some embodiments, the phenotype is inhibition of growth or proliferation. In some embodiments, the phenotype is death.

An "isolated" nucleic acid molecule, as used herein, is a nucleic acid molecule that is identified and isolated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it is produced. Preferably, the isolated nucleic acid does not bind to all components associated with the production environment. Isolated nucleic acid molecules encoding the polypeptides and antibodies herein, in a form different from the form or environment in which they are found in nature. Thus, isolated nucleic acid molecules are distinct from nucleic acids encoding the polypeptides and antibodies herein that naturally occur in cells.

Unless otherwise indicated, "nucleotide sequences encoding amino acid sequences" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may contain one or more introns in some versions.

As used herein, the term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing an exogenous nucleic acid into a host cell (e.g., a T cell). A "transfected" or "transformed" or "transduced" cell is a cell transfected, transformed or transduced with an exogenous nucleic acid. The cell includes a primary subject cell and its progeny.

As used herein, "treatment" is a method for obtaining beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing remission (in part or in whole), reducing the dosage of one or more other drugs required to treat the disease, delaying the progression of the disease, improving the quality of life, and/or prolonging survival. "treating" also includes reducing the pathological consequences of cancer.

As used herein, "individual" or "subject" refers to a mammal, including but not limited to: human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

As used herein, the term "autologous" refers to any material derived from the same individual that is subsequently reintroduced into the individual.

"allogenic" refers to grafts derived from different individuals of the same species. By "allogeneic T cells" is meant T cells from a donor that have a tissue HLA type matched to the recipient. Typically, matching is based on variability of three or more loci of HLA genes, and preferably perfect matching at these loci. In some cases, allogeneic donors may be related (usually siblings with close HLA matches), syngeneic (single egg "in ovo" twins of patients), or unrelated (donors with no consanguinity and found to be very close in HLA match). HLA genes are divided into two classes (type I and type II). In general, mismatches in the type I gene (i.e., HLA-A, HLA-B or HLA-C) increase the risk of transplant rejection. Mismatches to HLAII type genes (i.e., HLA-DR or HLA-DQB1) increase the risk of GvHD.

As used herein, "patient" includes any person having a disease (e.g., cancer or viral infection). The terms "subject", "individual" and "patient" are used interchangeably herein. The term "donor subject" or "donor" refers herein to a subject whose cells are obtained for further engineering in vitro. The donor subject can be a patient to be treated with the cell population generated by the methods described herein (i.e., an autologous donor), or can be an individual who donates a blood sample (e.g., a lymphocyte sample) and, after the cell population is generated by the methods described herein, will be used to treat a different individual or patient (i.e., an allogeneic donor). Those subjects that receive cells prepared by the present methods may be referred to as "recipients" or "recipient subjects.

As used herein, the term "stimulus" refers to a primary response induced by attachment of a cell surface moiety (e.g., a ligand, receptor, or molecule that binds to a cell surface moiety). For example, in the case of a receptor, such stimulation requires the attachment of the receptor (e.g., the binding of a ligand or molecule to the receptor), and subsequent signaling events. With respect to stimulation of T cells, such stimulation refers to the attachment of a T cell surface moiety, which in one embodiment subsequently induces a signaling event, such as binding to the TCR/CD3 complex. In addition, the stimulatory event may activate the cell and up-or down-regulate the expression or secretion of the molecule, such as down-regulating TGF- β. Thus, even in the absence of a direct signaling event, ligation of cell surface moieties may result in recombination of the cytoskeletal structure, or coalescence of cell surface moieties, each of which may be used to enhance, modify, or alter subsequent cellular responses.

As used herein, the term "activation" refers to the state of a cell after sufficient attachment of cell surface moieties to induce significant biochemical or morphological changes. In the context of T cells, such activation refers to a state in which T cells have been sufficiently stimulated to induce cell proliferation. Activation of T cells may also induce cytokine production and regulatory properties or cytolytic effector functions. In the context of other cells, the term infers up-or down-regulation of a particular physicochemical process. The term "activated T cell" means that the T cell is currently undergoing cell division, cytokine production, regulatory properties or cytolytic effector function, and/or has recently undergone an "activation" process.

When the term "comprising" is used in the present description and claims, it does not exclude other elements or steps.

It is to be understood that the embodiments of the present application described herein include embodiments "consisting of … …" and/or "consisting essentially of … …".

Reference herein to "about" a value or parameter includes (and describes) variations that are directed to that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

As used herein, reference to a "non" value or parameter generally means and describes "excluding" that value or parameter. For example, the method is not used to treat type X cancer means that the method is used to treat cancers other than type X.

The term "about X-Y" as used herein has the same meaning as "about X to about Y".

For the recitation of numerical ranges of nucleotides herein, each intervening number is explicitly contemplated. For example, for the range of 19-21nt, the number 20nt is considered in addition to 19nt and 21nt, and for the range of MOI, each intervening number, whether integer or fractional, is explicitly considered.

As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Methods of identifying target genes that modulate sensitivity or resistance of T cells to NK cell killing

The present application provides methods of identifying target genes in T cells (e.g., allogeneic T cells or CAR-T cells (e.g., allogeneic CAR-T cells)) that modulate T cell activity, such as in response to NK cell treatment. In some embodiments, there are providedA method is provided for identifying a target gene in a T cell that modulates T cell activity, comprising: a) Providing a T cell library comprising a plurality of T cells, wherein each of the plurality of T cells has a mutation (e.g., an inactivating mutation) at a hit in the genome, wherein hits of at least two of the plurality of T cells are different from each other; b) treating the T cell library with NK cells; c) obtaining T cells from a T cell library that are sensitive or resistant to NK cell killing; and d) identifying the hit gene in the T cell, thereby identifying a target gene in the T cell that modulates T cell activity. In some embodiments, the T cell library is generated by genome-wide gene editing of an initial population of T cells. In some embodiments, the T cell library is generated by contacting an initial population of T cells (e.g., allogeneic T cells or CAR-T cells (e.g., allogeneic CAR-T cells)) with: i) a sgRNA library comprising a plurality of sgRNA constructs, wherein each sgRNA construct comprises or encodes a sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary (e.g., any of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site of a hit gene in a genome; and optionally, ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein under conditions that allow introduction of the sgRNA construct and optionally the Cas component into the initial population of T cells. In some embodiments, the Cas protein is Cas 9. In some embodiments, each sgRNA comprises a guide sequence fused to a second sequence, wherein the second sequence comprises a repeat-trans-repeat stem loop that interacts with Cas 9. In some embodiments, the second sequence of each sgRNA further comprises stem loop 1, stem loop 2, and/or stem loop 3. In some embodiments, each sgRNA further includes an iBAR sequence ("sgRNA) ^iBAR "), wherein each sgRNA ^iBAR Can be manipulated with the Cas protein to modify (e.g., cleave or regulate expression) the hit gene. In some embodiments, each sgRNA ^iBAR The iBAR sequence of (a) is inserted into the loop region of the repeat-trans-repeat stem loop. In some embodiments, each sgRNA ^iBAR Comprising in the 5 'to 3' direction a first stem sequence and a second stem sequenceWherein the first stem sequence is hybridized to the second stem sequence to form a double-stranded rna (dsrna) region that interacts with the Cas protein, and wherein the iBAR sequence is located between the 3 'end of the first stem sequence and the 5' end of the second stem sequence. In some embodiments, each guide sequence comprises from about 17 to about 23 nucleotides.

In some embodiments, the multiple sets of sgrnas are included by contacting a population of naive T cells (e.g., allogeneic T cells or CAR-T cells (e.g., allogeneic CAR-T cells)) with i) ^iBAR sgRNA of constructs ^iBAR Library contact to generate a library of T cells, wherein each set of sgrnas ^iBAR The construct comprises three or more (e.g., 4) sgrnas ^iBAR Constructs, each comprising or encoding a sgRNA ^iBAR Wherein each sgRNA ^iBAR Comprising a leader sequence and an iBAR sequence, three or more (e.g., 4) sgRNAs ^iBAR The guide sequences of the constructs are identical and complementary to the same target site in the genome (e.g., any one of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary), with three or more (e.g., 4) sgrnas ^iBAR The iBAR sequences of each of the constructs are different from each other, with each set of sgrnas ^iBAR The guide sequence of the construct is complementary to a different target site in the genome (e.g., a different hit gene, or different sites within the same hit gene), and wherein each sgRNA ^iBAR Operable with Cas9 protein to modify a target site; and optionally, ii) allowing the sgRNA to grow ^iBAR A Cas9 component comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, under conditions in which the construct and optional Cas component are introduced into the initial T cell population. In some embodiments, the T cell population is generated by contacting an initial T cell population with i) a population comprising multiple sets of sgrnas ^iBAR sgRNA of construct ^iBAR Library contact to generate a library of T cells, wherein each set of sgrnas ^iBAR The construct comprises three or more (e.g., 4) sgrnas ^iBAR Constructs, each comprising or encoding a sgRNA ^iBAR Wherein each sgRNA ^iBAR Comprising a leader sequence, a second sequence, and an iBAR sequence, three or more (e.g., 4) sgRNA ^iBAR The guide sequences of the constructs are identical and complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to the same target site in the genome, with three or more (e.g., 4) sgrnas ^iBAR The iBAR sequence of each of the constructs is different from each other, wherein the guide sequence is fused to a second sequence, wherein the second sequence comprises a repeat-trans-repeat stem loop that interacts with the Cas9 protein, wherein the iBAR sequence is inserted into a loop region of the repeat-trans-repeat stem loop, wherein each set of sgrnas ^iBAR The guide sequence of the construct is complementary to different target sites in the genome, and wherein each sgRNA ^iBAR Operable with Cas9 protein to modify a target site; and optionally, ii) allowing the sgRNA to grow ^iBAR A Cas9 component comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein under conditions in which the construct and optional Cas9 component are introduced into the initial T cell population. In some embodiments, each iBAR sequence comprises from about 1 to about 50 nucleotides. In some embodiments, each set of sgrnas ^iBAR The construct comprises 4 sgrnas ^iBAR Construct, and wherein 4 sgrnas ^iBAR The iBAR sequences of each of the constructs differ from each other. In some embodiments, the sgRNA ^iBAR The library comprises at least about 100 sgRNAs ^iBAR Constructs. In some embodiments, different groups of sgrnas ^iBAR At least two sgRNAs in the construct ^iBAR The iBAR sequences of the constructs are identical (e.g., first and second sets of sgrnas ^iBAR Constructs in these two groups of sgrnas ^iBAR With at least 1, 2, 3, 4, or more iBAR sequences shared between constructs). In some embodiments, at least two sets of sgrnas ^iBAR The iBAR sequences of the constructs were identical. In some embodiments, the sgRNA ^iBAR The library is contacted with the initial population of T cells at an MOI of greater than about 2 (e.g., at least about 3, 5, or 10). In some embodiments, comprising a plurality of sgrnas ^iBAR sgRNA of constructs ^iBAR The library comprises or encodes sgrnas with guide sequences complementary to target sites of each annotated gene in the genome ^iBAR . In some casesIn embodiments, the sgRNA ^iBAR At least about 95% (e.g., at least about any of 96%, 97%, 98%, 99% or more) of the sgrnas in the library, such as at least about 99% ^iBAR The construct is introduced into the initial T cell population. In some embodiments, the T cell library is for each sgRNA ^iBAR Having an average coverage of at least about 100-fold (e.g., at least about any of 200-, 400-, 500-, 1,000-fold, or more). In some embodiments, the T cell library is for each group of sgrnas ^iBAR Have an average coverage of at least about 400-fold (e.g., at least about any of 800-, 1,000-, 2,000-, 4,000-fold, or more). In some embodiments, the T cell library is directed to sgrnas ^iBAR The library has an average coverage of at least about 100-fold (e.g., at least about any of 200-, 400-, 500-, 1,000-fold, or more). In some embodiments, the library of T cells has an average coverage per gene of at least about 800-fold (e.g., at least about any of 1,200-, 1,600-, 2,000-, 3,000-, 4,000-, 10,000-fold, or more). In some embodiments, the T cell library further comprises a B2M mutation (e.g., an inactivated B2M mutation). In some embodiments, the B2M mutation (e.g., an inactivated B2M mutation) is generated by contacting a T cell library or an initial population of T cells used to generate the T cell library with a B2M sgRNA construct (e.g., a viral vector or virus) that comprises or encodes a B2M sgRNA, the construct comprising a guide sequence that is complementary (e.g., at least any one of about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site in a B2M gene. In some embodiments, the B2M sgRNA construct is contacted with the T cell library or an initial population of T cells used to generate the T cell library at an MOI of greater than about 2 (e.g., at least about 3, 5, or 10).

In some embodiments, sgrnas described herein are used ^iBAR The library screening method can improve target identification and data reproducibility and reduce False Discovery Rate (FDR) through statistical analysis. In a traditional CRISPR/Cas-based screening method using a pooled sgRNA library, a cell library is constructedA high quality library of cells expressing grnas is generated using a low MOI in the process to ensure that each cell has on average less than one sgRNA or paired guide RNAs ("pgrnas"). Since the sgRNA molecules in the library are randomly integrated into the transfected cells, a sufficiently low MOI ensures that each cell expresses a single sgRNA, thereby minimizing the screened FDR. To further reduce FDR and improve data reproducibility, deep coverage of grnas and multiple biological replications is often required to obtain a hit gene with high statistical significance. Traditional screening methods face difficulties when large numbers of whole genome screens are required, when the cellular material used to construct the library is limited, or when more challenging screens (i.e., in vivo screens) are performed that make it difficult to arrange experimental replicates or control MOI. Use of sgRNAs described herein ^iBAR Library screening methods overcome the difficulty by including iBAR sequences in each sgRNA, which enables the collection of internal copies within each sgRNA group with the same guide sequence but different iBAR sequences. This iBAR method can reduce experimental noise. For example, as shown in WO2020125762, iBAR with 4 nucleotides per sgRNA can provide sufficient internal copies to evaluate different sgrnas against the same genomic locus ^iBAR Data consistency between constructs. The high degree of agreement between two independent experiments in WO2020125762 indicates that one experimental replicate is sufficient for CRISPR/Cas screening using the iBAR method. Since the library coverage increases significantly with high MOI during viral transduction of host cells, the number of cells in the initial cell population can be reduced more than 20-fold to reach the same library coverage as shown by the whole genome human library constructed in WO 2020125762. Likewise, sgRNA was used ^iBAR The workload of each whole genome screen of (a) can be proportionally reduced. Using sgrnas with different iBAR sequences, the performance of each leader sequence can be followed multiple times in the same experiment by counting the leader sequences and corresponding iBAR nucleotide sequences, thereby greatly reducing FDR and improving efficiency and performance. The transduction efficiency and library coverage can be further improved by using high viral titers during the viral transduction step, e.g., MOI>1 (e.g., MOI)>1.5、MOI>2，MOI>2.5、MOI>3、 MOI>3.5、MOI>4、MOI>4.5、MOI>5、MOI>5.5、MOI>6、MOI>6.5、MOI>7、 MOI>7.5、MOI>8、MOI>8.5、MOI>9、MOI>9.5 or MOI>10; e.g., an MOI of about any one of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10).

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., an allogeneic T cell or CAR-T cell (e.g., an allogeneic CAR-T cell)) that modulates T cell activity, comprising: a) providing a T cell library comprising sgrnas described herein that target one or more hits in the genome ^iBAR A library; b) treating the T cell library with NK cells; c) obtaining T cells from a T cell library that are sensitive or resistant to NK cell killing; and d) identifying the hit gene in the T cell, thereby identifying a target gene in the T cell that modulates T cell activity. In some embodiments, there is provided a method of identifying a target gene in a T cell that modulates T cell activity, comprising: a) providing a T cell library comprising sgrnas described herein that target one or more hits in a genome ^iBAR A library; b) treating the T cell library with NK cells; c) obtaining T cells from a T cell library that are sensitive or resistant to NK cell killing; and d) identifying a hit gene in the T cell, thereby identifying a target gene in the T cell that modulates T cell activity; wherein the treatment with NK cells comprises: i) an initial processing step comprising contacting a library of T cells with NK cells; ii) an optional first enrichment step comprising sorting the mixture of treated cells to obtain a first subpopulation of T cells susceptible to or resistant to NK cell killing; iii) an optional first recovery step comprising culturing a first subpopulation of T cells; and iv) optionally a second treatment step comprising contacting the first subpopulation of T cells with NK cells; and/or wherein the T cells are obtained from a library of T cells sensitive or resistant to NK cell killing, comprising: i) a sorting step comprising sorting the cells obtained from step b) to obtain a second subpopulation of T cells sensitive or resistant to NK cell killing; and ii) optionally a second recovery step comprising culturing a second subpopulation of T cells prior to harvesting the cells. In that In some embodiments, the sgRNA ^iBAR Library targeting each annotated gene in the genome (i.e., sgRNA) ^iBAR The library was a whole genome sgRNA ^iBAR A library). In some embodiments, the T cell library is for a whole genome sgRNA ^iBAR The library has an average coverage of at least about 100-fold (e.g., at least about 400-fold). In some embodiments, identifying the hit genes in the T cells obtained from step c) comprises: i) identifying sgRNAs in T cells obtained from step c) ^iBAR A sequence; and ii) identification and sgRNA ^iBAR The targeting sequence of (2) corresponds to the hit gene. In some embodiments, identifying the target gene comprises: i) obtaining sgRNAs in the final T cell subpopulation obtained from step c) ^iBAR A sequence; ii) sequence count based on sgRNA ^iBAR Ordering the respective guide sequences of the sequences, wherein the ordering comprises based on the sgRNA corresponding to the guide sequence ^iBAR Data consistency between iBAR sequences in the sequence adjusts the ordering of each pilot sequence; and iii) identifying the hit genes corresponding to the guide sequences that are ranked above a predetermined threshold level. In some embodiments, the method is a positive screen. In some embodiments, the method is a negative screen. In some embodiments, the sequence counts obtained from the final T cell subpopulation obtained in step c) are compared to corresponding sequence counts obtained from a control T cell subpopulation to provide a fold change (e.g., an actual fold change, or a derivative of a fold change, such as a log2 or log10 fold change). In some embodiments, the control T cell subpopulation is obtained from the same T cell library cultured under the same conditions without undergoing NK cell treatment, and optionally subjected to the same acquisition method in step c). In some embodiments, the T cell library further comprises a B2M mutation (e.g., an inactivated B2M mutation). In some embodiments, the B2M mutation (e.g., an inactivated B2M mutation) is generated by contacting a T cell library or an initial population of T cells used to generate a T cell library with a B2M sgRNA construct (e.g., a viral vector or virus) described herein. In some embodiments, treating the T cell library with NK cells in step b) comprises culturing in the presence of NK cells T cell libraries.

In some embodiments, there is provided a method of identifying a target gene that modulates the activity of a T cell (e.g., an allogeneic T cell or CAR-T cell (e.g., an allogeneic CAR-T cell)) comprising: a) Providing a T cell library comprising a plurality of T cells, wherein each of the plurality of T cells has a mutation (e.g., an inactivating mutation) at a hit in the genome, wherein hits of at least two of the plurality of T cells are different from each other; b) treating the T cell library with NK cells; c) obtaining T cells from the T cell library that are sensitive or resistant to NK cell killing (treated T cell population); and d) identifying the target gene based on the difference between the hit gene mutation signature in the T cells (or treated T cell population) obtained from step c) and the control T cells (or control T cell population). In some embodiments, the control T cells (or control T cell population) are obtained from the same T cell library cultured under the same conditions without treatment with NK cells, and optionally subjected to the same obtaining method in step c). In some embodiments, treating the T cell library with NK cells comprises culturing the T cell library in the presence of NK cells. In some embodiments, the characteristics of the hit gene mutations in the T cells (or treated T cell population) obtained from step c) and the control T cells (or control T cell population) are identified by next generation sequencing. In some embodiments, the T cell library is subjected to two or more (e.g., 2, 3, 4 or more) separate different NK cell treatments and/or obtaining methods in steps b) and c), and the target gene is identified based on the difference between the characteristics of each treatment. In some embodiments, the sequence counts comprising the hit gene mutations are subjected to median ratio normalization followed by mean-variance modeling. In some embodiments, the method comprises: comparing the sequence count comprising the hit gene mutation obtained from the T cell (or treated T cell population) obtained from step c) with the sequence count comprising the hit gene mutation obtained from a control T cell (or control T cell population), wherein i) in at least one NK cell treatment with FDR ≦ 0.01, or in at least two separate different NK cell treatments with FDR ≦ 0.05, the corresponding mutation is identified as depleted (and/or having at least about 2-fold depletion) in the T cells (or treated T cell population) resistant to NK cell killing obtained from step c), such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more depletion) of the hit gene, is identified as a target gene whose mutation sensitizes the T cell to NK cell killing; ii) in at least one NK cell treatment with FDR ≦ 0.05, or in at least two separate different NK cell treatments with FDR ≦ 0.15, the respective mutation thereof being identified as a hit gene that is enriched (and/or has at least about 2-fold enrichment, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more enrichment) in the T-cells (or treated T-cell population) obtained from step c) that are resistant to NK cell killing, as a target gene whose mutation renders the T-cells resistant to NK cell killing; iii) in at least one NK cell treatment with FDR ≦ 0.05, or in at least two separate distinct NK cell treatments with FDR ≦ 0.15, whose respective mutation is identified as a hit that depletes (and/or has at least about 2-fold depletion, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more depletion) in the T cells (or treated T cell population) that are sensitive to NK cell killing obtained from step c), identified as a target gene whose mutation renders the T cells resistant to NK cell killing; and/or iv) in at least one NK cell treatment with FDR ≦ 0.01, or in at least two separate different NK cell treatments with FDR ≦ 0.05, the respective mutation thereof being identified as a hit gene enriched (and/or having at least about 2-fold enrichment, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more enrichment) in the T-cells (or treated T-cell population) sensitive to NK cell killing obtained from step c), as a target gene whose mutation renders the T-cells sensitive to NK cell killing.

In some embodiments, there is provided a method of identifying a target gene that modulates the activity of a T cell (e.g., an allogeneic T cell or a CAR-T cell (e.g., an allogeneic CAR-T cell)) in the T cell, comprising: a) providing a T cell library comprising a plurality of T cells, wherein each of the plurality of T cells has a mutation (e.g., an inactivating mutation) at a hit gene in the genome, wherein the hit genes in at least two of the plurality of T cells are different from each other, wherein the T cell library is generated by contacting an initial population of T cells with: i) a sgRNA library comprising a plurality of sgRNA constructs, wherein each sgRNA construct comprises or encodes a sgRNA, and wherein each sgRNA comprises a guide sequence complementary to a target site of a corresponding hit gene; and ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein under conditions that allow introduction of the sgRNA construct and the Cas component into the initial population of T cells and generation of mutations at the hits; b) treating the T cell library with NK cells; c) obtaining T cells (or a treated population of T cells) from a T cell library that are susceptible to or resistant to NK cell killing; and d) identifying the target gene based on the difference between the sgRNA or the mutated characteristics of the hit gene in the T cells (or the treated T cell population) obtained from step c) and the control T cells (or the control T cell population). In some embodiments, the sgRNA library and the Cas component are introduced sequentially into the initial T cell population. In some embodiments, the control T cell (or control T cell population) is obtained from the same T cell library cultured under the same conditions without NK cell treatment, and optionally subjected to the same obtaining method in step c). In some embodiments, treating the T cell library with NK cells comprises culturing the T cell library in the presence of NK cells. In some embodiments, the characteristics of the sgRNA or hit gene mutations in the T cells (or treated T cell population) obtained from step c) and the control T cells (or control T cell population) are identified by next generation sequencing. In some embodiments, the T cell library is subjected to two or more (e.g., 2, 3, 4 or more) separate different NK cell treatments and/or obtaining methods in steps b) and c), and the target gene is identified based on the difference between the characteristics of each treatment. In some embodiments, sequence counts comprising sgRNA or hit gene mutations are subjected to median ratio normalization followed by mean-variance modeling. In some embodiments, the method comprises: comparing the sequence count comprising the sgRNA or hit mutations obtained from the T cells (or treated T cell population) obtained from step c) with the sequence count comprising the sgRNA or hit mutations obtained from a control T cell (or control T cell population), wherein i) in at least one NK cell treatment with FDR ≦ 0.01, or in at least two separate different NK cell treatments with FDR ≦ 0.05, whose corresponding sgRNA guide sequence or mutation is identified as depleted (and/or having at least about 2-fold depletion) in the T cells (or treated T cell population) obtained from step c) that are resistant to NK cell killing, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more depletion) of the target gene, identified as a target gene whose mutation sensitizes the T cell to NK cell killing; ii) in at least one NK cell treatment with FDR ≦ 0.05, or in at least two separate different NK cell treatments with FDR ≦ 0.15, the respective sgRNA guide sequences or mutations thereof are identified as hit genes that are enriched (and/or have at least about 2-fold enrichment, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more enrichment) in the T cells (or treated T cell population) obtained from step c) that are resistant to NK cell killing, as target genes whose mutations render the T cells resistant to NK cell killing; iii) in at least one NK cell treatment with FDR ≦ 0.05, or in at least two separate different NK cell treatments with FDR ≦ 0.15, whose respective sgRNA guide sequences or mutations were identified as hits that are depleted (and/or have at least about 2-fold depletion, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more depletion) in the T-cells (or treated T-cell population) sensitive to NK cell killing obtained from step c), as target genes whose mutations render the T-cells resistant to NK cell killing; and/or iv) in at least one NK cell treatment with FDR ≦ 0.01, or in at least two separate different NK cell treatments with FDR ≦ 0.05, the corresponding sgRNA guide sequences or mutations thereof are identified as hits enriched (and/or having at least about 2-fold enrichment, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more enrichment) in the T cells sensitive to NK cell killing obtained from step c) (or the treated T cell population), as target genes whose mutations render the T cells sensitive to NK cell killing.

In some embodiments, identification of T cells is providedA method of modulating a target gene for the activity of an allogeneic T cell or CAR-T cell (e.g., an allogeneic CAR-T cell)) in said T cell (e.g., comprising: a) Providing a T cell library comprising a plurality of T cells, wherein each of the plurality of T cells has a mutation (e.g., an inactivating mutation) at a hit gene in the genome, wherein the hit genes in at least two of the plurality of T cells are different from each other, wherein the T cell library is generated by contacting an initial population of T cells with: i) comprising multiple sets of sgRNAs ^iBAR sgRNA of constructs ^iBAR Library, wherein each group of sgRNAs ^iBAR The construct comprises three or more (e.g., 4) sgrnas ^iBAR Constructs, each construct comprising or encoding a sgRNA ^iBAR Wherein each sgRNA ^iBAR Comprising a guide sequence complementary to a target site in a corresponding hit gene, three or more (e.g., 4) of the sgRNAs ^iBAR The leader sequences of the constructs are identical, with three or more (e.g., 4) sgrnas ^iBAR The iBAR sequence of each of the constructs is different from each other, and wherein each group of sgrnas ^iBAR The leader sequence of the construct is complementary to a different target sequence in the hit gene; and ii) allowing the sgRNA to bind ^iBAR A Cas component comprising a Cas protein (e.g., Cas9) or a nucleic acid encoding the Cas protein, under conditions in which the construct and Cas component are introduced into an initial population of T cells and mutations are generated; b) treating the T cell library with NK cells; c) obtaining from the T cell library T cells (or a T cell population of the treated T cell population) that are sensitive or resistant to NK cell killing; and d) identifying the target gene based on the difference between the sgRNA or hit gene mutation signature in the T cell (or treated T cell population) obtained from step c) and the control T cell (or control T cell population). In some embodiments, the sgRNA is used to generate sgRNA ^iBAR The library and Cas component are introduced sequentially into the initial T cell population. In some embodiments, each sgRNA ^iBAR The iBAR sequence of (a) is inserted into the loop region of the repeat-trans-repeat stem loop. In some embodiments, the control T cell (or control T cell population) is obtained from the same T cell library cultured under the same conditions without NK cell treatment, and optionally subjected to the same obtaining method in step c). At a certain levelIn embodiments, treating the T cell library with NK cells comprises culturing the T cell library in the presence of NK cells. In some embodiments, the sgrnas in the T cells (or treated T cell population) obtained from step c) and the control T cells (or control T cell population) ^iBAR Or hit gene mutation, was characterized by next generation sequencing. In some embodiments, the T cell library is subjected to two or more (e.g., 2, 3, 4 or more) separate different NK cell treatments and/or obtaining methods in steps b) and c), and the target gene is identified based on the difference between the characteristics of each treatment. In some embodiments, the pairs comprise sgrnas ^siBAR Or hit the gene mutation sequence count for median ratio normalization, followed by mean-variance modeling. In some embodiments, the sgRNA corresponding to the guide sequence is based on ^iBAR Data consistency between iBAR sequences in the sequence adjusts the variance of each pilot sequence. In some embodiments, the sgrnas corresponding to each guide sequence are determined based on the direction of fold change of each iBAR sequence ^iBAR Data consistency between iBAR sequences in a sequence, wherein the variance of the pilot sequence increases if the fold changes of the iBAR sequences are in different directions relative to each other. In some embodiments, the method comprises: will comprise sgRNAs obtained from the T cells (or treated T cell population) obtained from step c) ^iBAR Or hit gene mutation and a sequence count comprising sgrnas obtained from a control T cell (or a control T cell population) ^iBAR Or hit the sequence count of the gene mutation for comparison, wherein i) in at least one NK cell treatment with FDR ≦ 0.01, or in at least two separate different NK cell treatments with FDR ≦ 0.05, its corresponding sgRNA ^iBAR The leader sequence or mutation is identified as a hit that is depleted (and/or has at least about 2-fold depletion, such as at least any of about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more depletion) in the T cells (or treated T cell population) obtained from step c) that are resistant to NK cell killing, as a target gene whose mutation renders the T cells susceptible to NK cell killing; ii) in at least one NK cell treatment with FDR ≦ 0.05, or at least two separate treatments with FDR ≦ 0.15In different NK cell processing of (1), its corresponding sgRNA ^iBAR The leader sequence or mutation is identified as a hit gene that is enriched in (and/or has at least about a 2-fold enrichment, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more enrichment) the T cells obtained from step c) that are resistant to NK cell killing (or the treated population of T cells), as a target gene whose mutation renders the T cells resistant to NK cell killing; iii) in at least one NK cell treatment with FDR ≦ 0.05, or in at least two separate different NK cell treatments with FDR ≦ 0.15, their corresponding sgRNAs ^iBAR The leader sequence or mutation is identified as a hit gene depleted (and/or having at least about 2-fold depletion, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more depletion) in the T cells (or treated T cell population) obtained from step c) that are susceptible to NK cell killing, as a target gene whose mutation renders the T cells resistant to NK cell killing; and/or iv) in at least one NK cell treatment with FDR ≦ 0.01, or in at least two separate different NK cell treatments with FDR ≦ 0.05, their corresponding sgRNAs ^iBAR The leader sequence or mutation is identified as a hit gene that is enriched in (and/or has at least about a 2-fold enrichment, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more enrichment) the T cells (or treated T cell population) obtained from step c) that are susceptible to NK cell killing, and as a target gene whose mutation renders the T cells susceptible to NK cell killing. In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., an allogeneic T cell or CAR-T cell (e.g., an allogeneic CAR-T cell)) that modulates T cell activity, comprising: a) providing a T cell library comprising sgrnas described herein that target one or more hits in the genome ^iBAR A library; b) treating the T cell library with NK cells; c) obtaining T cells from a T cell library that are resistant to NK cell killing; and d) identifying the hit gene in the T cell obtained in step c), thereby identifying a target gene in the T cell that modulates T cell activity; wherein steps b) and c) comprise: i) an initial processing step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 0.5:1Contact for about 72 hours; ii) an enrichment step comprising sorting as T cells (e.g., B2M negative (or defective), or CD3 ⁺ ) And a mixture of viable treated cells, thereby obtaining a first T cell subpopulation that is resistant to NK cell killing; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; iv) a second treatment step comprising contacting the recovered first T cell subset with NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3: 1; and v) a sorting step, including sorting as a T cell (e.g., B2M negative (or defective), or CD3 ⁺ ) And a final mixture of viable treated cells, thereby obtaining a second T cell subpopulation of cells that are resistant to NK killing. In some embodiments, identifying hits in the T cells obtained from step c) comprises: i) identifying sgRNAs in T cells obtained from step c) ^iBAR A sequence; and ii) identification of sgRNA ^iBAR The leader sequence of (a) corresponds to the hit gene. In some embodiments, there is provided a method of identifying a target gene in a T cell that modulates T cell activity, comprising: a) providing a T cell library comprising sgrnas described herein that target one or more hits in the genome ^iBAR A library; b) treating the T cell library with NK cells; c) obtaining T cells from a T cell library that are resistant to NK cell killing; wherein steps b) and c) comprise: i) an initial processing step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 0.5:1 for about 72 hours; ii) an enrichment step comprising sorting as T cells (e.g., B2M negative (or defective), or CD3 ⁺ ) And a mixture of viable treated cells, thereby obtaining a first subpopulation of T cells that are resistant to NK cell killing; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; iv) a second treatment step comprising contacting the restored first T cell subset with NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3: 1; and v) a sorting step comprising sorting as a T cell (e.g., B2M negative (or defective), or CD3 ⁺ ) And a final mixture of viable treated cells, thereby obtaining cells of a second T cell subpopulation that are resistant to NK killing; and d) identifying target genes in T cells that modulate T cell activityWherein identifying the target gene comprises: i) obtaining sgRNAs in the final T cell subpopulation resulting from step c) ^iBAR A sequence; ii) sequence count based on sgRNA ^iBAR Sequencing respective guide sequences of the sequences, wherein the sequencing comprises sequencing based on sgRNA corresponding to the guide sequences ^iBAR Data consistency between iBAR sequences in the sequence adjusts the ordering of each pilot sequence; and iii) identifying the hit genes corresponding to the leader sequences ranked above a predetermined threshold level. In some embodiments, the sgRNA ^iBAR The library targets each annotated gene in the genome. In some embodiments, the T cell library is for a whole genome sgRNA ^iBAR The library has an average coverage of at least about 100-fold (e.g., at least about 400-fold). In some embodiments, the method is a positive screen. In some embodiments, the method is a negative screen. In some embodiments, the sequence counts are normalized for median ratio and then modeled for mean-variance. In some embodiments, the targeting sequence is based on sgRNA corresponding to the guide sequence ^iBAR Data consistency between iBAR sequences in the sequence, the variance of each pilot sequence is adjusted. In some embodiments, the sgrnas corresponding to each guide sequence are determined based on the direction of fold change of each iBAR sequence ^iBAR Data consistency between iBAR sequences in a sequence, wherein the variance of the pilot sequence increases if the fold changes of the iBAR sequences are in different directions relative to each other (e.g., increasing versus decreasing, increasing versus constant, or decreasing versus constant). In some embodiments, the sequence counts obtained from the final T cell subpopulation obtained in step c) are compared to corresponding sequence counts obtained from a control T cell subpopulation to provide a fold change (e.g., an actual fold change, or a derivative of a fold change, such as a log2 or log10 fold change). In some embodiments, the control T cell subpopulation is obtained from the same T cell library cultured under the same conditions and not subjected to NK cell treatment, and optionally subjected to the same obtaining method in step c). In some embodiments, the T cell library further comprises a B2M mutation (e.g., a deactivated B2M mutation). In some embodiments, the B2M mutation (e.g., c) E.g., an inactivated B2M mutation) is generated by contacting a T cell library or an initial population of T cells used to generate a T cell library with a B2M sgRNA construct (e.g., a viral vector or virus) as described herein. In some embodiments, treating the T cell library with NK cells in step b) comprises culturing the T cell library in the presence of NK cells.

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., an allogeneic T cell or CAR-T cell (e.g., an allogeneic CAR-T cell)) that modulates T cell activity, comprising: a) providing a T cell library comprising sgrnas described herein that target one or more hits in the genome ^iBAR A library; b) treating the T cell library with NK cells; c) obtaining T cells from a T cell library that are resistant to NK cell killing; and d) identifying the hit gene in the T cell obtained in step c), thereby identifying a target gene in the T cell that modulates T cell activity; wherein steps b) and c) comprise: i) a treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 0.5:1 for about 10 days; and ii) a sorting step comprising sorting as T cells (e.g., B2M negative (or defective), or CD3 ⁺ ) And a mixture of viable treated cells, thereby obtaining a subpopulation of T cells that is resistant to NK cell killing. In some embodiments, identifying hits in the T cells obtained from step c) comprises: i) identifying sgRNAs in T cells obtained from step c) ^iBAR Sequencing; and ii) identification and sgRNA ^iBAR The targeting sequence of (2) corresponds to the hit gene. In some embodiments, there is provided a method of identifying a target gene in a T cell that modulates T cell activity, comprising: a) providing a T cell library comprising sgrnas described herein that target one or more hits in the genome ^iBAR A library; b) treating the T cell library with NK cells; c) obtaining T cells from a T cell library that are resistant to NK cell killing; wherein steps b) and c) comprise: i) a treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 0.5:1 for about 10 days; and ii) a sorting step comprising sorting as T cells (e.g., B2M negative (or defective), or CD3 ⁺ ) And the living channel(ii) a mixture of keratinocytes, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing; and d) identifying a target gene in the T cell that modulates T cell activity, wherein identifying the target gene comprises: i) obtaining sgRNAs in the final T cell subpopulation resulting from step c) ^iBAR A sequence; ii) sequence count based on sgRNA ^iBAR Sequencing respective guide sequences of the sequences, wherein the sequencing comprises sequencing based on sgRNA corresponding to the guide sequences ^iBAR Data consistency between iBAR sequences in the sequence adjusts the ordering of each pilot sequence; and iii) identifying the hit genes corresponding to the leader sequences ranked above a predetermined threshold level. In some embodiments, the sgRNA ^iBAR The library targets each annotated gene in the genome. In some embodiments, the T cell library is for a whole genome sgRNA ^iBAR The library has an average coverage of at least about 100-fold (e.g., at least about 400-fold). In some embodiments, the method is a positive screen. In some embodiments, the method is a negative screen. In some embodiments, the sequence counts are normalized for median ratio and then modeled for mean-variance. In some embodiments, the targeting sequence is based on sgRNA corresponding to the guide sequence ^iBAR Data consistency between iBAR sequences in the sequence, adjusting the variance of each pilot sequence. In some embodiments, the sgrnas corresponding to each guide sequence are determined based on the direction of fold change of each iBAR sequence ^iBAR Data consistency between iBAR sequences in a sequence, wherein the variance of the pilot sequence increases if the fold changes of the iBAR sequences are in different directions relative to each other (e.g., increasing versus decreasing, increasing versus invariant, or decreasing versus invariant). In some embodiments, the sequence counts obtained from the final T cell subpopulation obtained in step c) are compared to corresponding sequence counts obtained from a control T cell subpopulation to provide a fold change (e.g., an actual fold change, or a derivative of a fold change, such as a log2 or log10 fold change). In some embodiments, the control T cell subpopulation is obtained from the same T cell library cultured under the same conditions and not subjected to NK cell treatment, and optionally subjected to the same acquirer in step c)The method is carried out. In some embodiments, the T cell library further comprises a B2M mutation (e.g., a deactivated B2M mutation). In some embodiments, the B2M mutation (e.g., an inactivated B2M mutation) is generated by contacting a T cell library or an initial population of T cells used to generate a T cell library with a B2M sgRNA construct (e.g., a viral vector or virus) described herein. In some embodiments, treating the T cell library with NK cells in step b) comprises culturing the T cell library in the presence of NK cells.

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., an allogeneic T cell or CAR-T cell (e.g., an allogeneic CAR-T cell)) that modulates T cell activity, comprising: a) providing a T cell library comprising sgrnas described herein that target one or more hits in the genome ^iBAR A library; b) treating the T cell library with NK cells; c) obtaining T cells from a T cell library that are resistant to NK cell killing; and d) identifying the hit gene in the T cell obtained in step c), thereby identifying a target gene in the T cell that modulates T cell activity; wherein steps b) and c) comprise: i) a treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 1:1 for about 48 hours; ii) a sorting step comprising sorting as T cells (e.g., B2M negative (or defective), or CD3 ⁺ ) And a mixture of viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing; and iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours prior to harvesting the cells. In some embodiments, identifying hits in the T cells obtained from step c) comprises: i) identifying sgRNAs in T cells obtained from step c) ^iBAR Sequencing; and ii) identification and sgRNA ^iBAR The leader sequence of (a) corresponds to the hit gene. In some embodiments, there is provided a method of identifying a target gene in a T cell that modulates T cell activity, comprising: a) providing a T cell library comprising sgrnas described herein that target one or more hits in the genome ^iBAR A library; b) treating the T cell library with NK cells; c) obtaining T cells from a T cell library that are resistant to NK cell killing; wherein steps b) and c) comprise: i) treatment ofA step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 1:1 for about 48 hours; ii) a sorting step comprising sorting as T cells (e.g., B2M negative (or defective), or CD3 ⁺ ) And a mixture of viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing; and iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours prior to harvesting the cells; and d) identifying a target gene in the T cell that modulates T cell activity, wherein identifying the target gene comprises: i) obtaining sgRNAs in the final T cell subpopulation resulting from step c) ^iBAR A sequence; ii) sequence count based on sgRNA ^iBAR Ordering the respective guide sequences of the sequences, wherein the ordering comprises based on the sgRNA corresponding to the guide sequence ^iBAR Data consistency between iBAR sequences in the sequence adjusts the ordering of each pilot sequence; and iii) identifying the hit genes corresponding to the leader sequences ranked above a predetermined threshold level. In some embodiments, the sgRNA ^iBAR The library targets each annotated gene in the genome. In some embodiments, the T cell library is for a whole genome sgRNA ^iBAR The library has an average coverage of at least about 100-fold (e.g., at least about 400-fold). In some embodiments, the method is a positive screen. In some embodiments, the method is a negative screen. In some embodiments, the sequence counts are normalized for median ratio, followed by mean-variance modeling. In some embodiments, the targeting sequence is based on sgRNA corresponding to the guide sequence ^iBAR Data consistency between iBAR sequences in the sequence, the variance of each guide sequence is adjusted. In some embodiments, the sgrnas corresponding to each guide sequence are determined based on the direction of fold change of each iBAR sequence ^iBAR Data consistency between iBAR sequences in a sequence, wherein the variance of the pilot sequence increases if the fold changes of the iBAR sequences are in different directions relative to each other (e.g., increasing versus decreasing, increasing versus invariant, or decreasing versus invariant). In some embodiments, the sequence count obtained from the final T cell subpopulation obtained in step c) is compared to the corresponding sequence count obtained from a control T cell subpopulation To provide fold changes (e.g., actual fold changes, or derivatives of fold changes, such as log2 or log10 fold changes). In some embodiments, the control T cell subpopulation is obtained from the same T cell library cultured under the same conditions and not subjected to NK cell treatment, and optionally subjected to the same obtaining method in step c). In some embodiments, the T cell library further comprises a B2M mutation (e.g., an inactivated B2M mutation). In some embodiments, the B2M mutation (e.g., an inactivated B2M mutation) is generated by contacting a T cell library or an initial population of T cells used to generate a T cell library with a B2M sgRNA construct (e.g., a viral vector or virus) described herein. In some embodiments, treating the T cell library with NK cells in step b) comprises culturing the T cell library in the presence of NK cells.

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., an allogeneic T cell or CAR-T cell (e.g., an allogeneic CAR-T cell)) that modulates T cell activity, comprising: a) providing a T cell library comprising sgrnas described herein that target one or more hits in the genome ^iBAR A library; b) treating the T cell library with NK cells; c) obtaining T cells from a T cell library that are resistant to NK cell killing; and identifying the hit gene in the T cell obtained in step c), thereby identifying a target gene in the T cell that modulates T cell activity; wherein steps b) and c) comprise: i) a treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 1:1 for about 48 hours; ii) an enrichment step comprising sorting as T cells (e.g., B2M negative (or defective), or CD3 ⁺ ) And a mixture of viable treated cells, thereby obtaining a first subpopulation of T cells that are resistant to NK cell killing; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and) a sorting step comprising sorting as a T cell (e.g., B2M negative (or defective), or CD3 ⁺ ) And surviving the recovered first T cell subset, thereby obtaining a second T cell subset NK cells that are resistant to NK cell killing. In some embodiments, identifying hits in the T cells obtained from step c) comprises: i) identification fromsgRNA in T cells obtained in step c) ^iBAR A sequence; and ii) identification of sgRNA ^iBAR The targeting sequence of (2) corresponds to the hit gene. In some embodiments, there is provided a method of identifying a target gene in a T cell that modulates T cell activity, comprising: a) providing a T cell library comprising sgrnas described herein that target one or more hits in the genome ^iBAR A library; b) treating the T cell library with NK cells; c) obtaining T cells from a T cell library that are resistant to NK cell killing; wherein steps b) and c) comprise: i) a treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 1:1 for about 48 hours; ii) an enrichment step comprising sorting as T cells (e.g., B2M negative (or defective), or CD3 ⁺ ) And a mixture of viable treated cells, thereby obtaining a first T cell subpopulation that is resistant to NK cell killing; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and iv) a sorting step comprising sorting as a T cell (e.g., B2M negative (or defective), or CD3 ⁺ ) And surviving the recovered first subpopulation of T cells, thereby obtaining second subpopulation of T cells NK cells that are resistant to NK cell killing; and d) identifying a target gene in the T cell that modulates T cell activity, wherein identifying the target gene comprises: i) obtaining sgRNAs in the final T cell subpopulation resulting from step c) ^iBAR A sequence; ii) sequence count based on sgRNA ^iBAR Ordering the respective guide sequences of the sequences, wherein the ordering comprises based on the sgRNA corresponding to the guide sequence ^iBAR Data consistency between iBAR sequences in the sequence adjusts the ordering of each pilot sequence; and iii) identifying the hit genes corresponding to the leader sequences that are ranked above a predetermined threshold level. In some embodiments, the sgRNA ^iBAR The library targets each annotated gene in the genome. In some embodiments, the T cell library is for a whole genome sgRNA ^iBAR The library has an average coverage of at least about 100-fold (at least about 400-fold). In some embodiments, the method is a positive screen. In some embodiments, the method is a negative screen. In some embodiments, the sequence counts are normalized by median ratio, howeverAnd then performing mean-variance modeling. In some embodiments, the targeting sequence is based on sgRNA corresponding to the guide sequence ^iBAR Data consistency between iBAR sequences in the sequence, adjusting the variance of each pilot sequence. In some embodiments, the sgrnas corresponding to each guide sequence are determined based on the direction of fold change of each iBAR sequence ^iBAR Data consistency between iBAR sequences in a sequence, wherein the variance of the pilot sequence increases if the fold changes of the iBAR sequences are in different directions relative to each other (e.g., increasing versus decreasing, increasing versus invariant, or decreasing versus invariant). In some embodiments, the sequence counts obtained from the final T cell subpopulation obtained in step c) are compared to corresponding sequence counts obtained from a control T cell subpopulation to provide a fold change (e.g., an actual fold change, or a derivative of a fold change, such as a log2 or log10 fold change). In some embodiments, the control T cell subpopulation is obtained from the same T cell library cultured under the same conditions and not subjected to NK cell treatment, and optionally subjected to the same obtaining method in step c). In some embodiments, the T cell library further comprises a B2M mutation (e.g., an inactivated B2M mutation). In some embodiments, the B2M mutation (e.g., an inactivated B2M mutation) is generated by contacting a T cell library or an initial population of T cells used to generate a T cell library with a B2M sgRNA construct (e.g., a viral vector or virus) described herein. In some embodiments, treating the T cell library with NK cells in step b) comprises culturing the T cell library in the presence of NK cells.

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., an allogeneic T cell or CAR-T cell (e.g., an allogeneic CAR-T cell)) that modulates T cell activity, comprising: a) providing a T cell library comprising sgrnas described herein that target one or more hits in the genome ^iBAR A library; b) subjecting a T cell library to at least two different treatments with NK cells as described herein; c) obtaining from a T cell library T cells (or T cell subsets) sensitive or resistant to NK cell killing per treatment of step b); and d) identifying regulatory T cell activity in T cellsA sexual target gene; wherein identifying the target gene comprises: i) obtaining sgRNA in the T cells (or T cell subpopulations) obtained in step c) for each NK cell treatment ^iBAR A sequence; ii) sequence count to sgRNA based on each NK cell treatment ^iBAR Ordering respective ones of the sequences, wherein the ordering comprises ordering based on sgRNAs corresponding to the guide sequences ^iBAR Data consistency between iBAR sequences in the sequence adjusts the ordering of each pilot sequence; and iii) identifying for each NK cell processing hits corresponding to leader sequences that are ranked above a predetermined threshold level; wherein (1) hits identified as depleted from (and/or having at least about 2-fold depletion, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more depletion) the final T cell subpopulation (from step c) in at least one NK cell treatment at FDR ≦ 0.01, or in at least two different respective NK cell treatments at FDR ≦ 0.05, are identified as target genes whose mutation (e.g., inactivation) renders the T cells susceptible to NK cell killing; (2) hits identified as enriched from a final T cell subpopulation (from step c) that is resistant to NK cell killing in at least one NK cell treatment with an FDR ≦ 0.05 or in at least two separate distinct NK cell treatments with an FDR ≦ 0.15 (and/or with at least about 2-fold enrichment, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more enrichment), are identified as target genes whose mutation (e.g., inactivation) renders the T cells resistant to NK cell killing; (3) hits identified as depleted (and/or having at least about 2-fold depletion, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more depletion) from the final T cell subpopulation (from step c) that is susceptible to NK cell killing in at least one NK cell treatment with an FDR ≦ 0.05 or in at least two separate different NK cell treatments with an FDR ≦ 0.15 are identified as target genes whose mutation (e.g., inactivation) renders the T cells resistant to NK cell killing; and/or (4) is enriched from the final T cell subpopulation (from step c) identified as being sensitive to NK cell killing in at least one NK cell treatment with FDR ≦ 0.01, or in at least two different respective NK cell treatments with FDR ≦ 0.05 (and/or has at least one About 2-fold enrichment, such as at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more of any of the enrichment) of the hit gene, is identified as its mutation (e.g., inactivation) makes T cells sensitive to NK cell killing of the target gene. In some embodiments, the sgRNA ^iBAR The library targets each annotated gene in the genome. In some embodiments, the T cell library is for a whole genome sgRNA ^iBAR The library has an average coverage of at least about 100-fold (at least about 400-fold). In some embodiments, the method is a positive screen. In some embodiments, the method is a negative screen. In some embodiments, the sequence counts are normalized for median ratio and then modeled for mean-variance. In some embodiments, the targeting sequence is based on sgRNA corresponding to the guide sequence ^iBAR Data consistency between iBAR sequences in the sequence, adjusting the variance of each pilot sequence. In some embodiments, the sgrnas corresponding to each guide sequence are determined based on the direction of fold change of each iBAR sequence ^iBAR Data consistency between iBAR sequences in a sequence, wherein the variance of the pilot sequence increases if the multiple changes of the iBAR sequences are in different directions relative to each other (e.g., increasing versus decreasing, increasing versus constant, or decreasing versus constant). In some embodiments, the sequence counts obtained from the final T cell subpopulation obtained for each NK cell treatment of step c) are compared to the corresponding sequence counts obtained from the control T cell subpopulation to provide a fold change (e.g., an actual fold change, or a derivative of a fold change, such as a log2 or log10 fold change). In some embodiments, the control T cell subpopulation is obtained from the same T cell library cultured under the same conditions and without the corresponding NK cell treatment in step b), and optionally subjected to the same corresponding obtaining method as in step c). In some embodiments, the T cell library further comprises a B2M mutation (e.g., an inactivated B2M mutation). In some embodiments, the B2M mutation (e.g., an inactivated B2M mutation) is produced by contacting a T cell library or an initial population of T cells used to generate a T cell library with a B2M sgRNA construct (e.g., a viral vector or virus) described herein And (4) producing. In some embodiments, treating the T cell library with NK cells in step b) comprises culturing the T cell library in the presence of NK cells.

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., an allogeneic T cell or CAR-T cell (e.g., an allogeneic CAR-T cell)) that modulates T cell activity, comprising: a) providing a T cell library comprising sgrnas described herein that target one or more hits in the genome ^iBAR A library; b-c) subjecting the T cell library to at least two of 4 separate NK cell treatment assays (e.g., killing), thereby obtaining from the T cell library T cells that are resistant to NK cell killing from each assay; and d) identifying hits in the T cells obtained from each of the assays of steps b-c), thereby identifying target genes in the T cells that modulate T cell activity; wherein the 4 trials are: (I) test I: i) an initial processing step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 0.5:1 for about 72 hours; ii) an enrichment step comprising sorting a mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a first T cell sub-population that is resistant to NK cell killing; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; iv) a second treatment step comprising contacting the recovered first T cell subset with NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3: 1; and v) a sorting step comprising sorting the final mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a second T cell sub-population that is resistant to NK cell killing; (II) run II: i) a treatment step comprising contacting the T cell library with NK cells for about 10 days at a ratio of NK cells to T cells of about 0.5: 1; and ii) a sorting step comprising sorting as T cells (e.g., B2M negative (or defective), or CD3 ⁺ ) And a mixture of viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing; (III) run III: i) a treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 1:1 for about 48 hours; ii) a sorting step comprising sorting as T cells (e.g., B2M negative (or defective), or CD3 ⁺ ) And a mixture of viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing; and iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours prior to harvesting the cells; and (IV) test IV: i) a treatment step comprising contacting a T cell library with NK cells for about 48 hours at a ratio of NK cells to T cells of about 1: 1; ii) an enrichment step comprising sorting as T cells (e.g., B2M negative (or defective), or CD3 ⁺ ) And a mixture of viable treated cells, thereby obtaining a first subpopulation of T cells that are resistant to NK cell killing; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and iv) a sorting step comprising sorting as T cells (e.g., B2M negative (or defective), or CD3 ⁺ ) And surviving the recovered first subpopulation of T cells, thereby obtaining a second subpopulation of T cells that are resistant to NK cell killing. In some embodiments, identifying hits in the T cells obtained from step c) comprises: i) identifying sgRNA in T cells obtained from step c) ^iBAR Sequencing; and ii) identification and sgRNA ^iBAR The targeting sequence of (2) corresponds to the hit gene. In some embodiments, identifying the target gene comprises identifying hits from at least two of the 4 separate trials, wherein: i) hits identified as depleted from (and/or having at least about 2-fold depletion, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more depletion) in at least one NK cell treatment with FDR ≦ 0.01, or in at least two assays with FDR ≦ 0.05 (e.g., any of FDR ≦ 0.04, 0.03, 0.02, 0.01, 0.005, 0.001, or less), are identified as target genes whose mutation (e.g., inactivation) sensitizes T cells to NK cell killing; and/or ii) hits identified as enriched from (and/or having at least about 2-fold enrichment, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more enrichment) a final T cell subpopulation in at least one trial having FDR ≦ 0.05 or at least two trials having FDR ≦ 0.15 (e.g., any of FDR ≦ 0.14, 0.13, 0.12, 0.11, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, or less), are identified as having mutations (e.g., Inactivation) of target genes that render T cells resistant to NK cell killing. In some embodiments, the sgRNA ^iBAR The library targets each annotated gene in the genome. In some embodiments, the library of T cells is directed to whole genome sgrnas ^iBAR The library has an average coverage of at least about 100-fold (e.g., at least about 400-fold). In some embodiments, the method is a positive screen. In some embodiments, the method is a negative screen. In some embodiments, the sequence counts are normalized for median ratio, and then subjected to mean-variance modeling. In some embodiments, the targeting sequence is based on sgRNA corresponding to the guide sequence ^iBAR Data consistency between iBAR sequences in the sequence, adjusting the variance of each pilot sequence. In some embodiments, the sgrnas corresponding to each guide sequence are determined based on the direction of fold change of each iBAR sequence ^iBAR Data consistency between iBAR sequences in a sequence, wherein the variance of the pilot sequence increases if the fold changes of the iBAR sequences are in different directions relative to each other (e.g., increasing versus decreasing, increasing versus invariant, or decreasing versus invariant). In some embodiments, the sequence counts obtained from the final T cell subpopulation resulting from each assay of steps b-c) are compared to the corresponding sequence counts obtained from the control T cell subpopulation to provide a fold change (e.g., an actual fold change, or a fold change, such as a log2 or log10 fold change). In some embodiments, the control T cell subpopulation is obtained from the same T cell library cultured under the same conditions without NK cell treatment in the corresponding assay of step b-c), and optionally the same acquisition method step b-c) is performed in the corresponding assay. In some embodiments, the T cell library further comprises a B2M mutation (e.g., an inactivated B2M mutation). In some embodiments, the B2M mutation (e.g., an inactivated B2M mutation) is generated by contacting a T cell library or an initial population of T cells used to generate a T cell library with a B2M sgRNA construct (e.g., a viral vector or virus) described herein. In some embodiments, treating the T cell library with NK cells in step b-c) comprises culturing the T cell library in the presence of NK cells.

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., an allogeneic T cell or CAR-T cell (e.g., an allogeneic CAR-T cell)) that modulates T cell activity, comprising: a) providing a T cell library comprising sgrnas described herein that target one or more hits in the genome ^iBAR A library; b-c) subjecting the T cell library to at least two of 4 separate NK cell treatment assays (e.g., killing), thereby obtaining from the T cell library T cells that are resistant to NK cell killing by each assay; of these 4 trials were: (I) test I: i) an initial processing step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 0.5:1 for about 72 hours; ii) an enrichment step comprising sorting a mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a first T cell subpopulation that is resistant to NK cell killing; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; iv) a second treatment step comprising contacting the restored first T cell subpopulation with NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3: 1; and v) a sorting step comprising sorting the final mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a second T cell subpopulation that is resistant to NK cell killing; (II) run II: i) a treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 0.5:1 for about 10 days; and ii) a sorting step comprising sorting as T cells (e.g., B2M negative (or defective), or CD3 ⁺ ) And a mixture of viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing; (III) run III: i) A treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 1:1 for about 48 hours; ii) a sorting step comprising sorting as T cells (e.g., B2M negative (or defective), or CD3 ⁺ ) And a mixture of viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing; and iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours prior to harvesting the cells; and (IV) test IV: i) a treatment step comprising contacting T cells with a ratio of NK cells to T cells of about 1:1Contacting the pool with NK cells for about 48 hours; ii) an enrichment step comprising sorting as T cells (e.g., B2M negative (or defective), or CD3 ⁺ ) And a mixture of viable treated cells, thereby obtaining a first T cell subpopulation that is resistant to NK cell killing; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and iv) a sorting step comprising sorting as a T cell (e.g., B2M negative (or defective), or CD3 ⁺ ) And surviving the recovered first T cell subset, thereby obtaining NK cells of a second T cell subset resistant to killing; and d) identifying a target gene in the T cell that modulates T cell activity, wherein identifying the target gene comprises: i) obtaining sgRNAs in the final T cell subpopulation obtained from each assay of steps b-c) ^iBAR A sequence; ii) sequence count pairs sgRNA on a per assay basis ^iBAR Ordering respective ones of the sequences, wherein the ordering comprises ordering based on sgRNAs corresponding to the guide sequences ^iBAR Data consistency between iBAR sequences in the sequence adjusts the ordering of each pilot sequence; and iii) identifying the hit genes corresponding to the leader sequences for each trial that are ranked above a predetermined threshold level; wherein (1) in at least one NK cell treatment with FDR ≦ 0.01, hits identified as depleted from (and/or having at least about 2-fold depletion, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more depletion) from a final T cell subpopulation in at least two assays for FDR ≦ 0.05 (e.g., any of FDR ≦ 0.04, 0.03, 0.02, 0.01, 0.005, 0.001, or less) are identified as having a mutation (e.g., inactivation) of a target gene that renders T cells susceptible to NK cell killing; and/or (2) a hit gene identified as enriched from (and/or having at least about 2-fold enrichment, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more enrichment) in the final T cell subpopulation in at least one assay of FDR ≦ 0.05 or at least two assays of FDR ≦ 0.15 (e.g., any of FDR ≦ 0.14, 0.13, 0.12, 0.11, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01 or less), identified as a target gene whose mutation (e.g., inactivation) renders the T cell resistant to NK cell killing. In some embodiments, the composition is prepared by sgRNA ^iBAR The library targets each annotated gene in the genome. In some embodiments, the T cell library is for a whole genome sgRNA ^iBAR The library has an average coverage of at least about 100-fold (e.g., at least about 400-fold). In some embodiments, the method is a positive screen. In some embodiments, the method is a negative screen. In some embodiments, the sequence counts are normalized by median ratio and then modeled as mean-variance. In some embodiments, the targeting sequence is based on sgRNA corresponding to the guide sequence ^iBAR Data consistency between iBAR sequences in the sequence adjusts the variance of each pilot sequence. In some embodiments, the sgrnas corresponding to each guide sequence are determined based on the direction of fold change of each iBAR sequence ^iBAR Data consistency between iBAR sequences in a sequence, wherein the variance of the pilot sequence increases if the fold changes of the iBAR sequences are in different directions relative to each other (e.g., increasing versus decreasing, increasing versus invariant, or decreasing versus invariant). In some embodiments, the sequence counts obtained from the final T cell subpopulation resulting from each assay of steps b-c) are compared to the corresponding sequence counts obtained from the control T cell subpopulation to provide a fold change (e.g., an actual fold change, or a fold change, such as a log2 or log10 fold change). In some embodiments, the control T cell subpopulation is obtained from the same T cell library cultured under the same conditions without NK cell treatment in the corresponding assay of step b-c), and optionally subjected to the same acquisition method in the corresponding assay of step b-c). In some embodiments, the T cell library further comprises a B2M mutation (e.g., an inactivated B2M mutation). In some embodiments, the B2M mutation (e.g., an inactivated B2M mutation) is generated by contacting a T cell library or an initial population of T cells used to generate a T cell library with a B2M sgRNA construct (e.g., a viral vector or virus) described herein. In some embodiments, treating the T cell library with NK cells in step b-c) comprises culturing the T cell library in the presence of NK cells.

In some embodiments, any of the methods of identification described herein further comprises validating the target gene by: a) modifying a T cell (e.g., an allogeneic T cell or CAR-T cell (e.g., an allogeneic CAR-T cell)) by making a mutation (e.g., an inactivating mutation) in a target gene of the T cell; b) determining the sensitivity or resistance of the modified T cells to NK cell killing. In some embodiments, the method further comprises generating a mutation (e.g., an inactivating mutation) in B2M of the T cell.

Also provided are modified T cells (e.g., modified allogeneic T cells, or modified CAR-T cells (such as modified allogeneic CAR-T cells)) obtained by inactivating one or more target genes identified by any of the methods described herein.

^iBAR Single-stranded guide RNA (sgRNA) library and sgRNA library

In some embodiments, the invention uses CRISPR/Cas guide RNAs (e.g., single stranded guide RNAs) and constructs encoding CRISPR/Cas guide RNAs to generate mutations (e.g., inactivating mutations) in one or more hits of a genome. In some embodiments, the mutation is generated by cleavage of the hit (e.g., with CRISPR/Cas 9). In some embodiments, the mutation is generated by modulating (e.g., repressing or reducing) the expression of the hit gene (e.g., with CRISPR/dCas fused to a repressor domain).

In some embodiments, a sgRNA library is provided that includes one or more (e.g., 1, 2, 3, 4, 5, 10, 100, 1,000, 10,000, 20,000, or more) sgRNA constructs, wherein each sgRNA construct (e.g., a lentiviral or lentiviral vector encoding a sgRNA) comprises or encodes a sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary (e.g., at least any one of about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site in a corresponding hit gene. In some embodiments, the sgRNA library comprises a plurality (e.g., 2, 3, 4, 5, 10, 100, 1,000, 10,000, 20,000, or more) of sgRNA constructs, wherein at least two hits that are complementary to the guide sequence are different from each other. In some embodiments, the sgRNA construct comprises (or consists of) a sgRNA. In some embodiments, the sgRNA construct encodes a sgRNA. In some embodiments, the sgRNA construct is a plasmid encoding the sgRNA. In some embodiments, the sgRNA construct is a viral vector (e.g., a lentiviral vector) encoding the sgRNA. In some embodiments, the sgRNA construct is a virus (e.g., a lentivirus) that encodes the sgRNA. In some embodiments, each sgRNA comprises a guide sequence fused to a second sequence, wherein the second sequence comprises a repeat-trans-repeat stem loop that interacts with a Cas protein (e.g., Cas 9). In some embodiments, the second sequence of each sgRNA further comprises stem loop 1, stem loop 2, and/or stem loop 3. In some embodiments, each guide sequence comprises from about 17 to about 23 nucleotides. In some embodiments, the sgRNA library comprises at least about 100 sgRNA constructs, such as at least about 200, 300, 400, 1,000, 1,600, 4,000, 10,000, 15,000, 16,000, 19,000, 20,000, 38,000, 50,000, 100,000, 150,000, 155,000, 200,000, or more sgRNA constructs. In some embodiments, a sgRNA library comprising a plurality of sgRNA constructs, comprising or encoding sgrnas having a guide sequence complementary to a target site of each annotated gene in the genome (hereinafter also referred to as a "whole genome sgRNA library"). In some embodiments, the sgRNA library comprises at least two sgRNA constructs comprising or encoding sgrnas with guide sequences complementary to at least two different target sites of the same hit gene, i.e. the sgRNA library has an average at least two-fold coverage of the hit gene. In some embodiments, the sgRNA library comprises at least two (e.g., 2, 3, 4, 5, or more) sgRNA constructs comprising or encoding sgrnas that have guide sequences complementary to at least two different target sites within the same hit gene of each annotated gene in the genome, i.e., the sgRNA library has an average at least two-fold coverage of the whole genome. In some embodiments, the sgRNA library further comprises one or more (e.g., 1, 2, 3, 4, 5, 10, 100, 1,000, 2,000, 10,000, or more) "negative control sgRNA constructs," wherein each negative control sgRNA construct (e.g., a lentiviral or lentiviral vector encoding a negative control sgRNA) comprises or encodes a negative control sgRNA, and wherein each negative control sgRNA comprises a guide sequence that is complementary to an unrelated sequence that is not in the genome, is complementary to a control gene (e.g., known to respond the same or similar between a test group and a control group upon gene inactivation), or is complementary to a sequence unrelated to any annotated gene in the genome. In some embodiments, the sgRNA library further comprises negative control sgRNA constructs in an amount of about 3% to about 30% of the number of hit gene sgRNA constructs in the sgRNA library. In some embodiments, the sgRNA library further comprises about 1,000 negative control sgRNA constructs.

In some embodiments, the sgRNA further comprises an Internal Barcode (iBAR) sequence (such sgrnas are hereinafter referred to as "sgrnas) ^iBAR "). In some embodiments, the iBAR is located in the sgRNA such that the resulting sgRNA ^iBAR Can be manipulated with a Cas protein (e.g., Cas9) to modify (e.g., cleave or modulate expression) with sgrnas ^iBAR The targeting sequence of (a) is complementary. Thus, in some embodiments, the sgRNA library described herein is a sgRNA ^iBAR A library. In some embodiments, the sgRNA ^iBAR The library comprises one or more (e.g., 1, 2, 3, 4, 5, 10, 100, 1,000, 2,000, 10,000, or more) sgrnas ^iBAR Construct, wherein each sgRNA ^iBAR Constructs comprise or encode sgrnas ^iBAR Wherein each sgRNA ^iBAR Comprises a leader sequence and an iBAR sequence, and wherein each leader sequence is complementary to a target site in a corresponding hit gene of the genome (e.g., at least about any one of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary). In some embodiments, the sgRNA ^iBAR The library comprises a plurality (e.g., 2, 3, 4, 5, 10, 100, 1,000, 10,000, or more) of sgrnas ^iBAR A construct wherein at least two hit genes complementary to the leader sequence are different from each other. In some embodiments, each sgRNA (or sgRNA) ^iBAR ) Comprising a leader sequence fused to a second sequence, wherein the second sequence comprisesContains repeat-trans-repeat stem loops that interact with a Cas protein (e.g., Cas 9). In some embodiments, each sgRNA (or sgRNA) ^iBAR ) Also comprises stem-loop 1, stem-loop 2 and/or stem-loop 3. In some embodiments, each sgRNA ^iBAR The iBAR sequence of (a) is inserted into the loop region of the repeat-trans-repeat stem loop. In some embodiments, each sgRNA ^iBAR Comprising in a 5 'to 3' direction a first stem sequence and a second stem sequence, wherein the first stem sequence hybridizes to the second stem sequence to form a double stranded rna (dsrna) region that interacts with a Cas protein, and wherein the iBAR sequence is located between the 3 'end of the first stem sequence and the 5' end of the second stem sequence. In some embodiments, each sgRNA ^iBAR Comprising in the 5 'to 3' direction: leader sequence, repeat-trans-repeat stem loop, stem loop 1, stem loop 2 and stem loop 3 with insertion of iBAR sequence in the loop region. In some embodiments, compositions comprising multiple sets of sgrnas are provided ^iBAR sgRNA of constructs ^iBAR Library, wherein each group of sgRNAs ^iBAR The construct comprises three or more (e.g., 3, 4, 5 or more, such as 4) sgrnas ^iBAR Constructs (e.g., encoding sgrnas) ^iBAR Lentivirus or lentivirus vector of (a), each comprising or encoding a sgRNA ^iBAR Wherein each sgRNA ^iBAR Comprising a leader sequence and an iBAR sequence, three or more sgRNAs ^iBAR The guide sequences of the constructs are identical, wherein three or more sgrnas ^iBAR The iBAR sequence of each of the constructs is different from each other, and wherein each group of sgrnas ^iBAR The leader sequences of the constructs are complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to different target sites of corresponding hit genes in the genome (e.g., different hit genes, or different sites within the same hit gene). In some embodiments, each set of sgrnas ^iBAR The construct comprises 4 sgrnas ^iBAR Construct, and wherein 4 sgrnas ^iBAR The iBAR sequences of each of the constructs differ from each other. Thus, in some embodiments, a recombinant vector comprising multiple sets of sgrnas is provided ^iBAR sgRNA of constructs ^iBAR Library, wherein each group of sgRNAs ^iBAR The construct comprises 4 sgrnas ^iBAR Constructs, each construct comprising or encoding a sgRNA ^iBAR Wherein each sgRNA ^iBAR Comprises a guide sequence and an iBAR sequence, wherein 4 sgRNAs ^iBAR The guide sequences of the constructs were identical, of which 4 sgrnas ^iBAR The iBAR sequence of each of the constructs is different from each other, and wherein each group of sgrnas ^iBAR The targeting sequences of the constructs are complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to different target sites of corresponding hit genes in the genome (e.g., different hit genes, or different sites within the same hit gene). In some embodiments, the sgRNA ^iBAR The library comprises at least about 100 (e.g., at least about 200, 400, 1,000, 1,200, 1,600, 4,000, 10,000, 15,000, 19,000, 20,000, 38,000, 50,000, 100,000, 150,000, 155,000, 200,000, or more) sgrnas of the set ^iBAR Constructs. In some embodiments, the sgrnas of different groups are different from each other ^iBAR At least two sgRNAs in the construct ^iBAR The iBAR sequences of the constructs are identical (e.g., first and second sets of sgrnas ^iBAR Two sets of sgrnas of constructs ^iBAR With at least 1,2, 3, 4, or more iBAR sequences shared between constructs). In some embodiments, at least two sets of sgrnas ^iBAR The iBAR sequences of the constructs were identical. In some embodiments, a plurality of sets of sgrnas are included ^iBAR sgRNA of constructs ^iBAR The library comprises or encodes sgrnas with guide sequences complementary to target sites of each annotated gene in the genome ^iBAR (hereinafter also referred to as "whole genome sgRNA) ^iBAR Library "). In some embodiments, the sgRNA ^iBAR The library comprises at least two groups (e.g., at least 2, 3, 4, 5, or more groups) of sgrnas ^iBAR A construct comprising or encoding a sgRNA having guide sequences complementary to at least two (e.g., at least 2, 3, 4, 5 or more, such as 2) different target sites of the same hit gene ^iBAR I.e. sgRNA ^iBAR The library has an average of at least two-fold coverage of the hit geneThe capping rate. In some embodiments, for each hit gene, the sgRNA ^iBAR The library contained 2 groups of sgrnas ^iBAR Construct comprising or encoding sgRNAs with guide sequences complementary to 2 different target sites of the same hit gene ^siBAR . In some embodiments, the sgRNA ^iBAR The library comprises at least two groups (e.g., at least 2, 3, 4, 5, or more groups) of sgrnas ^iBAR A construct comprising or encoding a sgRNA having a guide sequence complementary to at least two (e.g., at least 2, 3, 4, 5 or more, such as 2) different target sites within the same hit gene of each annotated gene in the genome ^iBAR I.e. sgRNA ^iBAR The library has an average of at least two-fold coverage of the whole genome. In some embodiments, each guide sequence comprises from about 17 to about 23 nucleotides. In some embodiments, each iBAR sequence comprises from about 1 to about 50 (e.g., about 6) nucleotides. In some embodiments, the sgRNA ^iBAR Constructs comprise sgrnas ^iBAR (or consist of) thereof. In some embodiments, the sgRNA ^iBAR Construct encoding sgRNA ^iBAR . In some embodiments, the sgRNA ^iBAR The construct encodes sgRNA ^iBAR The plasmid of (1). In some embodiments, the sgRNA ^iBAR The construct encodes sgRNA ^iBAR A viral vector (e.g., a lentiviral vector) of (a). In some embodiments, the sgRNA ^iBAR The construct encodes sgRNA ^iBAR A virus (e.g., lentivirus). Set of different sgrnas with different iBAR sequences ^iBAR Constructs can be used in single gene editing and screening experiments to provide duplicate data. In some embodiments, the sgRNA ^iBAR The library also comprises one or more sets of "negative control sgrnas ^iBAR Construct ", wherein each group of negative control sgrnas ^iBAR The construct comprises three or more (e.g., 3, 4, 5 or more, such as 4)) negative control sgrnas ^iBAR Constructs (e.g., encoding negative control sgrnas) ^iBAR A lentivirus or lentivirus vector of (a), each comprising or encoding a negative control sgRNA ^iBAR Wherein each negative control sgRNA ^iBAR Comprising a leader sequence and an iBAR sequence, wherein three or more negative control sgRNAs ^iBAR The guide sequence construct of (a), wherein three or more negative control sgrnas are identical ^iBAR The iBAR sequences of each of the constructs were different from each other, and wherein each group of negative control sgrnas ^iBAR The leader sequence of the construct is complementary to a target site unrelated to any annotated gene in the genome, to a control gene (e.g., known to respond identically or similarly between test and control groups upon gene inactivation), or to an unrelated sequence not in the genome. In some embodiments, the sgRNA ^iBAR The library also comprises a consensus sgRNA ^iBAR Library hit gene sgRNA ^iBAR Negative control sgRNA in an amount of about 3% to about 30% of the number of constructs ^iBAR And constructing a body. In some embodiments, the sgRNA ^iBAR The library also contained about 1,000 negative control sgrnas ^iBAR Constructs.

In some embodiments, constructs comprising one or more sgrnas (e.g., sgrnas) are provided ^iBAR Construct) of sgRNA library (e.g., sgRNA ^iBAR Library), wherein each sgRNA construct (e.g., a lentivirus or lentiviral vector encoding the sgRNA) comprises or encodes a sgRNA (e.g., a sgRNA) ^iBAR ) And wherein each sgRNA (e.g., sgRNA) ^iBAR ) Comprising a guide sequence that is complementary (e.g., at least any one of about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site in a target gene selected from the group consisting of: TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34 and PACS 2. In some embodiments, the sgRNA library further comprises a sgRNA construct (e.g., a lentiviral or lentiviral vector encoding the sgRNA) comprising or encoding a sgRNA whose guide sequence is complementary to a target site in B2M.

In some embodiments, a recombinant vector comprising multiple sets of sgrnas is provided ^iBAR sgRNA of constructs ^iBAR Library, wherein each group of sgrnas ^iBAR The construct comprises three or more (e.g., 3, 4, 5 or more, such as 4) sgrnas ^iBAR Constructs (e.g., encoding the sgrnas ^iBAR Lentivirus or lentivirus vector of (a), each comprising or encoding a sgRNA ^iBAR Wherein each sgRNA ^iBAR Comprising a leader sequence and an iBAR sequence, three or more sgRNAs ^iBAR The leader sequences of the constructs are identical, with three or more sgrnas ^iBAR The iBAR sequences of each of the constructs are different from each other, with each set of sgrnas ^iBAR The guide sequences of the constructs are complementary (e.g., at least about any one of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to different target sites in corresponding hit genes in the genome (e.g., different hit genes, or different sites within the same hit gene), and wherein each sgRNA is complementary ^iBAR Can be manipulated with Cas9 protein to modify the target site. In some embodiments, provided are compositions comprising multiple sets of sgrnas ^iBAR sgRNA of constructs ^iBAR Library, wherein each set of sgRNAs ^iBAR The construct comprises 4 sgrnas ^iBAR Constructs, each construct comprising or encoding a sgRNA ^iBAR Wherein each sgRNA ^iBAR Comprises a guide sequence and an iBAR sequence, wherein 4 sgRNAs ^iBAR The guide sequences of the constructs were identical, of which 4 sgrnas ^iBAR The iBAR sequences of each of the constructs are different from each other, with each set of sgrnas ^iBAR The guide sequences of the constructs are complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to different target sites in corresponding hit genes in the genome (e.g., different hit genes, or different sites within the same hit gene), and wherein each sgRNA is complementary ^iBAR Cas9 protein can be manipulated to modify target sites. In some embodiments, each sgRNA ^iBAR The sequence comprises a guide sequence fused to a second sequence, wherein the second sequence comprises a repeat-trans-repeat stem loop that interacts with Cas 9. In some embodiments, each sgRNA ^iBAR The second sequence of the sequence further comprises stem loop 1, stem loop 2 and/or stem loop 3. In some embodiments, the iBAR sequence is inserted into a loop region of a repeat-trans-repeat stem loop, and/or a loop region of stem loop 1, stem loop 2, or stem loop 3. In some embodiments, each iBAR sequence comprises about 1-50 (e.g., about 6) nucleotides. In some embodiments, each sgRNA ^iBAR The construct is an RNA, a plasmid, a viral vector (e.g., a lentiviral vector), or a virus (e.g., a lentivirus). In some embodiments, the sgRNA ^iBAR The library comprises at least about 100 (e.g., at least about 200, 400, 1,000, 1,200, 1,600, 4,000, 10,000, 15,000, 19,000, 20,000, 38,000, 50,000, 100,000, 150,000, 155,000, 200,000, or more) sgrnas of the group ^iBAR Constructs. In some embodiments, the sgrnas of different groups are different from each other ^iBAR At least two sgRNAs in the construct ^iBAR The iBAR sequences of the constructs are identical (e.g., first and second sets of sgrnas ^iBAR Two sets of sgrnas of the construct ^iBAR Having at least 1,2, 3, 4, or more shared iBAR sequences between constructs). In some embodiments, at least two sets of sgrnas ^iBAR The iBAR sequences of the constructs were identical. In some embodiments, a plurality of sets of sgrnas are included ^iBAR sgRNA of constructs ^iBAR Library comprising or encoding sgRNAs with guide sequences complementary to target sites of each annotated gene in the genome ^iBAR . In some embodiments, the sgRNA ^iBAR The library comprises at least two groups (e.g., 2, 3, 4, 5, or more groups) of sgrnas ^iBAR Constructs comprising or encoding sgrnas with guide sequences complementary to at least two (e.g., 2, 3, 4, 5 or more, such as 2) different target sites within the same hit gene of each annotated gene in the genome ^iBAR . In some embodiments, each guide sequence comprises from about 17 to about 23 nucleotides.

In some embodiments, a recombinant vector comprising multiple sets of sgrnas is provided ^iBAR sgRNA of constructs ^iBAR Library, wherein each group of sgrnas ^iBAR The construct comprises three or more (e.g., 3, 4, 5 or more, such as 4) sgRNA ^iBAR Constructs, each construct comprising or encoding a sgRNA ^iBAR Wherein each sgRNA ^iBAR Comprising a leader sequence, a second sequence, and an iBAR sequence, wherein three or more sgRNAs ^iBAR The guide sequences of the constructs are identical, wherein three or more sgrnas ^iBAR The iBAR sequence of each of the constructs is different from each other, wherein the guide sequence is fused to said second sequence, wherein the second sequence comprises a repeat-trans-repeat stem loop interacting with the Cas9 protein, wherein the iBAR sequence is inserted in a loop region of the repeat-trans-repeat stem loop, wherein each set of sgrnas ^iBAR The guide sequences of the constructs are complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to different target sites of corresponding hit genes in the genome (e.g., different hit genes, or different sites within the same hit gene), and wherein each sgRNA is complementary ^iBAR Can be manipulated with Cas9 protein to modify the target site. In some embodiments, a recombinant vector comprising multiple sets of sgrnas is provided ^iBAR sgRNA of constructs ^iBAR Library, wherein each group of sgRNAs ^iBAR The construct comprises 4 sgrnas ^iBAR Constructs, each construct comprising or encoding a sgRNA ^iBAR Wherein each sgRNA ^iBAR Comprises a guide sequence, a second sequence and an iBAR sequence, wherein 4 sgRNAs ^iBAR The guide sequences of the constructs were identical, of which 4 sgrnas ^iBAR The iBAR sequence of each of the constructs is different from each other, wherein the guide sequence is fused to said second sequence, wherein the second sequence comprises a repeat-trans-repeat stem loop interacting with the Cas9 protein, wherein the iBAR sequence is inserted into a loop region of the repeat-trans-repeat stem loop, wherein each set of sgrnas ^iBAR The guide sequence of the construct is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to different target sites of corresponding hit genes in the genome (e.g., different hit genes, or different sites within the same hit gene), and wherein each sgRNA is complementary ^iBAR Can be manipulated with Cas9 protein to modify the target site. In some embodiments, each sg isRNA ^iBAR The second sequence of the sequence further comprises stem loop 1, stem loop 2 and/or stem loop 3, e.g., fused to the 3' end of the repeat-trans-repeat stem loop sequence. In some embodiments, each iBAR sequence comprises about 1-50 (e.g., 6) nucleotides. In some embodiments, each sgRNA ^iBAR The construct is an RNA, a plasmid, a viral vector (e.g., a lentiviral vector), or a virus (e.g., a lentivirus). In some embodiments, the sgRNA ^iBAR The library comprises at least about 100 (e.g., at least about 200, 400, 1,000, 1,200, 1,600, 4,000, 10,000, 15,000, 19,000, 20,000, 38,000, 50,000, 100,000, 150,000, 155,000, 200,000, or more) sgrnas of the group ^iBAR Constructs. In some embodiments, the sgrnas of different groups are different from each other ^iBAR At least two sgRNAs in the construct ^iBAR The iBAR sequences of the constructs are identical (e.g., first and second sets of sgrnas ^iBAR Two sets of sgrnas of the construct ^iBAR With at least 1,2, 3, 4, or more iBAR sequences shared between constructs). In some embodiments, at least two sets of sgrnas ^iBAR The iBAR sequences of the constructs were identical. In some embodiments, a plurality of sets of sgrnas are included ^iBAR sgRNA of constructs ^iBAR Library comprising or encoding sgRNAs with guide sequences complementary to target sites of each annotated gene in the genome ^iBAR . In some embodiments, the sgRNA ^iBAR The library comprises at least two groups (e.g., 2, 3, 4, 5, or more groups) of sgrnas ^iBAR A construct comprising or encoding a sgRNA having guide sequences complementary to at least two (e.g., 2, 3, 4, 5 or more, such as 2) different target sites within the same hit gene of each annotated gene in the genome ^iBAR . In some embodiments, each guide sequence comprises from about 17 to about 23 nucleotides.

In some embodiments, a sgRNA is provided ^iBAR A construct comprising a guide sequence and encoding that targets (e.g., has at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity thereto) a target site in a corresponding hit gene of a genomeRepeats-a reverse-repeat duplex and a leader hairpin of a tetracyclic sequence in which the iBAR is embedded in the tetracyclic as an internal copy. In some embodiments, the iBAR comprises a sequence of 1 nucleotide ("nt") -50nt (e.g., 1nt-40nt, 1nt-30nt, 1nt-25nt, 2nt-20nt, 3nt-18nt, 3nt-16nt, 3nt-14nt, 3nt-12nt, 3nt-10nt, 3nt-9nt, 4nt-8nt, 5nt-7 nt; preferably, 3nt, 4nt, 5nt, 6nt, 7nt) consisting of A, T, C and G nucleotides. In some embodiments, the guide sequence is about any one of 17-23, 18-22, or 19-21 nucleotides in length and can bind to a Cas nuclease (e.g., Cas9) upon transcription of the hairpin sequence. In some embodiments, the sgRNA ^iBAR The construct further comprises sequences encoding stem loop 1, stem loop 2 and/or stem loop 3. In some embodiments, each sgRNA ^iBAR The construct is an RNA, a plasmid, a viral vector (e.g., a lentiviral vector), or a virus (e.g., a lentivirus).

Also provided are sgRNA molecules encoded by any of the sgRNA constructs or libraries described herein. Also provided is a sgRNA encoded by any of the sgrnas described herein ^iBAR sgRNA encoded by a construct, panel or library ^iBAR A molecule. Also provided are recombinant vectors comprising any one of the sgRNAs or sgRNAs ^iBAR Compositions and kits of constructs, molecules, groups or libraries.

In some embodiments, provided are sgrnas or sgrnas comprising any of the herein described ^iBAR An isolated T cell (e.g., an allogeneic T cell or a CAR-T cell (e.g., an allogeneic CAR-T cell)) of a construct, molecule, panel, or library. In some embodiments, a library of T cells is provided, wherein each T cell comprises one or more sgRNA constructs from a sgRNA library described herein, or from a sgRNA described herein ^iBAR One or more sgRNAs of a library ^iBAR Constructs. In some embodiments, the T cell library comprises a sgRNA library or sgrnas described herein that targets each annotated gene in the genome ^iBAR A library. In some embodiments, the host cell comprises or expresses one or more components of a CRISPR/Cas system, such as can be associated with a sgRNA or sgRNA ^iBAR Construct operating togetherAnd (3) a Cas protein. In some embodiments, the Cas protein is Cas9 nuclease.

iBAR sequence

Group of sgRNAs ^iBAR The construct comprises three or more sgrnas ^iBAR Constructs, each construct comprising a different iBAR sequence. In some embodiments, a set of sgrnas ^iBAR The construct comprises three sgrnas ^iBAR Constructs, each construct comprising a different iBAR sequence. In some embodiments, a set of sgrnas ^iBAR The construct comprises 4 sgrnas ^iBAR Constructs, each construct comprising a different iBAR sequence. In some embodiments, a set of sgrnas ^iBAR The construct comprises 5 sgrnas ^iBAR Constructs, each construct comprising a different iBAR sequence. In some embodiments, a set of sgrnas ^iBAR The construct comprises 6 or more sgrnas ^iBAR Constructs, each construct comprising a different iBAR sequence.

The iBAR sequence may be of any suitable length. In some embodiments, each iBAR sequence is about 1 to 50 nucleotides ("nt") in length, such as any one of about 1nt to 40nt, 1nt to 30nt, 1nt to 20nt, 2nt to 20nt, 3nt to 18nt, 3nt to 16nt, 3nt to 14nt, 3nt to 12nt, 3nt to 10nt, 3nt to 9nt, 3nt to 8nt, 4nt to 8nt, or 5nt to 7 nt. In some embodiments, each iBAR sequence is about any of 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, or 8nt in length. In some embodiments, each sgRNA ^iBAR The iBAR sequences in the constructs were of the same length. In some embodiments, the sgrnas are different ^iBAR The iBAR sequences of the constructs were of different lengths. In some embodiments, a set of sgrnas ^iBAR The iBAR sequences in the constructs are of the same length. In some embodiments, a set of sgrnas ^iBAR The iBAR sequences in the constructs are of different lengths. In some embodiments, a set of sgrnas ^iBAR iBAR sequence and another set of sgRNAs in the construct ^iBAR The iBAR sequences in the constructs are of different lengths. In some embodiments, the iBAR sequence is about 6nt, hereinafter referred to as "iBAR ₆ ". In some embodiments, the sgRNA ^iBAR Each iBAR sequence within the library is about 6 nt.

The iBAR sequence may have any suitable sequence. In some embodiments, the iBAR sequence is a DNA sequence consisting of any one of A, T, C and/or G nucleotides. In some embodiments, the iBAR sequence is an RNA sequence consisting of any one of A, U, C and/or G nucleotides. In some embodiments, the iBAR sequence has unconventional or modified nucleotides other than A, T/U, C and G. In some embodiments, each iBAR sequence is 6 nucleotides long, consisting of A, T, C and a G nucleotide. In some embodiments, the encoded sgRNA ^iBAR The iBAR sequence of (1) is 6 nucleotides long and consists of A, U, C and G nucleotides.

In some embodiments, the sgRNA is ^iBAR Each set of sgRNAs in the library ^iBAR The set of iBAR sequences with which the constructs are related differ from each other. In some embodiments, the sgrnas of different groups are different from each other ^iBAR At least two sgRNAs in the construct ^iBAR The iBAR sequences of the constructs are identical (e.g., first and second sets of sgrnas ^iBAR Two sets of sgrnas of the construct ^iBAR Constructs having at least 1, 2, 3, 4 or more shared iBAR sequences between them, but the same set of sgrnas ^iBAR Each sgRNA in the construct ^iBAR The iBAR sequences of the constructs differ from each other). In some embodiments, the sgRNA ^iBAR At least two (e.g., at least about 2, 3, 4, 5, 10, 50, 100, 1000, or more) sets of sgrnas in the library ^iBAR The iBAR sequences of the constructs were identical. In some embodiments, one or more identical iBAR sequences are used for sgrnas ^iBAR Each set of sgRNAs in the library ^iBAR One or more sgrnas of a construct ^iBAR Constructs (but same set of sgrnas) ^iBAR Each sgRNA in the construct ^iBAR The iBAR sequences of the constructs differ from each other). In some embodiments, the same set of iBAR sequences is used for sgrnas ^iBAR Each set of sgRNAs in the library ^iBAR Constructs. In some embodiments, there is no need for a different set of sgrnas ^iBAR Constructs design different iBAR groups. In some embodiments, an immobilized iBAR group is used for sgrnas ^iBAR LibrariesAll sgrnas in (a) ^iBAR And (4) a construct group. In some embodiments, multiple iBAR sequences are randomly assigned to an sgRNA ^iBAR Different sets of sgRNAs in the library ^iBAR Constructs. iBAR strategy (MAGECK) with up-to-date analytical tools as described herein ^iBAR (ii) a Zhu et al, Genome biol.2019; 20:200) can facilitate large-scale CRISPR/Cas screening for biomedical discovery in various environments.

The iBAR sequence may be inserted (including appended) into any suitable region of the guide RNA (e.g., sgRNA) that does not affect the efficiency of the gRNA in guiding a Cas nuclease (e.g., Cas9) to its target site. In some embodiments, the iBAR sequence is located at the 3' end of the sgRNA. In some embodiments, the iBAR sequence is located 5' to the sgRNA. In some embodiments, the iBAR sequence is located at an internal position of the sgRNA. For example, the sgRNA can comprise various stem loops that interact with the Cas nuclease in the CRISPR complex, and the iBAR sequence can be embedded into the loop region of any one of the stem loops. In some embodiments, each sgRNA ^iBAR The sequence comprises in the 5 'to 3' direction a first stem sequence and a second stem sequence, wherein the first stem sequence hybridizes to the second stem sequence to form a double-stranded rna (dsrna) region that interacts with a Cas protein (e.g., Cas9), and wherein the iBAR sequence is located between the 3 'end of the first stem sequence and the 5' end of the second stem sequence. In some embodiments, the sgRNA ^iBAR Also included are stem loop 1, stem loop 2 and/or stem loop 3, wherein the iBAR sequence is inserted into the loop region of stem loop 1, stem loop 2 and/or stem loop.

For example, the guide RNA of the CRISPR/Cas9 system can comprise a guide sequence that targets a genomic locus (e.g., hits a target site in a gene), as well as a guide hairpin sequence that encodes repeats a trans-repeat duplex and four loops. In some embodiments, ibars are inserted into four rings as internal copies. In the context of the endogenous CRISPR/Cas9 system, crRNA is hybridized with trans-activated crRNA (tracrRNA) to form a crRNA tracrRNA duplex, which is loaded onto Cas9 to direct cleavage of a homologous DNA sequence (PAM) with an appropriate protospacer sequence proximity motif. The endogenous crRNA sequence can be divided into a leader (20nt) and a repeat (12nt), while the endogenous tracrRNA sequence can be divided into a trans-repeat (14nt) and three tracrRNA stem loops. In some embodiments, the sgRNA binds to target DNA to form a T-shaped structure comprising a guide target heteroduplex, a repeat: a trans-repeat duplex, and stem loops 1-3. In some embodiments, the repeat and the trans-repeat moiety are linked by a tetracycle and the repeat and the trans-repeat form a repeat, the trans-repeat duplex, linked to stem loop 1 by a single nucleotide (A51), and

stem loops

1 and 2 are linked by a 5nt single-stranded linker (nucleotides 63-67). In some embodiments, the guide sequence (nucleotides 1-20) and the target DNA (nucleotides 10-200) form a guide: target heteroduplex by 20 Watson-Crick base pairs, and the repeat (nucleotides 21-32) and the trans-repeat (nucleotides 37-50) form a repeat: trans-repeat duplex by 9 Watson-Crick base pairs (U22: A49-A26: U45 and G29: C40-A32: U37). In some embodiments, the tracrRNA tails (nucleotides 68-81 and 82-96)

form stem loops

2 and 3 by 4 and 6 Watson-Crick base pairs (A69: U80-U72: A77 and G82: C96-G87: C91), respectively. Nishimasu et al describe the Crystal structure of an exemplary CRISPR/Cas9 system (Nishimasu et al, "Crystal structure of Cas9 in complex with guide RNA and target DNA." cell.2014; 156: 935-949), which is incorporated herein by reference in its entirety.

In some embodiments, the iBAR sequence is inserted into the four loops of the sgRNA or into the loop region of the stem loop of the repeat, trans-repeat. In some embodiments, each sgRNA within the library ^iBAR The iBAR sequence of (a) is inserted into the loop region of the repeat-trans-repeat stem loop. The four loops of the Cas9sgRNA backbone are located outside of the Cas9-sgRNA ribonucleoprotein complex, which is altered for various purposes without affecting the activity of its upstream guide sequence (Gilbert et al cell 159,647-661 (2014); Zhu et al methods Mol Biol 1656,175-181 (2017)). The applicant has previously demonstrated in WO2020125762 that IBAR (iBAR) is 6nt long ₆ ) Can be inserted into the four loops of a typical Cas9sgRNA backbone without affecting the gene editing efficiency of the sgRNA or increasing off-target effects, and in iBAR ₆ Without sequence preference. Exemplary iBAR ₆ Generating 4,096 barcode combinations provides sufficient variation for high throughput screening (see WO2020125762, fig. 1A).

Boot sequence

The guide sequence hybridizes to a target sequence (e.g., a target site in a hit gene) and directs sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a leader sequence and its corresponding target sequence is about or greater than any one of about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100% complementary) when optimally aligned using a suitable alignment algorithm. A guide sequence that is "complementary" to a target site or a gene hit may be fully or partially complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to the target site or gene hit. Any suitable algorithm for aligning sequences may be used to determine an optimal alignment, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wimsch algorithm, and Burrows-Wheeler transform-based algorithms. In certain embodiments, the leader sequence is about or more than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides in length. In some embodiments, the leader sequence comprises from about 17 to about 23 nucleotides. The ability of the guide sequence to direct sequence-specific binding of the CRISPR complex to the target sequence can be assessed by any suitable assay. For example, a CRISPR system component sufficient to form a CRISPR complex, including a guide sequence to be tested, can be provided to a host cell having a corresponding target sequence, such as by transfection with a vector encoding the CRISPR sequence component, and then assessing preferential cleavage within the target sequence. Similarly, cleavage of the target polynucleotide sequence can be assessed in the test tube by: components of a target sequence, CRISPR complex, comprising a guide sequence to be tested and a control guide sequence different from the test guide sequence are provided, and the rate of binding or cleavage at the target sequence is compared between the test and control guide sequence reactions.

In some embodiments, the leader sequence may be shortTo about 10 nucleotides and as long as about 30 nucleotides. In some embodiments, the leader sequence is any one of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. The synthetic leader sequence may be about 20 nucleotides in length, but may be longer or shorter. For example, the guide sequence of the CRISPR/Cas9 system may consist of 20 nucleotides that are complementary to the target sequence (e.g., target site in the hit gene), i.e., the guide sequence may be identical to the 20 nucleotides upstream of the PAM sequence, except for the a/U difference between DNA and RNA. In some embodiments, the leader sequence comprises from about 17 to about 23 nucleotides. In some embodiments, each sgRNA or sgRNA within a library ^iBAR Have the same length. In some embodiments, at least two sgrnas or sgrnas within the library ^iBAR Have different lengths. In some embodiments, a set of sgrnas ^iBAR The leader sequences within the constructs are of the same length. In some embodiments, a set of sgrnas ^iBAR The leader sequences within the constructs are of different lengths. In some embodiments, a set of sgrnas ^iBAR Guide sequences in constructs with another set of sgrnas ^iBAR The leader sequences in the constructs are of different lengths.

In some embodiments, a set of sgrnas ^iBAR The leader sequences in the constructs were identical. In some embodiments, a set of sgrnas ^iBAR The leader sequence in the constructs was identical, and each set of sgrnas ^iBAR The guide sequences in the constructs are complementary to different target sites in the genome (e.g., different hit genes, or different target sites within the same hit gene). In some embodiments, at least two sets of sgrnas ^iBAR The leader sequence of the construct is complementary to two different target sites of the same hit gene. In some embodiments, each hit in the genome is grouped into at least two (e.g., 2, 3, 4, or more, such as 2) groups of sgrnas in at least two (e.g., 2, 3, 4, or more, such as 2) different target sites ^iBAR At least two (e.g., 2, 3, 4 or more, such as 2) leader sequences of the construct are targeted. In some casesIn embodiments, each set of sgrnas ^iBAR The leader sequences within the constructs were complementary to the different hits in the genome.

sgRNA constructs or sgRNAs ^iBAR The leader sequence in the construct may be designed according to any method known in the art. The leader sequence may target a coding region, such as an exon or splice site, a 5 'untranslated region (UTR) or a 3' untranslated region (UTR) of a target gene. For example, the reading frame of a gene may be disrupted by Double Strand Break (DSB) -mediated indels at the target site of the guide RNA. Alternatively, a guide RNA targeted to the 5' end of the coding sequence can be used to efficiently generate gene knockouts. The leader sequence may be designed and optimized for certain sequence characteristics to achieve high editing activity at the target gene and low off-target effects. For example, the GC content of the guide sequence may range from about 20% to about 70%, and sequences comprising homopolymer segments (e.g., TTTT, GGGG) may be avoided.

The leader sequence may be designed to target any target genomic locus (e.g., any target site of any hit gene). In some embodiments, the leader sequence targets a protein-encoding gene. In some embodiments, the guide sequence targets a gene encoding an RNA, such as a small RNA (e.g., microRNA, piRNA, siRNA, snoRNA, tRNA, rRNA, and snRNA), ribosomal RNA, or long non-coding RNA (lincrna). In some embodiments, the leader sequence targets a non-coding region of the genome. In some embodiments, the guide sequence targets a chromosomal locus. In some embodiments, the guide sequence targets an extrachromosomal locus. In some embodiments, the guide sequence targets a mitochondrial gene. In some embodiments, the guide sequence is complementary to a target site of any annotated gene in a genome (e.g., a human genome). In some embodiments, the leader sequence targets a region of the genome that is free of any gene injection ("nongenic region"). sgRNA or sgRNA comprising or encoding such a guide sequence complementary to a non-genetic region ^iBAR The construct can be used as a negative control.

In some embodiments, the leader sequence is designed to suppress or inactivate the expression of any hit gene or target gene of interest. The hit or target gene may be an endogenous gene or a transgene. In some embodiments, the hit or target gene is known to be associated with a particular phenotype. In some embodiments, a hit gene or target gene is a gene that is not involved in a particular phenotype, such as a known gene that is not known to be associated with a particular phenotype, or an unknown gene that has not been characterized. In some embodiments, the region targeted by the leader sequence is located on a different chromosome as the hit gene or target gene.

Other sgRNAs or sgRNAs ^iBAR Assembly

In some embodiments, the sgRNA or sgRNA ^iBAR Comprising additional sequence elements that facilitate formation of a CRISPR complex with a Cas protein. In some embodiments, the sgRNA or sgRNA ^iBAR Comprising a second sequence comprising a repeat-trans-repeat stem loop. The repeat-trans-repeat stem loop comprises a tracr mate sequence fused to a tracr sequence that is complementary to the tracr mate sequence by a loop region.

Typically, in the context of an endogenous CRISPR/Cas9 system, formation of a CRISPR complex (comprising a guide sequence that hybridizes to a target sequence and complexes with one or more Cas proteins) results in cleavage of one or both strands within or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs) the target sequence. the tracr sequence, which may comprise or consist of all or part of a wild-type tracr sequence (e.g., any of about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridizing along at least part of the tracr sequence with all or part of a tracr mate sequence operably linked to a guide sequence. In some embodiments, the tracr sequence is sufficiently complementary to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex. As with the target sequence, it is believed that complete complementarity is not required, provided sufficient functionality is provided. In some embodiments, the tracr sequence has any of at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned. Determining the optimal arrangement is within the ability of those skilled in the art. For example, there are published and commercially available alignment algorithms and programs, such as, but not limited to: ClustalW, Smith-Waterman in Matlab, Bowtie, Geneius, Biopython, and SeqMan. In some embodiments, the tracr sequence is about or more than about any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 or more nucleotides in length. Any of the known tracr mate sequences and tracr sequences derived from naturally occurring CRISPR systems, such as those from the streptococcus pyogenes (s.pyogenes) CRISPR/Cas9 system, such as US8697359 and those described herein, may be used.

In some embodiments, the tracr sequence and the tracr mate sequence are contained within a single transcript such that hybridization between the two produces a transcript having secondary structure, such as a stem-loop (also known as a hairpin), referred to as a "repeat-trans-repeat stem-loop.

In some embodiments, the loop region of the stem loop in a sgRNA construct without the iBAR sequence is 4 nucleotides in length, and such loop region is also referred to as "tetracycle". In some embodiments, the loop region has a GAAA sequence. However, longer or shorter loop sequences may be used, or alternative sequences may be used, such as sequences comprising nucleotide triplets (e.g., AAA) and additional nucleotides (e.g., C or G). In some embodiments, the sequence of the loop region is CAAA or AAAG. In some embodiments, the iBAR is inserted into a loop region, such as a tetracycle. For example, the iBAR sequence may be inserted before the first nucleotide, between the first or second nucleotides, between the second and third nucleotides, between the third and 4 th nucleotides, or after the 4 th nucleotide in the tetracycle. In some embodiments, the iBAR sequence replaces one or more nucleotides in the loop region.

In some embodiments, the sgRNA ^iBAR Comprising at least two or more stem loops. In some embodiments, the sgRNA ^iBAR With two, three, 4 or 5 stem loops. In some embodiments, thesgRNA ^iBAR With up to 5 hairpins. In some embodiments, the sgRNA or sgRNA ^iBAR The construct also comprises a transcription termination sequence, such as a polyT sequence, for example 6T nucleotides.

In some embodiments, wherein the Cas protein is Cas9, each sgRNA or sgRNA ^iBAR Comprising a guide sequence fused to a second sequence comprising a repeat-reverse-repeat stem loop that interacts with Cas 9. In some embodiments, the iBAR sequence is inserted into the loop region of a repeat-trans-repeat stem loop. In some embodiments, the iBAR sequence replaces one or more nucleotides in the loop region of the repeat-trans-repeat stem loop. In some embodiments, each sgRNA or sgRNA ^iBAR Also comprises stem-loop 1, stem-loop 2 and/or stem-loop 3. In some embodiments, the iBAR sequence is inserted into the loop region of stem loop 1. In some embodiments, the iBAR sequence replaces one or more nucleotides in the loop region of stem loop 1. In some embodiments, the iBAR sequence is inserted into the loop region of stem loop 2. In some embodiments, the iBAR sequence replaces one or more nucleotides in the loop region of stem loop 2. In some embodiments, the iBAR sequence is inserted into the loop region of stem loop 3. In some embodiments, the iBAR sequence replaces one or more nucleotides in the loop region of stem loop 3.

In some embodiments, each sgRNA ^iBAR Comprising in a 5 'to 3' direction a first stem sequence and a second stem sequence, wherein the first stem sequence hybridizes to the second stem sequence to form a double stranded rna (dsrna) region that interacts with a Cas protein, and wherein the iBAR sequence is located between the 3 'end of the first stem sequence and the 5' end of the second stem sequence.

In the CRISPR/Cas9 system, guide RNAs can be used to direct Cas9 nuclease to cleave genomic DNA. For example, the guide RNA can consist of a nucleotide spacer of a variable sequence (guide sequence) that targets the CRISPR/Cas system nuclease to a genomic location in a sequence-specific manner and an invariant hairpin sequence that is constant between different guide RNAs and allows the guide RNA to bind to the Cas nuclease. In some embodiments, the CRISPR/Cas guide RNA comprises a CRISPR/Cas variable guide sequence that is homologous or complementary to a target genomic sequence (e.g., a target site for a hit gene) in a host cell and an invariant hairpin sequence that is capable of binding a Cas nuclease (e.g., Cas9) when transcribed, wherein the hairpin sequence encodes repeats a trans-repeat duplex and a tetracycle, and the iBAR is embedded in this tetracycle region.

The guide sequence of the CRISPR/Cas9 guide RNA can be any one of about 17-23, 18-22, or 19-21 nucleotides in length. The guide sequence can target the Cas nuclease to a genomic locus in a sequence-specific manner, and can be designed according to general principles known in the art. Invariant guide RNA hairpin sequences may be provided according to common general knowledge in the art, e.g., as disclosed by Nishimasu et al (Nishimasu H, et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. cell. 2014; 156: 935-949). Any invariant hairpin sequences can be used as long as they are capable of binding to the Cas nuclease after transcription.

Previous studies have shown that although sgrnas with 48-nttracrRNA tails, referred to as sgrnas (+48), are the smallest regions, sgrnas (+67) and sgrnas (+85) with extended tracrRNA tails can increase Cas9 cleavage activity in vivo for Cas 9-catalyzed in vitro DNA cleavage (Jinek et al, 2012) (Hsu et al, 2013). In some embodiments, the sgRNA or sgRNA ^iBAR Including stem-loop 1, stem-loop 2 and/or stem-loop 3. The stem loop 1, stem loop 2 and/or stem loop 3 regions can increase editing efficiency in the CRISPR/Cas9 system. In some embodiments, the sgRNA comprises in the 5 'to 3' direction: leader sequence, repeat-trans-repeat stem loop, stem loop 1, stem loop 2 and stem loop 3. In some embodiments, the sgRNA ^iBAR Comprising in the 5 'to 3' direction: leader sequence, repeat-trans-repeat stem loop, stem loop 1, stem loop 2 and stem loop 3 with insertion of iBAR sequence in the loop region.

Carrier and carrier

In some embodiments, the sgRNA construct comprises one or more regulatory elements operably linked to a guide RNA sequence. In some embodiments, the sgRNA ^iBAR The construct comprises one or more guide RNA sequences operably linked to andregulatory elements of iBAR sequences. Exemplary regulatory elements include, but are not limited to: promoters, enhancers, Internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). For example, such regulatory elements are described in: goeddel, GENE EXPRESSION TECHNOLOGY: METHOD IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells, as well as those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).

sgRNA or sgRNA ^iBAR The construct may be present in a vector. In some embodiments, the vector is suitable for replication and integration in a eukaryotic cell, such as a mammalian cell (e.g., a T cell). In some embodiments, the sgRNA or sgRNA ^iBAR The construct is an expression vector, such as a viral vector or a plasmid. Examples of viral vectors include, but are not limited to: adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, retroviral vectors, herpes simplex virus vectors and derivatives thereof. Viral vector technology is well known in the art and is described, for example, in: sambrook et al (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,2001, New York), and other virology and Molecular biology manuals. One skilled in the art will appreciate that the design of an expression vector may depend on factors such as the choice of host cell to be transformed, the level of expression desired, and the like. In some embodiments, the sgRNA or sgRNA ^iBAR The construct is a lentiviral vector. In some embodiments, the sgRNA or sgRNA ^iBAR The construct is a virus. In some embodiments, the sgRNA or sgRNA ^iBAR The construct is an adenovirus or adeno-associated virus. In some embodiments, the sgRNA or sgRNA ^iBAR The construct is a lentivirus. In some embodiments, the vector further comprises a selectable marker. In some embodiments, the vector further comprises one or more nucleotide sequences encoding one or more elements of a CRISPR/Cas system, such as a nucleotide encoding a Cas nuclease (e.g., Cas9)And (3) sequence. In some embodiments, a vector system is provided comprising one or more vectors encoding a nucleotide sequence encoding one or more elements of a CRISPR/Cas system, and comprising a sgRNA or sgRNA described herein ^iBAR A vector of any one of the constructs. The carrier may comprise one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., a promoter and/or enhancer) that regulate the expression of the polypeptide of interest, and/or one or more selectable marker genes (e.g., an antibiotic resistance gene, or a fluorescent protein-encoding gene).

Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged into retroviral particles using techniques known in the art. Recombinant viruses can then be isolated and delivered to engineered mammalian cells in vitro or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In some embodiments, a lentiviral vector is used. In some embodiments, a self-inactivating lentiviral vector is used. Self-inactivating lentiviral vectors can be packaged into lentiviruses using protocols known in the art. The resulting lentiviruses can be used to transduce mammalian cells (e.g., primary human T cells) using methods known in the art. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, as they allow long-term stable integration of transgenes and propagation in progeny cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.

In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a transposon, such as the sleeping beauty transposon system or the PiggyBac transposon system. In some embodiments, the carrier is a polymer-based non-viral carrier including, for example, poly (lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA), poly (ethyleneimine) (PEI), and dendrimers. In some embodiments, the vector is a cationic lipid-based non-viral vector, such as cationic liposomes, lipid nanoemulsions, and Solid Lipid Nanoparticles (SLNs). In some embodiments, the vector is a peptide-based gene non-viral vector, such as poly-L-lysine. Any known non-viral vector suitable for gene editing can be used for sgRNA or sgRNA ^iBAR The encoding nucleic acid is introduced into an immune effector cell (e.g., a T cell). See, for example, Yin h. et al. nature rev. genetics (2014)15: 521-; "The Sleeping Beauty transposon system: a non-viral vector for gene therapy" "hum. mol. Gene. (2011) R1: R14-20; andZhao S.et al, "PiggyBac transposon vectors: the tools of the human gene editing." Transl. Lung Cancer Res. (2016)5(1): 120-. In some embodiments, the sgRNA or sgRNA described herein is encoded by a physical method ^iBAR Any one or more of the nucleic acids of (a) are introduced into a T cell, and the physical methods include, but are not limited to: electroporation, sonoporation, photoporation, magnetic transfection, hydroperforation.

In some embodiments, the sgRNA or sgRNA is encoded ^iBAR And one or more nucleic acids encoding one or more elements of the CRISPR/Cas system (e.g., a Cas nuclease, such as Cas9) are located in different vectors (e.g., viral vectors, such as lentiviral vectors). In some embodiments, the sgRNA or sgRNA is encoded ^iBAR And one or more nucleic acids encoding one or more elements of the CRISPR/Cas system are on the same vector. In some embodiments, the sgRNA or sgRNA is encoded ^iBAR And one or more nucleic acids encoding one or more elements of the CRISPR/Cas system are operably controlled by separate promoters. In some embodiments, the sgRNA or sgRNA is encoded ^iBAR And one or more nucleic acids encoding one or more elements of the CRISPR/Cas system are operably controlled by the same promoter. In some embodiments, the sgRNA or sgRNA is encoded ^iBAR And one or more nucleic acids encoding one or more elements of the CRISPR/Cas system are linked by one or more linking sequences, such as an IRES.

The nucleic acid may be cloned into the vector using any molecular cloning method known in the art, including, for example, the use of restriction endonuclease sites and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter. Various promoters have been explored for gene expression in mammalian cells, and any promoter known in the art can be used in the present invention. Promoters can be broadly classified as constitutive promoters or regulated promoters, such as inducible promoters.

In some embodiments, the sgRNA or sgRNA is encoded ^iBAR And/or one or more nucleic acids encoding one or more elements of a CRISPR/Cas system (e.g., Cas9) are operably linked to a constitutive promoter. Constitutive promoters allow constitutive expression of a heterologous gene (also referred to as a transgene) in a host cell. Exemplary promoters contemplated herein include, but are not limited to: cytomegalovirus very early promoter (CMV IE), human elongation factor-1 α (hEF1 α), ubiquitin C promoter (UbiC), phosphoglycerate kinase Promoter (PGK), simian virus 40 early promoter (SV40), chicken β -actin promoter and CMV early enhancer (CAGG), Rous Sarcoma Virus (RSV) promoter, polyoma enhancer/herpes simplex thymidine kinase (MC1) promoter, β actin (β -ACT) promoter, "myeloproliferative sarcoma virus enhancer, negative control region deleted, d1587rev primer binding site substituted (MND)" promoter. The efficiency of such constitutive promoters in promoting transgene expression has been widely compared in a number of studies.

In some embodiments, the sgRNA or sgRNA is encoded ^iBAR And/or one or more nucleic acids encoding one or more elements of a CRISPR/Cas system (e.g., Cas9) are operably linked to an inducible promoter. Inducible promoters belong to the class of regulated promoters. The inducible promoter may be induced by one or more conditions, such as the physical condition of the engineered T cell, the microenvironment or physiological state of the engineered T cell, an inducer (i.e., inducer), or a combination thereof. In some embodiments, the induction conditions do not induce engineered T cells in and/or receive T cell therapyExpression of an endogenous gene in the subject. In some embodiments, the induction conditions are selected from: inducers, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox status, tumor environment, and activation status of engineered T cells. In some embodiments, the inducible promoter can be the NFAT promoter,

A promoter or an NF-. kappa.B promoter.

Libraries

The sgRNA library described herein comprises one or more sgRNA constructs, wherein each sgRNA construct comprises or encodes a sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site of a corresponding hit gene in the genome. The sgRNA libraries described herein can be designed to target one or more genomic loci (e.g., multiple target sites in one or more hit genes in the genome) as needed for gene screening. In some embodiments, a single sgRNA construct is designed to target each hit gene. In some embodiments, multiple (e.g., at least about 2, 3, 4, 5, 10, 20, 100, or more) sgRNA constructs can be designed with different guide sequences targeting a single hit gene. For example, such multiple sgRNA constructs can comprise or encode guide sequences that target different target sites of a single hit gene, such as 2 (or about 3 to about 12) different target sites of a single hit gene.

Comprising one or more sgRNAs ^iBAR sgRNA library of constructs, also referred to herein as sgrnas ^iBAR Library, wherein each sgRNA construct comprises or encodes an iBAR sequence. sgRNA described herein ^iBAR The library comprises one or more sgrnas ^iBAR Construct, wherein each sgRNA ^iBAR Constructs comprise or encode sgrnas ^iBAR And wherein each sgRNA ^iBAR Comprising complementarity to a target site of a corresponding hit gene in the genome (e.g., at least about 50%, 60%, 70%, 80%, 90%, 95)%, 96%, 97%, 98%, 99% or 100% complement). The sgrnas described herein are useful for screening genes ^iBAR The library can be designed to target one or more genomic loci (e.g., multiple target sites in one or more hit genes in the genome). In some embodiments, a single sgRNA is designed ^iBAR Constructs were made to target each hit. In some embodiments, multiple sgrnas can be designed (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more) with different leader sequences targeting a single gene of interest ^iBAR Constructs. For example, such multiple sgrnas ^iBAR The construct may comprise or encode targeting sequences that target different target sites of a single hit gene (e.g., 2 different target sites of a single hit gene).

In some embodiments, a sgRNA described herein ^iBAR The library comprises one or more sets of sgRNAs ^iBAR Construct, wherein each group of sgRNAs ^iBAR The construct comprises three or more (e.g., 3, 4, 5 or more, such as 4) sgrnas ^iBAR Constructs, each construct comprising or encoding a sgRNA ^iBAR Wherein each sgRNA ^iBAR Comprising a leader sequence and an iBAR sequence, three or more sgRNAs ^iBAR The guide sequences of the constructs are identical, wherein three or more sgrnas ^iBAR The iBAR sequence of each of the constructs is different from each other, and wherein each group of sgrnas ^iBAR The leader sequences of the constructs are complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to different target sites of corresponding hit genes in the genome (e.g., different hit genes, or different sites within the same hit gene). In some embodiments, each sgRNA group ^iBAR The construct comprises 4 sgrnas ^iBAR Construct, and wherein 4 sgrnas ^iBAR The iBAR sequences of each of the constructs differ from each other. In some embodiments, a single set of sgrnas is designed ^iBAR Constructs were made to target each hit. In some embodiments, the sgRNA ^iBAR The library comprises a plurality of groups (examples)E.g., at least about 2, 3, 4, 5, 10, 20, or more) sgrnas of a group ^iBAR Constructs with different leader sequences for a single hit gene. In some embodiments, the sgRNA ^iBAR The library comprises at least 2 (e.g. 2) groups of sgRNAs ^iBAR Constructs designed to target at least 2 (e.g., 2) different target sites per hit gene, wherein each set of sgrnas ^iBAR The construct comprises 4 sgrnas ^iBAR Constructs. In some embodiments, the sgRNA ^iBAR The library comprises at least about 100 sgRNAs ^iBAR Constructs, such as at least about 200, 300, 400, 800, 1,000, 2,000, 3,000, 5,000, 10,000, 15,000, 19,000, 20,000, 40,000, 50,000, 100,000, 150,000, 200,000 or more sgrnas of a group ^iBAR Constructs. In some embodiments, the sgRNA ^iBAR The library comprises at least about 100, such as about 18,000 to about 20,000 groups of sgRNAs ^iBAR Constructs. In some embodiments, the sgRNA ^iBAR The library comprises about 36,000 to about 40,000 groups of sgrnas ^iBAR Constructs.

In some embodiments, the sgRNA library or sgRNA ^iBAR The library comprises at least about 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 400, 500, 1,000, 2,000, 4,000, 5,000, 10,000, 15,000, 19,000, 20,000, 38,000, 39,000, 40,000, 50,000, 100,000, 150,000, 155,000, 200,000 or more sgRNA constructs or sgrnas ^iBAR Any one of the constructs. In some embodiments, the sgRNA library or sgRNA ^iBAR The library comprises at least about 150,000 sgRNA constructs or sgRNAs ^iBAR Constructs. In some embodiments, the sgRNA library comprises about 15,000 to about 200,000 sgRNA constructs, such as about 18,000 to about 20,000, about 38,000 to about 40,000, about 18,000 to about 50,000, about 50,000 to about 100,000, about 10,000 to about 200,000, about 140,000 to about 180,000, or about 150,000 to about 160,000 sgRNA constructs. In some embodiments, the sgRNA ^iBAR The library comprises about 15,000 to about 200,000 sgRNAs ^iBAR Constructs, such as about 18,000 to about 50,000, about 18,000 to about 20,000, about 38,000 to about 40,000, about 50,000 to about 100,000,about 10,000 to about 200,000, about 140,000 to about 180,000, or about 150,000 to about 160,000 sgRNAs ^iBAR Constructs. In some embodiments, the sgRNA ^iBAR The library comprises at least about 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 400, 500, 1,000, 2,000, 5,000, 10,000, 19,000, 20,000, 38,000, 50,000, 100,000, 150,000, 200,000, or more groups of sgrnas ^iBAR Any one of the constructs. In some embodiments, the sgRNA library or sgRNA ^iBAR The library targets any of at least about 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 15,000, 19,000, 20,000, 38,000, 50,000 or more genes in a cell or organism. In some embodiments, the organism is a human. In some embodiments, the sgRNA library or sgRNA ^iBAR The library is a whole genome library of protein-encoding genes and/or non-encoding RNAs. In some embodiments, the sgRNA library or sgRNA ^iBAR The library is a whole genome library for each annotated gene. Thus, in some embodiments, a sgRNA library comprising a plurality of sgRNA constructs, comprises or encodes sgrnas having guide sequences complementary to a target site of each annotated gene in a genome, such as target sites of 19,114 annotated genes in a human genome. In some embodiments, comprising a plurality of sgrnas ^iBAR sgRNA of constructs ^iBAR Library comprising or encoding sgRNAs with guide sequences complementary to target sites of each annotated gene in the genome ^iBAR Such as the target sites of 19,114 annotated genes in the human genome. In some embodiments, the sgRNA library or sgRNA ^iBAR The library targets at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of any of the genes in the cell or organism. In some embodiments, the sgRNA library or sgRNA ^iBAR Libraries are targeted libraries that target selected genes in signaling pathways or associated with cellular processes, such as killing, cell proliferation, cell cycle, transcriptional regulation mediated by immune effector cells (e.g., NK cells)Ubiquitination, apoptosis, immune responses such as autoimmunity, tumor metastasis, malignant transformation of tumors, and the like. In some embodiments, the sgRNA library or sgRNA ^iBAR The libraries are used for whole genome screening associated with a particular regulatory phenotype, such as susceptibility or resistance to immune effector cell (e.g., NK cell) mediated killing. In some embodiments, the sgRNA library or sgRNA ^iBAR The libraries are used for genome-wide screening to identify at least one target gene associated with a particular regulated phenotype, such as a target gene in a T cell that modulates T cell activity in response to NK cell treatment. In some embodiments, the sgRNA library or sgRNA ^iBAR The libraries are designed to target eukaryotic genomes, such as mammalian genomes. Exemplary target genomes include the genomes of rodents (mice, rats, hamsters, guinea pigs), domesticated animals (e.g., cows, sheep, cats, dogs, horses, or rabbits), non-human primates (e.g., monkeys), fish (e.g., zebrafish), non-vertebrates (e.g., Drosophila melanogaster (Drosophila melanogaster), and Caenorhabditis elegans (Caenorhabditis elegans)), and humans.

sgRNA library or sgRNA ^iBAR The guide sequences of the library can be designed using any known algorithm that recognizes CRISPR/Cas target sites in a user-defined list with high target specificity in the human genome, such as genomic target scanning (GT-Scan) (see O' Brien et al, Bioinformatics (2014)30:2673-2675), DeepCRISPR, CasFinder, CHOPCHOP, CRISPR Scan, and the like. In some embodiments, at least about 100, 400, 500, 1,000, 5,000, 10,000, 15,000, 19,000, 20,000, 50,000, 100,000, 150,000, 155,000, 200,000 or more sgRNA constructs or sgrnas ^iBAR Constructs can be generated on a single array. In some embodiments, at least about 19,000, 20,000, 38,000, 50,000, 100,000, 150,000, 155,000, 200,000, or more sgrnas construct or sgrnas ^iBAR Any of the constructs can be generated on a single array, providing sufficient coverage to screen the human genome for all genes in its entirety. This method can also be used to synthesize multiple sgRNA libraries or sgRNAs in parallel ^iBAR The library was expanded to allow for whole genome screening. The exact number of sgRNA constructs in the sgRNA library, or sgRNA ^iBAR sgRNA in library ^iBAR Construct (or sgRNA) ^iBAR Set of constructs) the exact number may depend on whether the screen is: 1) targeted genes are also regulatory elements, 2) targeted to the entire genome or to a subset of genomic genes.

In some embodiments, the sgRNA library or sgRNA ^iBAR The library is designed to target each PAM sequence that overlaps a gene in the genome, where the PAM sequence corresponds to a Cas protein. In some embodiments, the sgRNA library or sgRNA ^iBAR The library is designed to target a subset of PAM sequences found in the genome, where the PAM sequences correspond to the Cas protein.

In some embodiments, the sgRNA library comprises one or more control sgRNA constructs that do not target any genomic locus in the genome. In some embodiments, sgRNA constructs that do not target a putative genomic gene can be included in the sgRNA library as a negative control. In some embodiments, the sgRNA ^iBAR The library comprises one or more control sgrnas that do not target any genomic locus in the genome ^iBAR Constructs. In some embodiments, sgrnas that do not target putative genomic genes ^iBAR The construct can be included in the sgRNA as a negative control ^iBAR In a library.

Any nucleic acid synthesis and/or molecular cloning methods known in the art can be used to prepare the sgRNA constructs and libraries described herein. In some embodiments, the sgRNA library is synthesized by electrochemical methods on an array (e.g., CustomArray, Twist, Gen9), DNA printing (e.g., Agilent), or solid phase synthesis of individual oligonucleotides (e.g., by IDT). The sgRNA construct can be amplified by PCR and cloned into an expression vector (e.g., a lentiviral vector). In some embodiments, the lentiviral vector further encodes one or more components of a CRISPR/Cas-based genetic editing system, such as a Cas protein, e.g., Cas 9.

The present invention provides in some embodimentsEncoding any sgRNA construct, sgRNA described herein ^iBAR Construct, sgRNA ^iBAR Construct group, sgRNA library, sgRNA ^iBAR An isolated nucleic acid of a library or B2M sgRNA construct. Also provided are sgrnas comprising a nucleic acid encoding any of the sgrnas described herein ^iBAR Construct, sgRNA ^iBAR Construct group, sgRNA library, sgRNA ^iBAR Vectors (e.g., non-viral vectors, or viral vectors such as lentiviral vectors) and viruses (e.g., lentiviruses) of any nucleic acid of the library and/or the B2M sgRNA construct.

Cas protein

sgRNA constructs or sgRNAs described herein ^iBAR The construct can be designed to operate with any of the naturally occurring or engineered CRISPR/Cas systems known in the art. In some embodiments, the sgRNA construct or sgRNA ^iBAR The constructs are operable with a type I CRISPR/Cas system. In some embodiments, the sgRNA construct or sgRNA ^iBAR The constructs are operable with a type II CRISPR/Cas system. In some embodiments, the sgRNA construct or sgRNA ^iBAR The constructs are operable with a type III CRISPR/Cas system. Exemplary CRISPR/Cas systems can be found in WO2013176772, WO2014065596, WO2014018423, WO2016011080, US8697359, US8932814, US10113167B2, the entire contents of which are incorporated herein by reference.

In certain embodiments, the sgRNA construct or sgRNA ^iBAR The constructs can be manipulated with Cas proteins derived from CRISPR/Cas type I, type II, or type III systems that have RNA-guided polynucleotide binding and/or nuclease activity. Examples of such Cas proteins are described, for example, in WO2014144761, WO2014144592, WO2013176772, US20140273226 and US20140273233, which are incorporated herein by reference in their entirety.

In certain embodiments, the Cas protein is derived from a type II CRISPR-Cas system. In certain embodiments, the Cas protein is or is derived from a Cas9 protein. In certain embodiments, the Cas protein is or is derived from a bacterial Cas9 protein, including those identified in WO 2014144761.

In some embodiments, the sgRNA construct or sgRNA ^iBAR The constructs can be manipulated with Cas9 (also known as Csn1 and Csx12), homologues thereof, or modified forms thereof. In some embodiments, the sgRNA construct or sgRNA ^iBAR The construct can be operable with two or more (e.g., 2, 3, 4, 5, or more) Cas proteins. In some embodiments, the sgRNA construct or sgRNA ^iBAR The construct can be manipulated with Cas9 protein from streptococcus pyogenes (s. pyogenes) or streptococcus pneumoniae (s. pneumoniae). Cas enzymes are known in the art; for example, the amino acid sequence of a streptococcus pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW 2.

Cas proteins (also referred to herein as "Cas nucleases") provide desired activities, such as target binding, target nicking, or cleavage activity. In certain embodiments, the desired activity is target binding. In certain embodiments, the desired activity is target nicking or target cleavage. In certain embodiments, the desired activity further comprises a function provided by a polypeptide covalently fused to the Cas protein or the nuclease-deficient Cas protein. Examples of such desired activities include transcriptional regulatory activity (activation or inhibition), epigenetic modifying activity or target visualization/identification activity.

In some embodiments, the sgRNA construct or sgRNA ^iBAR The constructs can be manipulated with Cas nucleases that cleave the target sequence, including double-stranded and single-stranded cleavage. In some embodiments, the sgRNA construct or sgRNA ^iBAR The constructs can be manipulated with catalytically inactive Cas ("dCas"). In some embodiments, the sgRNA construct or sgRNA ^iBAR The constructs can operate with dCas of a CRISPR activation ("CRISPRa") system, wherein the dCas is fused to a transcriptional activator. In some embodiments, the sgRNA construct or sgRNA ^iBAR The constructs can be operated with dCas of the CRISPR interference (CRISPRi) system. In some embodiments, the dCas are fused to a repression domain (e.g., a KRAB domain). Such CRISPR/Cas systems can be used to modulate (e.g., induce, inhibit, increase, or decrease) gene expression。

In certain embodiments, the Cas protein is a mutant of a wild-type Cas protein (e.g., Cas9) or a fragment thereof. Cas9 proteins typically have at least two nuclease (e.g., dnase) domains. For example, a Cas9 protein may have a RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC and HNH domains work together to cleave both strands at the target site, forming a double-strand break in the target polynucleotide. (Jinek et al, Science 337: 816-21). In certain embodiments, the mutant Cas9 protein is modified to include only one functional nuclease domain (RuvC-like or HNH-like nuclease domain). For example, in certain embodiments, a mutant Cas9 protein is modified such that one of the nuclease domains is deleted or mutated such that it is no longer functional (i.e., nuclease activity is absent). In some embodiments where one of the nuclease domains is inactive, the mutant is capable of introducing a nick into a double-stranded polynucleotide (such proteins are referred to as "nickases") but is incapable of cleaving the double-stranded polynucleotide. In certain embodiments, the Cas protein is modified to increase nucleic acid binding affinity and/or specificity, alter enzymatic activity, and/or alter another property of the protein. In certain embodiments, the Cas protein is truncated or modified to optimize the activity of the effector domain. In certain embodiments, both the RuvC-like nuclease domain and the HNH-like nuclease domain are modified or eliminated such that the mutant Cas9 protein is unable to nick or cleave the target polynucleotide. In certain embodiments, target identification activity is more or less maintained relative to Cas9 protein lacking some or all nuclease activity of the wild-type counterpart.

In certain embodiments, the Cas protein is a fusion protein comprising a naturally occurring Cas or a variant thereof fused to another polypeptide or effector domain. The other polypeptide or effector domain may be, for example, a cleavage domain, a transcription activation domain, a transcription repression domain, or an epigenetic modification domain. In certain embodiments, the fusion protein comprises a modified or mutated Cas protein, wherein all nuclease domains have been inactivated or deleted. In certain embodiments, the RuvC and/or HNH domains of the Cas protein are modified or mutated such that they no longer have nuclease activity.

In certain embodiments, the effector domain of the fusion protein is a cleavage domain obtained from any endonuclease or exonuclease having the desired property.

In certain embodiments, the effector domain of the fusion protein is a transcriptional activation domain. Generally, the transcription activation domain interacts with transcription control elements and/or transcriptional regulatory proteins (i.e., transcription factors, RNA polymerases, etc.) to increase and/or activate transcription of a gene. In certain embodiments, the transcriptional activation domain is a herpes simplex virus VP16 activation domain, VP64 (which is a tetrameric derivative of VP 16), nfkb Bp65 activation domain,

p53 activation domains

1 and 2, CREB (cAMP response element binding protein) activation domain, E2A activation domain, or NFAT (nuclear factor of activated T cells) activation domain. In certain embodiments, the transcriptional activation domain is Gal4, Gcn4, MLL, Rtg3, Gln3, Oaf1, Pip2, Pdr1, Pdr3, Pho4, or Leu 3. The transcriptional activation domain may be wild-type, or a modified or truncated version of the original transcriptional activation domain.

In certain embodiments, the effector domain of the fusion protein is a transcription repression domain, such as an inducible cAMP early repression (icor) domain, Kruppel associated box a (KRAB-a) repression domain, YY1 glycine-rich repression domain, Sp 1-like repressor, e (spi) repressor, i.κ.b repressor, or MeCP 2.

In certain embodiments, the effector domain of the fusion protein is an epigenetic modification domain that alters gene expression by modifying a histone structure and/or a chromosomal structure, such as a histone acetyltransferase domain, histone deacetylase domain, histone methyltransferase domain, histone demethylase domain, DNA methyltransferase domain, or DNA demethylase domain.

In certain embodiments, the Cas protein further comprises at least one additional domain, such as a Nuclear Localization Signal (NLS), a cell penetration or translocation domain, and a marker domain (e.g., a fluorescent protein marker).

The Cas protein may be introduced into the T cell as (i) the Cas protein, or (ii) mRNA encoding the Cas protein, or (iii) linear or circular DNA encoding the protein. The Cas protein or construct encoding the Cas protein may or may not be purified in the composition. Methods of introducing a protein or nucleic acid construct into a host cell are well known in the art and are applicable to all methods described herein that require introduction of a Cas protein or a construct thereof into a T cell. In certain embodiments, the Cas protein is delivered into the T cell as a protein. In certain embodiments, the Cas protein is constitutively expressed from mRNA or DNA in the host T cell. In certain embodiments, expression of Cas protein from mRNA or DNA is inducible or inducible in a host T cell. In certain embodiments, the Cas protein may be introduced into the host T cell as a Cas protein sgRNA complex using recombinant techniques known in the art. Exemplary methods of introducing Cas proteins or constructs thereof are described, for example, in WO2014144761WO2014144592 and WO2013176772, which are incorporated herein by reference in their entirety.

In some embodiments, the methods use the CRISPR/Cas9 system. Cas9 is a nuclease from a microbial type II CRISPR (clustered regularly interspaced short palindromic repeats) system that has been shown to cleave DNA when paired with a single-stranded guide rna (sgrna). The sgRNA directs Cas9 to a complementary region in the target genomic gene, which may lead to a site-specific Double Strand Break (DSB), which can be repaired in an error-prone manner by a cellular non-homologous end joining (NHEJ) mechanism. Wild-type Cas9 cleaves mainly the genomic locus, the gRNA sequence followed by the PAM sequence (-NGG). NHEJ-mediated Cas 9-induced repair of DSBs induces extensive mutations initiated at the cleavage site, which are usually small (<10bp) insertions/deletions (indels), but may include larger (>100bp) insertions/deletions.

T cell library

The T cell libraries described herein comprise a plurality (e.g., at least about 2, 3, 4, 5, 10, 100, 1 × 10) ³ 、1×10 ⁴ 、1×10 ⁵ 、1×10 ⁶ 、1×10 ⁷ 、2×10 ⁷ 、3.5×10 ⁷ 、1×10 ⁸ Or any of more) T cells (e.g., cytotoxic)A sexual T lymphocyte or "CTL"), wherein each of the plurality of T cells has a mutation (e.g., an inactivating mutation) at a hit gene in a genome (e.g., a human genome), and wherein the hit genes of at least two of the plurality of T cells are different from each other. In some embodiments, the T cell library further comprises a B2M mutation (e.g., an inactivated B2M mutation).

In some embodiments, the T cell library comprises a plurality of T cells having at least about any of 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1,0002,000, 5,000, 10,000, 20,000, 50,000 or more hits in a cell or organism. In some embodiments, the organism is a human. In some embodiments, the T cell library comprises a plurality of T cells having a mutation (e.g., an inactivating mutation) in about 15,000 to about 50,000 hits, such as about 18,000 to about 20,000 hits. In some embodiments, the library of T cells comprises at least about 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 1 x 10 ⁴ 、2×10 ⁴ 、5×10 ⁴ 、 1×10 ⁵ 、2×10 ⁵ 、1×10 ⁶ 、5×10 ⁶ 、1×10 ⁷ 、1.5×10 ⁷ 、2×10 ⁷ 、3.5×10 ⁷ 、1×10 ⁸ 、1×10 ⁹ 、 1×10 ¹⁰ Any one of the one or more T cells. In some embodiments, at least two T cells within the T cell library have a mutation (e.g., an inactivating mutation) at a different target site (e.g., a different hit gene, or a different site within the same hit gene). In some embodiments, each T cell within the T cell library has a mutation (e.g., an inactivating mutation) at a different hit gene. In some embodiments, each T cell within the T cell library has a mutation (e.g., an inactivating mutation) at a different target site (e.g., can be within the same hit gene, or within a different hit gene). In some embodiments, the T cell library does not comprise T cells having a mutation (e.g., an inactivating mutation) at the same hit gene, such as an inactivating mutation at the same target site of the same hit gene, or at the same target site of the same hit gene Inactivating mutations at different target sites of the hit gene. In some embodiments, the T cell library does not comprise T cells having a mutation (e.g., inactivating mutation) at the same target site. In some embodiments, a plurality (e.g., at least about 2, 3, 4, 5, 10, 100, 500, 1,000, 2,000, 5,000, 10,000, 2 x 10, etc.) within the T cell library ⁷ Or more) T cells have a mutation (e.g., an inactivating mutation) at the same hit gene, such as an inactivating mutation at the same target site of the same hit gene, or an inactivating mutation at a different target site of the same hit gene. In some embodiments, the T cell library comprises a plurality of T cells that contain mutations (e.g., inactivating mutations) in at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95% or more of the hit genes of the genome. In some embodiments, the T cell library comprises a plurality of T cells (also referred to herein as a "whole genome T cell library") that comprise mutations (e.g., inactivating mutations) at all genes in the genome, such as all annotated gene human genomes. In some embodiments, for each annotated gene in the genome, or for each hit gene, there are at least two (e.g., 2, 3, 4, 5, or more, such as 2) T cells in the T cell library, each T cell comprising a mutation (e.g., an inactivating mutation) in a different target site of the same hit gene, e.g., T cell a comprises a mutation (e.g., an inactivating mutation) in target site a 'of gene X, and T cell B comprises a mutation (e.g., an inactivating mutation) in target site B' of gene X. In some embodiments, the T cell library is a targeted library comprising mutations at selected genes in a signal transduction pathway or associated with cellular processes (e.g., inactivating mutations), such as sensitivity or resistance to immune effector cell (e.g., NK cell) -mediated killing, cell proliferation, cell cycle, regulation of transcription, ubiquitination, apoptosis, immune responses such as autoimmunity, tumor metastasis, tumor malignant transformation, and the like. In some embodiments, the T cell library is used for whole genome screening associated with a particular regulatory phenotype, such as for immune effector cells (e.g., for immune effector cells) NK cell) mediated killing. In some embodiments, the T cell library is used in genome-wide screening to identify at least one target gene associated with a particular regulatory phenotype, such as a target gene in T cells that modulate the activity of T cells in response to NK cell processing. In some embodiments, the T cell library is a eukaryotic T cell library, such as a mammalian T cell library. Exemplary target gene sets encompassed by the T cell library include: the genomes of rodents (mouse, rat, hamster, guinea pig), domesticated animals (e.g., cattle, sheep, cat, dog, horse, or rabbit), non-human genomic primates (e.g., monkeys), fish (e.g., zebrafish), non-vertebrates (e.g., drosophila melanogaster and caenorhabditis elegans), and humans. In some embodiments, the T cell library is a human T cell library, such as a human whole genome T cell library.

In some embodiments, a plurality (e.g., about 2, 3, 4, 5, 10, 100, 500, 1000, 2000, 5000, 10000, or more) of T cells in a whole genome T cell library have a mutation (e.g., inactivating mutation) on the same hit gene, such a whole genome T cell library also being referred to as "having X-fold coverage of the genome" or "having X-fold coverage of each gene," where "X" is the number of T cells having a mutation (e.g., inactivating mutation) on the same hit gene. For example, a genome-wide T cell library comprising about 19,114T cells, each with a mutation at a different hit gene, such as an inactivating mutation, has an average of about 1-fold coverage of the human genome (about 19,114 annotated genes). Comprising about 1.9X 10 ⁷ A whole genome T cell library of individual T cells has an average coverage of about 1000-fold for the human genome, i.e., about 1000T cells have mutations (e.g., inactivating mutations) on the same hit gene. Comprises about 3.56X 10 ⁷ A whole genome T cell library of T cells, wherein about 2000T cells have a mutation (e.g., inactivating mutation) at the same hit gene (e.g., about 1000T cells have a mutation such as an inactivating mutation at a first target site of the same hit gene, about 1000T cells have a mutation such as an inactivating mutation at a second target site of the same hit gene(ii) a Or about 2000T cells having a mutation, such as an inactivating mutation, at the same target site of the same hit gene), has on average about 1000-fold coverage of the human genome. In some embodiments, a library of T cells described herein has an average coverage of at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, 10,000-fold, or more of a genome (e.g., a human genome). In some embodiments, the T cell libraries described herein have an average coverage of at least about 1,000-fold of the human genome. In some embodiments, a whole genome T cell library described herein has at least about 100-fold coverage of the human genome. In some embodiments, Cas9 ⁺ The sgRNA T cell library has an average coverage of about 100-fold to about 1000-fold for each sgRNA. In some embodiments, Cas9 described herein ⁺ The sgRNA (or mutagen-induced mutation) T cell library had an average coverage of about 300-fold to about 3000-fold per hit. In some embodiments, Cas9 ⁺ sgRNA ^iBAR T cell library for each sgRNA ^iBAR With an average coverage of about 25 times to about 250 times, such as about 100 times. In some embodiments, Cas9 ⁺ sgRNA ^iBAR T cell library for each group of sgRNAs ^iBAR Have an average coverage of about 100 times to about 1000 times, such as about 400 times. In some embodiments, Cas9 described herein ⁺ sgRNA ^iBAR The T cell library has an average coverage of about 300-fold to about 3000-fold (e.g., about any of 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1500, 1800, 2000, 2400, or 3000-fold) for each hit gene, such as about 300-fold.

Mutation of hit gene

In some embodiments, all annotated genes in the genome (e.g., the human genome) are selected as hits. In some embodiments, the hit gene is further selected based on expression of the encoded mRNA or protein within the T cell, or expression of the encoded protein on the T cell surface in a healthy T cell or a T cell in a disease state.

In some embodiments, the mutation of the hit gene is a pathogenic mutation or an inactivating mutation. An inactivating mutation described herein may be any mutation that results in complete abolishment or elimination of gene expression (transcription and/or translation) and/or function, such as an insertion, deletion (indel), substitution, frameshift, chromosomal rearrangement, or a combination thereof. In some embodiments, the inactivating mutation may completely eliminate transcription, translation, post-translational modifications, association with other molecules (e.g., other molecules in a protein complex), and/or function (e.g., signal transduction or receptor activation) of the gene. In some embodiments, the mutation at the hit gene is one of reducing (e.g., reducing by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more) or affecting (e.g., disrupting) one or more of: hit gene transcription, hit gene translation, hit gene mRNA processing, hit gene mRNA stability, hit gene mRNA function, hit gene protein function, association with other molecules (e.g., other molecules in a protein complex), and hit gene post-translational modifications. Mutations (e.g., inactivating mutations) of the hit gene may be within one or more regulatory regions, such as an enhancer, promoter, 5 'untranslated region (UTR), 3' UTR, or coding region (e.g., exon or splice site) of the hit gene. The hit gene described herein can be any genomic sequence, e.g., a protein-encoding gene, an RNA-encoding gene such as a small RNA (e.g., microRNA, piRNA, siRNA, snoRNA, tRNA, rRNA, and snRNA), ribosomal RNA, long non-coding RNA (lincrna), or a mitochondrial gene. The hit gene is known to be associated with a particular phenotype; or not related to a particular phenotype, such as an unknown known gene associated with a particular phenotype, or an unknown gene that has not yet been characterized. In some embodiments, the hit gene is a genomic sequence that does not encode anything or is not yet known to encode anything.

Pathogenic inactivating mutations (loss of function) of certain genes can be determined by examining experimental evidence in published scientific literature and examining key regions that may be disrupted, including but not limited to: frameshifts, missense mutations, truncation mutations, deletions, copy number variations, nonsense mutations, and gene losses or deletions. Pathogenic or inactivating mutations include, but are not limited to: homozygous deletions, biallelic (double hit) mutations, splice site mutations (e.g., second or additional splice site mutations), frameshift mutations, and nonsense mutations of the coding region, missense mutations that confirm the effect.

In some embodiments, the T cell library is generated by subjecting (e.g., contacting) a population of naive T cells (e.g., allogeneic T cells or CAR-T cells (e.g., allogeneic CAR-T cells)) to a mutagen. The mutagens can be divided into three classes: physical (e.g., gamma ray, ultraviolet radiation), chemical (e.g., ethyl methanesulfonate or EMS), and transposable elements (e.g., transposons, retrotransposons, T-DNA, retroviruses).

In some embodiments, the T cell library is generated by gene editing (e.g., whole genome gene editing) of a population of naive T cells (e.g., allogeneic T cells or CAR-T cells (e.g., allogeneic CAR-T cells)). Any known gene editing method can be used to generate the T cell libraries described herein, such as Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas based gene editing or genome engineering methods. See, e.g., Gaj et al, Trends biotechnol.2013; 31(7):397-405. In some embodiments, the T cell library is generated by whole genome gene editing of an initial population of T cells by a CRISPR/Cas-based method.

In some embodiments, the T cell library is generated by contacting a population of naive T cells (e.g., allogeneic T cells or CAR-T cells (e.g., allogeneic CAR-T cells)) with: i) sgRNA library or sgRNA described herein ^iBAR A library; and optionally ii) allowing the sgRNA construct or sgRNA to be introduced ^iBAR A Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein (e.g., Cas9) under conditions in which the construct and optional Cas component are introduced into the initial population of T cells. Thus, in some embodiments, the T cell library is generated by contacting an initial population of T cells with: i) comprising multiple sgRNA constructsA library of sgrnas of the constructs, wherein each sgRNA construct comprises or encodes a sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary (e.g., any of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site in a corresponding hit of the genome; and optionally, ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein under conditions that allow introduction of the sgRNA construct and optionally the Cas component into the initial population of T cells. In some embodiments, the T cell library is generated by contacting an initial population of T cells with: i) comprising multiple sets of sgRNAs ^iBAR sgRNA of construct ^iBAR Library, wherein each group of sgRNAs ^iBAR The construct comprises three or more (e.g., 3, 4, 5 or more, e.g., 4) sgrnas ^iBAR Constructs, each comprising or encoding a sgRNA ^iBAR Wherein three or more sgRNAs ^iBAR The guide sequences of the constructs are identical, wherein three or more sgrnas ^iBAR The iBAR sequence of each of the constructs is different from each other, and wherein each group of sgrnas ^iBAR The leader sequence of the construct is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a different target site in a corresponding hit gene of the genome (e.g., a different hit gene, or a different site within the same hit gene); and optionally, ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein under conditions that allow introduction of the sgRNA construct and optionally the Cas component into the initial population of T cells. In some embodiments, the conditions further allow for the generation of mutations at the hit genes. In some embodiments, each set of sgrnas ^iBAR The construct comprises 4 sgrnas ^iBAR Construct, and wherein 4 sgrnas ^iBAR The iBAR sequences of each of the constructs are different from each other. In some embodiments, the sgRNA library or sgRNA is used ^iBAR The library and Cas component are introduced simultaneously into the initial T cell population. In some embodiments, the sgRNA library or sgRNA is administered to a subject in need thereof ^iBAR The library and Cas component are introduced sequentially into the initial T cell population.In some embodiments, the sgRNA library or sgRNA ^iBAR The library and Cas component are introduced into the initial T cell population via separate vectors (e.g., lentiviral vectors) or separate viruses. In some embodiments, the sgRNA library or sgrnas are combined by the same vector or the same virus ^iBAR The library and Cas component are introduced into the initial T cell population. In some embodiments, the sgRNA library or sgrnas are administered by a lentiviral vector or lentivirus ^iBAR The library is introduced into an initial T cell population, and the Cas component is introduced into the initial T cell population as mRNA encoding the Cas component (e.g., Cas 9). In some embodiments, the initial T cell population has each carried a Cas component (e.g., a transgenic Cas9 or Cas9 introduced as mRNA; also referred to below as "Cas 9;) ⁺ T cells "), followed by sgRNA library or sgrnas ^iBAR The library is introduced into each cell, such as by a vector (e.g., a lentiviral vector) or a virus (e.g., a lentivirus).

In some embodiments, the T cell library comprises only sgrnas or sgrnas described herein ^iBAR Library, and not comprising a Cas component (e.g., Cas9), i.e., sgRNA library or sgrnas in a T cell library until further introduction of a Cas component (e.g., Cas9) (e.g., when sgRNA directed to B2M is introduced) ^iBAR The hit targeted by the library is inactivated. Comprising a sgRNA library or sgRNA as described herein ^iBAR T cell library of the library, hereinafter referred to as "sgRNA T cell library" or "sgRNA ^iBAR A T cell library ". In some embodiments, the T cell library comprises a library of sgrnas or sgrnas ^iBAR Libraries and Cas components (e.g., Cas9), i.e., T cell libraries, contain inactivated hit genes. In some embodiments, the initial population of T cells expresses a Cas protein. In some embodiments, the T cell library is prepared by contacting an initial population of T cells expressing a Cas protein with a sgRNA library or sgrnas described herein ^iBAR The library is contacted, which results in a T cell library containing inactivated hits. Comprising a sgRNA library or sgRNA as described herein ^iBAR Libraries and T cell libraries of Cas9 components (e.g., Cas9 protein or its encoding nucleic acid), hereinafter referred to as "Cas 9 ⁺ sgRNA T cell library "or" Cas9 ⁺ sgRNA ^iBAR A T cell library ". In some embodiments, the T cells in the initial T cell population comprise a B2M mutation (e.g., by introducing sgRNA and Cas9 against B2M), such as an inactivated B2M mutation, such T cells also referred to herein as "B2M ^- T cells ", the T cell library thus generated is referred to as" B2M ^- A T cell library ". In some embodiments, the T cells in the initial T cell population comprise sgRNA constructs directed to B2M (e.g., sgrnas directed to B2M or encoding vectors thereof). Comprising a sgRNA library or sgRNA as described herein ^iBAR Libraries and T-cell libraries for sgrnas of B2M, referred to as "B2M-sgRNA T-cell library" or "B2M-sgRNA ^iBAR A T cell library ". Comprising a sgRNA library or sgRNA as described herein ^iBAR Libraries, T cell libraries directed against sgrnas of B2M and Cas9 components (e.g., Cas9 protein or nucleic acid encoding same), hereinafter referred to as "Cas 9 ⁺ B2M ^- sgRNA T cell library ", or" Cas9 ⁺ B2M ^- sgRNA ^iBAR T cell library ", in which both B2M and the corresponding hit gene have been inactivated.

In some embodiments, at least about 50% (e.g., at least about any of 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more) of sgRNA constructs in a sgRNA library, or sgrnas ^iBAR sgRNA in libraries ^iBAR Construct, or sgRNA ^iBAR sgRNA in libraries ^iBAR Set of constructs introduced into the initial T cell population described herein, or B2M ^- T cell library, or Cas9 ⁺ B2M ^- T cell libraries. In some embodiments, at least about 95% (e.g., at least any one of about 96%, 97%, 98%, 99%, or more) of the sgRNA constructs in the sgRNA library, or the sgrnas ^iBAR sgRNA in libraries ^iBAR Construct, or sgRNA ^iBAR Multiple sets of sgrnas in a library ^iBAR Constructs introduced into the initial T cell population described herein, or B2M ^- T cell library, or Cas9 ⁺ B2M ^- T cell libraries. In some embodiments, the sgRNA library or sgRNA ^iBAR The efficiency of gene inactivation by hits in the library is at least about 80%, such as at least about any of 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, the sgRNA library or sgRNA ^iBAR The efficiency of gene inactivation by hits in the library is at least about 90%.

In some embodiments, the library of T cells comprises one or more (e.g., about 2, 3, 4, 5, 8, 10, 100, 250, 400, 500, 1,000, 2,000, 5,000, 10,000, or more) cells comprising the same sgRNA construct or the same sgRNA ^iBAR T cells of the construct that target the same hit gene. Such a T cell library is also referred to as "pair sgRNA/sgRNA ^iBAR With X-fold coverage "or" for each sgRNA/sgRNA ^iBAR Has X-fold coverage rate ", wherein 'X' expresses the same sgRNA or sgRNA ^iBAR The number of T cells of (a). In some embodiments, the T cell library is for each sgRNA or sgRNA ^iBAR Or sgRNA of each group ^iBAR Has an average coverage of about 1 to about 10,000 fold, e.g., for each sgRNA or sgRNA ^iBAR Or sgRNA of each group ^iBAR Has a coverage of from about 1 to about 5,000, from about 100 to about 10,000, from about 1,000 to about 5,000, from about 10 to about 100, from about 50 to about 500, from about 80 to about 200, from about 100 to about 400, from about 100 to about 800, from about 100 to about 1,000, from about 1 to about 1,000, from about 10 to about 1,000, or from about 300 to about 600 times. In some embodiments, the T cell library is for each sgRNA or sgRNA ^iBAR Or sgRNA of each group ^iBAR Have an average coverage of at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 100-fold, 400-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, 10,000-fold, or more. In some embodiments, the library of T cells has at least about 100-fold (e.g., about 400-fold) coverage for each sgRNA or mutation (e.g., mutagen-induced mutation). In some embodiments, each hit is targeted by about 2 to about 12 different sgrnas, or has a mutation at least 2 (e.g., about 2 to about 12) different target sites. In some embodiments, the library of T cells is directed toEach group of sgRNAs ^iBAR With a coverage of at least about 400 times (e.g., about 800 times). In some embodiments, the T cell library for each sgRNA ^iBAR With a coverage of at least about 100 times (e.g., about 200 times).

In some embodiments, the T cell library is for each sgRNA ^iBAR Having an average coverage of at least about 100-fold (e.g., at least about any of 200-, 400-, 500-, 1,000-, 4,000-, or more-fold). In some embodiments, the T cell library is for each group of sgrnas ^iBAR Having an average coverage of at least about 400-fold (e.g., at least about any of 800-, 1000-, 2000-, 4000-, 16,000-fold, or more). In some embodiments, the T cell library is directed to sgrnas ^iBAR The library has an average coverage of at least about 100-fold (e.g., at least about any of 200-, 400-, 500-, 1,000-, 4,000-fold, or more). In some embodiments, the library of T cells has an average coverage per hit of at least about 800-fold (e.g., at least about any of 1,000-, 1,600-, 2,000-, 2,400, 3,200-, 4,000-, 10,000, 16,000-fold, or more). In some embodiments, the sgRNA ^iBAR The library targets each annotated gene in the genome (i.e., sgRNA) ^iBAR The library was a whole genome sgRNA ^iBAR A library). In some embodiments, the library of T cells is directed to whole genome sgrnas ^iBAR The library has a coverage of at least about 100-fold (e.g., at least about any of 400-fold, 800-or 1,200-fold).

B2M mutation

Beta-2 microglobulin (B2M) is a component of MHC class I molecules (α 1, α 2, α 3) expressed on all nucleated cells. Host TCR α β cells recognize MHC class I molecules and distinguish between "self" and "foreign" cells. The activity of NK cells is regulated by a complex interaction of various cell surface inhibitory and activating receptors. Inhibitory receptors include the killer immunoglobulin-like receptor (KIR) and CD94/NKG2A, which recognize MHC or HLAI class molecules, allow NK cells to recognize autologous cells, and prevent them from attacking host tissues. Cells (e.g., T cells, such as allogeneic T cells) with reduced or absent expression of HLAI are targeted by NK cells as "foreign" resulting in rejection (Liu et al.

In some embodiments, a T cell library described herein further comprises a B2M mutation ("B2M-T cell library"). In some embodiments, the B2M mutation is an inactivating B2M mutation. The inactivating B2M mutation described herein may be any mutation that results in complete abolishment or elimination of B2M expression (transcription and/or translation) and/or function, such as an insertion, deletion (indel), substitution, frameshift, chromosomal rearrangement, or a combination thereof. In some embodiments, inactivating the B2M mutation may completely eliminate transcription, translation, post-translational modifications, binding to other molecules (e.g., other molecules in MHC class I molecules), and/or function (e.g., receptor recognition or antigen presentation) of B2M. In some embodiments, the B2M mutation is one or more of a decrease (e.g., a decrease of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more) or an effect (e.g., disruption) on: B2M transcription, B2M translation, B2M mRNA processing, B2M mRNA stability, B2M mRNA function, B2M protein function, B2M cell surface expression, and B2M post-translational modifications. In some embodiments, the B2M mutation is one or more of a decrease (e.g., a decrease of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more) or an effect (e.g., disruption) on: MHC class I molecules cell surface expression, assembly, function and/or ability to be recognized by NK cells. In some embodiments, the B2M mutation is a mutation in one or more regulatory regions of B2M, such as an enhancer, a promoter, a 5 'untranslated region (UTR), a 3' UTR, or a coding region (such as an exon or splice site). In some embodiments, the B2M mutation is a mutation that is not within the B2M gene or corresponding regulatory component but that affects B2M expression and/or function, such as a mutation in another molecule (e.g., a nucleic acid or protein) that affects B2 mrna splicing, B2M post-translational modifications, and the like. Has been reduced (e.g., by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%), Any of 95%, 96%, 97%, 98%, 99% or more) or cells that have eliminated B2M expression and/or function (e.g., T cells), also referred to herein as B2M negative or defective cells. In some embodiments, the T cell library comprises one or more mutations (e.g., inactivating mutations) in the B2M gene. In some embodiments, the T cells in the initial T cell population comprise a B2M mutation ("B2M") ^- T cell "), such as the inactivated B2M mutation. Such B2M-T cells are further used to construct a library of T cells as described herein. In some embodiments, a library of T cells as described herein, such as Cas9, is obtained ⁺ sgRNA/sgRNA ^iBAR T cell library, or sgRNA/sgRNA ^iBAR Following the T cell library, a B2M mutation (e.g., an inactivated B2M mutation) was introduced. In some embodiments, the B2M mutation (e.g., a deactivating B2M mutation) is generated by a mutagen, such as a physical mutagen (e.g., gamma rays, ultraviolet radiation), a chemical mutagen (e.g., ethyl methane sulfonate or EMS), or a transposable element (e.g., a transposon, retrotransposon, T-DNA, retrovirus). In some embodiments, the B2M mutation (e.g., an inactivated B2M mutation) is generated by B2M gene editing. In some embodiments, the B2M mutation (e.g., a deactivating B2M mutation) is generated by gene editing of a non-B2M gene that affects B2M expression and/or function. Any known gene editing method can be used to generate B2M described herein ^- T cell libraries, such as Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas based methods. In some embodiments, the B2M mutation (e.g., an inactivated B2M mutation) is generated by CRISPR/Cas-based gene editing of an initial population of T cells or a T cell library described herein. Thus, in some embodiments, the B2M mutation (e.g., an inactivated B2M mutation) is made by contacting an initial T cell population or a T cell library described herein (e.g., Cas9) ⁺ sgRNA/sgRNA ^iBAR T cell library, or sgRNA/sgRNA ^iBAR T cell library) with: i) one or more B2M sgRNA constructs, wherein each B2M sgRNA construct comprises or encodes a polypeptide comprising complementarity to a target site in a B2M gene (e.g., at least about 50%Any of 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complement) of a guide sequence (also referred to herein as "sgrnas for B2M", or "sgrnas targeting B2M"); and optionally, ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein (e.g., Cas9) under conditions that allow introduction of one or more B2M sgRNA constructs and optionally the Cas component into an initial population of T cells or a T cell library. In some embodiments, the B2M mutation (e.g., an inactivated B2M mutation) in the T cell library is generated with a B2M sgRNA construct. In some embodiments, the B2M mutation (e.g., an inactivated B2M mutation) in the T cell library is generated with two B2M sgRNA constructs, each construct comprising or encoding a B2M sgRNA containing guide sequences complementary to different target sites in the B2M gene. In some embodiments, the B2M sgRNA construct comprises a B2M sgRNA. In some embodiments, the B2M sgRNA construct encodes a B2M sgRNA. In some embodiments, the B2M sgRNA construct is a plasmid encoding B2M sgRNA. In some embodiments, the B2M sgRNA construct is a viral vector (e.g., a lentiviral vector) encoding a B2M sgRNA. In some embodiments, the B2M sgRNA construct is a virus (e.g., a lentivirus) encoding B2M sgRNA.

In some embodiments, the T cell libraries described herein are generated by: i) in allowing the sgRNA construct or sgRNA to be introduced ^iBAR Construction of an introduced into an initial T cell population (a "sgRNA T cell library" or "sgRNA ^iBAR T cell library ") with the sgRNA library or sgrnas described herein ^iBAR Library (e.g., by lentivirus) contact; ii) allowing introduction of the B2M sgRNA construct and the Cas component into a library comprising sgRNAs or sgRNAs ^iBAR Comprising a sgRNA library or sgrnas under conditions of T cells of the library (e.g., by electrotransformation) ^iBAR T cells of the library are contacted with a B2M sgRNA construct (e.g., B2M sgRNA) described herein and a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein (e.g., Cas9 mRNA), thereby generating Cas9 ⁺ B2M ^- sgRNA T cell library or Cas9 ⁺ B2M ^- sgRNA ^iBAR A library of T cells in which both B2M and the corresponding hit gene have been inactivated.

The guide sequence in the B2M sgRNA construct can be designed according to any method known in the art. The guide sequence may target a coding region, such as an exon or splice site, the 5 'UTR or the 3' UTR of B2M. For example, the reading frame of B2M may be disrupted by DSB insertion deletion mediated at the target site of B2M guide RNA. Alternatively, a guide RNA targeting the 5' end of the B2M coding sequence can be used to efficiently generate the B2M knockdown. The leader sequence may be designed and optimized for certain sequence features to achieve high editing activity at the target gene and low off-target effects. For example, the GC content of the guide sequence may range from about 20% to about 70%, and sequences comprising homopolymer segments (e.g., TTTT, GGGG) may be avoided.

In some embodiments, at least about 50% (e.g., at least about any of 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more) of B2M sgRNA constructs (e.g., sgrnas for B2M) and/or Cas components (e.g., Cas9 mRNA) are introduced to the initial T cell population, or Cas9 described herein ⁺ sgRNA/sgRNA ^iBAR T cell library, or sgRNA/sgRNA described herein ^iBAR T cell libraries. In some embodiments, at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) of B2M sgRNA constructs (e.g., sgrnas for B2M) and/or Cas components (e.g., Cas9 mRNA) are introduced into the sgrnas/sgrnas described herein ^iBAR T cell libraries. In some embodiments, the B2M inactivation efficiency (e.g., by B2M gene editing, such as by CRISPR/Cas with B2M sgRNA and a Cas component, such as Cas 9) is at least about 80%, such as at least any one of at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, the B2M inactivation efficiency is at least about 90%.

T cells and methods of making

In some embodiments, T-cell-containing cells are provided (e.g.E.g., an allogeneic T cell or CAR-T cell (e.g., an allogeneic CAR-T cell)) comprising a sgRNA or sgRNA described herein ^iBAR Any one of a construct, molecule, panel or library. In some embodiments, the T cell further comprises a B2M construct described herein, or one or more B2M mutations (e.g., an inactivated B2M mutation).

In some embodiments, a method of editing a genomic locus in a T cell is provided, comprising introducing into a host T cell (e.g., a primary T cell, or a T cell comprising a B2M mutation such as an inactivating B2M mutation) a guide RNA construct comprising a guide sequence targeting a genomic locus (e.g., a target site of a hit gene) and a guide RNA sequence encoding a repeat: an anti-repeat duplex and a tetracyclic guide hairpin sequence, wherein an iBAR is embedded in the tetracyclic as an internal replication, expressing the guide RNA targeting the genomic locus in the host T cell, thereby editing the targeted genomic locus (e.g., a hit gene) in the presence of a Cas nuclease (e.g., Cas 9). In some embodiments, the methods further comprise introducing the B2M sgRNA construct into a host T cell or a T cell comprising a guide RNA construct. In some embodiments, the method further comprises introducing a Cas component comprising the Cas protein or a nucleic acid encoding the Cas protein (e.g., as Cas9 mRNA) into the T cell.

In some embodiments, methods are provided by combining sgRNA libraries or sgrnas described herein ^iBAR Any of the libraries are transfected into a T cell library prepared from a plurality of host T cells (e.g., an initial population of T cells with or without a B2M mutation, such as an inactivated B2M mutation), wherein the sgRNA construct or sgRNA ^iBAR The construct is present in a viral vector (e.g., a lentiviral vector) or a virus (e.g., a lentivirus). In some embodiments, the initial T cell population is further transfected with a B2M sgRNA construct (e.g., mRNA, viral vector, or virus) described herein, or into a cell comprising a sgRNA library or sgrnas ^iBAR T cell library of the library. In some embodiments, the multiplicity of infection (MOI) between the viral vector or virus and the host T cell (e.g., initial T cell population or T cell library) during transfection is asAt least about 1. In some embodiments, the MOI is at least any one of about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or higher. In some embodiments, the MOI is about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. In some embodiments, the MOI is about any of 1-10, 1-3, 3-5, 5-10, 2-9, 3-8, 4-6, or 2-5. In some embodiments, the MOI between the viral vector or virus and the host T cell (e.g., the initial population of T cells or T cell library) during transfection is less than 1, such as less than any of about 0.8, 0.5, 0.3, or lower. In some embodiments, the MOI is from about 0.3 to about 1. In some embodiments, a viral sgRNA library or viral sgRNA ^iBAR The library is contacted with the initial population of T cells at an MOI of at least about 2, such as at least about 3. In some embodiments, the B2M sgRNA viral construct is contacted with the initial population of T cells or T cell library at an MOI of at least about 2, such as at least about 3.

In some embodiments, one or more vectors that cause expression of one or more elements of the CRISPR/Cas system are introduced into a host T cell (e.g., an initial population of T cells or a library of T cells) such that expression of the elements of the CRISPR system directs expression of the sgRNA molecules or sgrnas described herein at one or more target sites of one or more hits ^iBAR The molecules form a CRISPR complex. In some embodiments, the host T cell (e.g., the initial T cell population) has been introduced with a Cas nuclease (e.g., Cas9 mRNA), or engineered to stably express a CRISPR/Cas nuclease.

In some embodiments, the host T cell (e.g., the initial T cell population) is a T cell line, such as a pre-established T cell line. The host T cells and T cell lines may be human T cells or T cell lines, or they may be non-human, mammalian T cells or T cell lines. In some embodiments, the host T cell is difficult to transfect at low MOI (e.g., less than 1, 0.5, or 0.3) using viral vectors such as lentiviral vectors. In some embodiments, the host T cell is difficult to edit at low MOI (e.g., below 1, 0.5, or 0.3) using a CRISPR/Cas system. In some embodiments, a limited number of host T cells are obtained. In some embodiments, the host T cell is obtained from a blood sample of the individual.

Isolated culture of T cells

Prior to T cell expansion and genetic modification, a source of T cells is obtained from the individual. T cells can be obtained from a variety of sources, including Peripheral Blood Mononuclear Cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines available in the art may be used. In some embodiments, T cells can use any number of techniques known to those of skill in the art, such as FICOLL ^TM Isolated, obtained from a blood unit collected from a subject. In some embodiments, the cells from the circulating blood of the individual are obtained by apheresis techniques. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In some embodiments, cells collected by apheresis may be washed to remove the plasma fraction and placed in a suitable buffer or culture medium for subsequent processing steps. In some embodiments, the cells are washed with Phosphate Buffered Saline (PBS). In some embodiments, the wash solution lacks calcium and may lack magnesium or may lack many, if not all, divalent cations. In some embodiments, the initial activation step in the absence of calcium results in exaggerated activation. As one of ordinary skill in the art will readily appreciate, the washing step can be accomplished by methods known to those of skill in the art, such as by using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 Cell processor, Baxter CytoMate, or Cell Saver 5 by Haemonetics, Inc.) according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers, e.g., Ca-free ²⁺ And no Mg ²⁺ PBS, PlasmaLyteA or other saline solutions with or without buffer. Or, canTo remove unwanted components from the apheresis sample and to resuspend the cells directly in culture.

In some embodiments, the T cells are provided by a cord blood bank, a peripheral blood bank, or derived from induced pluripotent stem cells (ipscs), multipotent and pluripotent stem cells or human embryonic stem cells. In some embodiments, the T cell is derived from a cell line. In some embodiments, the T cells are obtained from a xenogeneic source, e.g., from mice, rats, non-human primates, and pigs. In some embodiments, the T cell is a human cell. In some aspects, the T cells are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, after blood collection, PBMCs are isolated from the donor blood sample, and then T cells are isolated from the PBMCs, for example, using immunomagnetic beads. In some embodiments, the cells comprise one or more T cell subsets, such as the whole T cell population, CD4 ⁺ Cell, CD8 ⁺ Cells and subpopulations thereof, such as those defined by the following properties: function, activation status, maturity, differentiation potential, expansion, recycling, localization and/or persistence ability, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion characteristics and/or degree of differentiation. With respect to the subject to be treated, the cells may be allogeneic and/or autologous. In certain instances, the T cells are allogeneic to one or more intended recipients. In some cases, T cells are suitable for transplantation, e.g., do not induce GvHD in a recipient. In some embodiments, the T cell is an allogeneic CAR-T cell. In some embodiments, the T cell (e.g., an allogeneic T cell) is modified to express a chimeric receptor, such as a CAR or an engineered TCR. In some embodiments, T cells (e.g., allogeneic T cells) are modified to knock out endogenous TCRs.

In T cells and/or CD4 ⁺ And/or CD8 ⁺ Among the subtypes and subpopulations of T cells, there is an initial T (T) _N ) Cells, effector T cells (T) _EFF ) Memory T cells and subtypes thereof, such as stem cell memory T (TSC) _M ) Central memory T (TC) _M ) Effect ofMemory T (T) _EM ) Or terminally differentiated effector memory T cells, Tumor Infiltrating Lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T Cells (CTLs), mucosa-associated invariant T (mait) cells, naturally occurring and adaptive regulatory T (treg) cells, helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α/β T cells, and δ/γ T cells.

In some embodiments, by lysing erythrocytes and removing monocytes, e.g., by passage through PERCOLL ^TM T cells were isolated from peripheral blood lymphocytes by gradient centrifugation or by countercurrent centrifugation elutriation. Specific T cell subsets, such as CD3, can be further isolated by positive or negative selection techniques ⁺ 、 CD28 ⁺ 、CD4 ⁺ 、CD8 ⁺ 、CD45RA ⁺ And CD45RO ⁺ T cells. For example, in some embodiments, by beads conjugated with anti-CD 3/anti-CD 28 (i.e., 3 x 28) -, such as

The CD3/CD28T were incubated together for a sufficient time to isolate T cells for positive selection of desired T cells. In some embodiments, the period of time is about 30 minutes. In further embodiments, the period of time ranges from 30 minutes to 36 hours or more, and all values therebetween. In another embodiment, the period of time is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the period of time is 10-24 hours. In some embodiments, the incubation time is 24 hours. To isolate T cells from leukemia patients, cell yield can be increased using longer incubation times, such as 24 hours. In comparison to other cell types, such as the isolation of Tumor Infiltrating Lymphocytes (TILs) from tumor tissue or immunocompromised individuals, in any case where T cells are rare, longer incubation times can be used to isolate T cells. In addition, the use of longer incubation times may improve the capture of CD8 ⁺ Efficiency of T cells. Thus, by simply shortening or extending the time allowed for T cells to bind to CD3/CD28 beads and/or by increasing or decreasingThe bead-poor to T cell ratio (as described further herein) can be used to preferentially select T cell subsets for or against at the start of the culture or at other time points in the process. In addition, by increasing or decreasing the ratio of anti-CD 3 and/or anti-CD 28 antibodies on beads or other surfaces, T cell subsets can be preferentially selected for or against at the start of culture or at other desired time points. The skilled person will appreciate that multiple rounds of selection may also be used. In some embodiments, it may be desirable to perform a selection procedure and use "unselected" cells during activation and expansion. "unselected" cells may also undergo further rounds of selection.

Enrichment of T cell populations by negative selection can be accomplished using a combination of antibodies directed against surface markers specific to the negatively selected cells. One approach is cell sorting and/or selection by negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells. For example, to enrich for CD4 by negative selection ⁺ Cells, monoclonal antibody cocktails generally include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD 8. In certain embodiments, it may be desirable for enrichment or positive selection to express CD4 in general ⁺ 、CD25 ⁺ 、 CD62Lhi、GITR ⁺ And FoxP3 ⁺ The regulatory T cell of (3). Alternatively, in certain embodiments, T regulatory cells are depleted by anti-CD 25 conjugated beads or other similar selection methods.

To isolate a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume of beads and cells mixed together (i.e., increase the concentration of cells) to ensure maximum contact of the cells and beads. For example, in one embodiment, a concentration of 20 hundred million cells/mL is used. In one embodiment, a concentration of 10 hundred million cells/mL is used. In a further embodiment, a concentration of greater than 100 million cells/mL is used. In another embodiment, 10, 15, 20, 25, 30, 35, 40, 45, or 50 million are usedCell concentration per mL. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/mL is used. In further embodiments, concentrations of 125 or 150 million cells/mL may be used. The use of high concentrations can improve cell yield, cell activation and cell expansion. In addition, the use of high cell concentrations can more effectively capture cells that may weakly express the target antigen of interest, such as CD28 negative T cells, or from samples where many tumor cells are present (i.e., leukemia blood, tumor tissue, etc.). Such cell populations may be of therapeutic value and need to be obtained. For example, CD8, which typically has weaker CD28 expression, can be more efficiently selected using high concentrations of cells ⁺ T cells.

In some embodiments, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surfaces (particles such as beads), particle-cell interactions are minimized. This will select cells expressing large amounts of the desired antigen to be bound to the particles. For example, CD4 ⁺ T cells express higher levels of CD28 and at dilute concentrations than CD8 ⁺ T cells are more efficiently captured. In some embodiments, the cell concentration used is 5X 10 ⁶ The volume is/mL. In some embodiments, the concentration used may be about 1 × 10 ⁵ To 1X 10/mL ⁶ mL, and any value in between.

In some embodiments, cells can be incubated on a spinner at different speeds for different lengths of time at 2-10 ℃, room temperature, or about 37 ℃.

T cells for stimulation may also be frozen after the washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and useful in this regard, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or media containing 10% dextran 40 and 5% glucose, 20% human serum albumin and 7.5% DMSO, or media containing 31.25% Plasmalyte-a, 31.25% glucose 5%, 0.45% NaCl, 10% dextran 40 and 5% glucose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing media containing, for example, Hespan and PlasmaLyteA, and then freezing the cells to-80 ℃ at a rate of 1 ° per minute and storing in the gas phase of a liquid nitrogen reservoir. Other controlled freezing methods may be used, and uncontrolled freezing immediately at-20 ℃ or in liquid nitrogen.

In some embodiments, cryopreserved cells are thawed and washed as described herein and allowed to stand at room temperature for one hour prior to activation.

The present application also contemplates collecting a blood sample or apheresis blood product from a subject at a time period prior to when expanded cells as described herein may be desired. Thus, the source of cells to be expanded can be collected at any necessary point in time, and the desired cells, such as T cells, isolated and frozen for later use in T cell therapy for the treatment of any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one embodiment, the blood sample or single blood sample is taken from a generally healthy subject. In certain embodiments, a blood sample or an apheresis is taken from a generally healthy subject at risk of developing a disease but who has not yet developed a disease, and the target cells are isolated and frozen for later use. In certain embodiments, the T cells may be expanded, frozen, and used at a later time. In certain embodiments, a sample is collected from a patient shortly after diagnosis of a particular disease as described herein, but prior to any treatment. In further embodiments, cells are isolated from a blood sample or a single blood sample of the subject prior to any number of relevant treatment modalities, including but not limited to: treatment with agents such as natalizumab, efuzumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies or other immunoablative agents such as camp ath, anti-CD 3 antibodies, cyclophosphamide, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporin and FK506), or inhibit p70S6 kinase (rapamycin) which is important for growth factor-induced signal transduction (Liu et al, Cell 66:807-815, 1991; Henderson et al, Immun 73:316-321, 1991; Bierer et al, curr. Opin. Immun.5:763-773, 1993). In further embodiments, cells are isolated and frozen for later use with (e.g., prior to, concurrent with, or subsequent to) the following treatments: bone marrow or stem cell transplantation, T cell ablation therapy with chemotherapeutic agents such as fludarabine, external-beam radiotherapy (XRT), cyclophosphamide or antibodies such as OKT3 or CAMPATH. In another embodiment, after B cell ablation therapy such as an agent that reacts with CD20 such as rituximab, the cells are previously isolated and may be frozen for later use in therapy.

In some embodiments, the T cells are obtained directly from the patient after treatment. In this regard, it has been observed that after certain cancer treatments, particularly treatments with immune system damaging drugs, the quality of T cells obtained may be optimal or improve their ability to expand in vitro shortly after treatment during which patients usually recover from treatment. Also, after ex vivo procedures using the methods described herein, these cells may be in a preferred state that enhances implantation and in vivo expansion. Therefore, it is contemplated in the context of the present invention to collect blood cells, including T cells, at this recovery stage. Furthermore, in certain embodiments, mobilization (e.g., with GM-CSF) and conditioning regimens can be used to create a condition in a subject in which the repopulation, recycling, regeneration, and/or expansion of a particular cell type is favored, particularly over a prescribed time window following treatment.

Activation and expansion of T cells

In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation step may comprise culturing, cultivating, stimulating, activating and/or propagating or amplifying. In some embodiments, the composition or cell is incubated in the presence of a stimulating condition or agent. Such conditions include those intended to induce proliferation, expansion, activation and/or survival of cells in the population to mimic antigen exposure and/or prepare the cells for genetic engineering. Conditions may include one or more of a specific medium, temperature, oxygen content, carbon dioxide content, time, agent such as nutrients, amino acids, antibiotics, ions, and/or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agent intended to activate a cell.

Methods that can be used to activate and expand T cells, whether before or after genetic modification of the T cells, are generally described, for example, in U.S. patent nos.: 6,352,694, respectively; 6,534,055, respectively; 6,905,680, respectively; 6,692,964, respectively; 5,858,358, respectively; 6,887,466, respectively; 6,905,681, respectively; 7,144,575, respectively; 7,067,318, respectively; 7,172,869, respectively; 7,232,566, respectively; 7,175,843, respectively; 5,883,223, respectively; 6,905,874; 6,797,514, respectively; 6,867,041, respectively; and U.S. patent application publication No. 20060121005.

Generally, T cells can be expanded by: contacting a surface having attached thereto an agent that stimulates a signal associated with the CD3/TCR complex and a ligand that stimulates a costimulatory molecule on the surface of a T cell. Specifically, a population of T cells can be stimulated as described herein, such as by contact with an anti-CD 3 antibody or antigen-binding fragment thereof, or an anti-CD 2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. To co-stimulate helper molecules on the surface of T cells, ligands that bind the helper molecules are used. For example, a population of T cells can be contacted with an anti-CD 3 antibody and an anti-CD 28 antibody under conditions suitable to stimulate T cell proliferation. To stimulate CD4 ⁺ T cells or CD8 ⁺ T cell proliferation requires the use of anti-CD 3 antibodies and anti-CD 28 antibodies. Examples of anti-CD 28 antibodies that can be used include 9.3, B-T3, XR-CD28(Diaclone, Besancon, France), other methods known in the art can be used (Berg et al, transfer Proc.30(8):3975-3977, 1998; Haanen et al, J. exp. Med.190(9):13191328,1999; Garland et al, J. Immunol meth. 227(1-2):53-63,1999).

In some embodiments, T cells are expanded as follows: by adding T cells to culture starting composition feeder cells, such as non-dividing Peripheral Blood Mononuclear Cells (PBMCs), (e.g., such that the resulting cell population comprises at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and culturing the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells may comprise gamma irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays of about 3000-. In some aspects, the feeder cells are added to the culture medium prior to addition of the T cell population.

In some embodiments, the primary and co-stimulatory signals of the T cell may be provided by different protocols. For example, the reagents that provide each signal may be in solution or bound to a surface. When bound to a surface, the agent may be bound to the same surface (i.e., in "cis" form), or bound to a separate surface (i.e., in "trans" form). Alternatively, one reagent may be bound to the surface while the other reagent is in solution. In one embodiment, the agent that provides the co-stimulatory signal binds to the cell surface, and the agent that provides the primary activation signal is in solution or bound to the surface. In certain embodiments, both agents may be in solution. In another embodiment, the agent may be in a soluble form and then cross-linked to a surface, such as a cell expressing an Fc receptor or an antibody or other binding agent that will bind to the agent. In this regard, see, e.g., U.S. patent application publications US20040101519 and US20060034810 for artificial antigen presenting cells (aapcs), which are believed to be useful for activating and expanding T cells in the present invention.

In some embodiments, the T cells are combined with beads coated with the agent, the beads and cells are subsequently separated, and the cells are then cultured. In an alternative embodiment, the agent-coated beads and cells are not isolated but are cultured together prior to culturing. In a further embodiment, the beads and cells are first concentrated by applying a force, such as a magnetic force, resulting in increased attachment of cell surface markers, thereby inducing cell stimulation.

For example, cell surface proteins can be linked by contacting T cells with paramagnetic beads (3 × 28 beads) to which anti-CD 3 and anti-CD 28 are attached. In one embodiment, the cell (e.g., 10) ⁴ To 10 ⁹ T cells) and beads (e.g.,

CD3/CD28T paramagnetic beads) in a buffer, preferably PBS (without divalent cations such as calcium and magnesium). Also, one of ordinary skill in the art will readily appreciate that any cell concentration may be used. For example, target cells may be very rare in a sample and comprise only 0.01% of the sample, or the entire sample (i.e., 100%) may include target cells of interest. Thus, any number of cells is within the scope of the invention. In certain embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact of the cells and particles. For example, in one embodiment, a concentration of about 20 hundred million cells/mL is used. In another embodiment, greater than 100 million cells/mL are used. In another embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 500 million cells/mL is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/mL is used. In further embodiments, concentrations of 125 or 150 million cells/mL may be used. The use of high concentrations can improve cell yield, cell activation and cell expansion. In addition, cells that may weakly express the antigen of interest, such as CD28 negative T cells, may be more efficiently captured using high cell concentrations. Such cell populations may have therapeutic value and in certain embodiments need to be obtained. For example, CD8, which typically has weaker CD28 expression, can be more efficiently selected using high concentrations of cells ⁺ T cells. In some embodiments, about 30 million cultured T cells are used for activation and expansion.

In some embodiments, the mixture may be incubated for a period of hours (about 3 hours) to about 14 days or any integer value of hours between the two. In another embodimentIn this case, the mixture may be cultured for 21 days. In one embodiment of the invention, the beads are cultured with the T cells for about 8 days. In another embodiment, the beads are cultured with the T cells for 2-3 days. Several stimulation cycles may also be required so that the time of culture of the T cells may be 60 days or more. Suitable conditions for T cell culture include appropriate media (e.g., minimal basal media or RPMI medium 1640 or X-vivo 15(Lonza)), which may contain factors required for proliferation and viability, including serum (e.g., fetal bovine or human serum)), interleukin-2 (IL-2), insulin, IFN- γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF β, and TNF α or any other additive known to the skilled artisan for cell growth. Other additives for cell growth include, but are not limited to: surfactants, plasma substitutes and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. The culture medium may comprise RPMI 1640, AIM-V, DMEM, MEM, alpha-MEM, F-12, X-Vivo 15 and X-Vivo 20, preferably supplemented with amino acids, sodium pyruvate and vitamins, serum-free or supplemented with appropriate amounts of serum (or plasma) or a defined set of hormones, and/or cytokines sufficient to allow T-cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in the experimental culture, and not in the cell culture to be injected into the subject. The target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37 ℃) and atmosphere (e.g., air + 5% CO) ₂ ). T cells exposed to different stimulation times may exhibit different characteristics. For example, the helper T cell population of a typical blood or apheresis peripheral blood mononuclear cell product (TH, CD 4) ⁺ ) Greater than the cytotoxic or suppressor T cell population (TC, CD 8). Expansion of T cells in vitro by stimulation of CD3 and CD28 receptors produced a population of T cells consisting primarily of TH cells before about days 8-9, while after about days 8-9, the population of T cells included an increasing population of TC cells. Thus, depending on the therapeutic objective, it may be advantageous to infuse a subject with a population of T cells comprising predominantly TH cells. Similarly, if a subpopulation of antigen-specific TC cells has been isolated, it may be beneficial to expand this subpopulation to a greater extent.

In addition, other phenotypic markers, in addition to the CD4 and CD8 markers, are significantly different but, to a large extent, reproducible during cell expansion. Thus, this reproducibility enables the tailoring of the activated T cell product for a particular purpose.

In some embodiments, the method comprises assessing the expression of one or more markers on the surface of the modified cell or cell to be engineered. In one embodiment, the method comprises assessing the surface expression of TCR or CD3 epsilon, for example by an affinity-based detection method, such as flow cytometry. In some aspects, when the method reveals surface expression of an antigen or other marker, the gene encoding the antigen or other marker is disrupted or otherwise expression is inhibited, e.g., using the methods described herein.

Isolation and enrichment of modified T cells

In some embodiments, the methods described herein further comprise isolating or enriching T cells comprising a mutation in the hit gene (e.g., an inactivating mutation) and/or a B2M mutation, such as an inactivating B2M mutation. In some embodiments, the methods described herein further comprise isolating or enriching a protein comprising a Cas component, sgRNA construct, sgRNA described herein ^iBAR T cells of the construct and/or B2M sgRNA construct. In some embodiments, the methods described herein further comprise isolating or enriching CD8 from the modified T cells ⁺ T cells.

In some embodiments, the isolation method includes, based on the presence or absence of one or more specific molecules in the cell, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids (e.g., sgrnas) ^iBAR B2M sgRNA and/or Cas-encoding nucleic acid), separate different cell types. In some embodiments, any known separation method based on such labels may be used. In some embodiments, the isolation is an affinity or immunoaffinity based isolation. For example, in some aspects, isolating comprises isolating cells and cell populations based on expression of the cells or expression levels of one or more markers (typically cell surface markers), e.g., by hybridization to a specific Antibodies or binding partners that bind such labels are incubated together, followed by a washing step, typically, and cells that are bound to the antibody or binding partner are separated from cells that are not bound to the antibody or binding partner. In some embodiments, isolating comprises isolating the cells and the population of cells based on expression of a selectable marker gene of the cells (e.g., an antibiotic resistance gene such as puromycin, or a fluorescent protein encoding gene). Such isolation steps may be based on positive selection, wherein cells that have bound the reagent, are resistant to the antibiotic, or express a fluorescent protein are retained for further use; and/or negative selection, wherein cells that are not bound to the antibody or binding partner or that do not express a fluorescent protein are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection may be particularly useful when no antibodies are available to specifically recognize cell types in the heterogeneous population, thereby making it desirable to perform the separation based on markers expressed by cells other than the desired population.

Isolation does not necessarily result in 100% enrichment or depletion of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell (e.g., a cell expressing a marker) refers to increasing the number or percentage of such cells, but need not result in the complete absence of cells that do not express the marker. Likewise, negative selection, removal, or depletion of a particular type of cell (e.g., a cell expressing a marker) refers to a reduction in the number or percentage of such cells, but need not result in complete removal of all such cells.

In some examples, multiple rounds of separation steps are performed, wherein fractions from a positive or negative selection of one step are subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step may deplete cells expressing multiple markers simultaneously, such as by incubating the cells with multiple antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can be positively selected simultaneously by incubating the cells with multiple antibodies or binding partners expressed on the various cell types.

For example, in some aspects, a particular subset of T cells, e.g., positive or high level expression of one or more surface markers such as CD28 ⁺ 、CD62L ⁺ 、CCR7 ⁺ 、CD27 ⁺ 、CD127 ⁺ 、CD4 ⁺ 、CD8 ⁺ 、 CD45RA ⁺ And/or CD45RO ⁺ T cells, isolated by positive or negative selection techniques. In some embodiments, T cells that do not express certain markers, such as the marker encoded by one or more hit genes and/or B2M, are isolated.

For example, CD3 ⁺ 、CD28 ⁺ T cells can be treated using CD3/CD28 conjugated magnetic beads (e.g.,

CD3/CD 28T Cell Expander) for positive selection.

In some embodiments, the isolation is performed by enriching a particular cell population by positive selection or depleting a particular cell population by negative selection. In some embodiments, positive or negative selection is accomplished by incubating the cells with one or more antibodies or other binding agents that specifically bind to the cells that are expressed on the positively or negatively selected cells, respectively (marker) ⁺ ) Or at a relatively high level (marker) ^{High (a)} ) The one or more surface markers of (a).

In some aspects, a sample or composition of cells to be isolated is incubated with small, magnetizable or magnetically responsive materials, such as magnetically responsive particles or microparticles such as paramagnetic beads (e.g., Dynabeads or MACS beads). Magnetically responsive materials, such as particles, are typically linked, directly or indirectly, to a binding partner, such as an antibody, that specifically binds a molecule, such as a surface marker, present on a cell, plurality of cells, or population of cells, that are desired to be isolated, such as cells that are desired to be selected negatively or positively.

In some embodiments, the magnetic particles or beads comprise a magnetically responsive material that is bound to a particular binding member, such as an antibody or other binding partner. There are many well known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in U.S. Pat. No. 4,452,773 to Molday and european patent specification EP452342B, which are incorporated herein by reference. Colloidal-sized particles, such as those described in U.S. patent No. US4,795,698 to Owen, and U.S. patent No. US5,200,084 to Liberti et al are other examples.

The incubation is typically performed under conditions such that the antibody or binding partner, or a molecule that specifically binds such antibody or binding partner, such as a secondary antibody or other agent (which is attached to the magnetic particle or bead), specifically binds to a cell surface molecule if present on the cells in the sample.

In some embodiments, the sample is placed in a magnetic field and those cells to which magnetically responsive or magnetizable particles are attached will be attracted to the magnet and separated from unlabeled cells. For positive selection, cells attracted by magnets are retained; for negative selection, cells that were not attracted (unlabeled cells) were retained. In some aspects, a combination of positive and negative selections are performed during the same selection step, wherein positive and negative fractions are retained and further processed, or subjected to further separation steps.

In certain embodiments, the magnetically responsive particles are coated in a primary or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, the magnetic particles are attached to the cells by coating with a primary antibody specific for one or more labels. In certain embodiments, cells other than beads are labeled with a primary antibody or binding partner, and then a cell-type specific secondary antibody or other binding partner (e.g., streptavidin) coated magnetic particle is added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

In some embodiments, the magnetically responsive particles are attached to cells that will be subsequently incubated, cultured, and/or engineered; in some aspects, the particles are attached to cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known, including, for example, the use of competitive unlabeled antibodies, magnetizable particles, or antibodies conjugated to cleavable linkers, and the like. In some embodiments, the magnetizable particles are biodegradable.

In some embodiments, the affinity-based selection is by Magnetic Activated Cell Sorting (MACS) (Miltenyi Biotec, Auburn, CA). Magnetically Activated Cell Sorting (MACS) systems enable high purity selection of cells with attached magnetized particles. In certain embodiments, MACS operates in a mode in which non-target species and target species are sequentially eluted after application of an external magnetic field. That is, the cells attached to the magnetized particles are fixed in place, while the unattached substances are eluted. Then, after this first elution step is completed, the substances trapped in the magnetic field and prevented from being eluted are released in such a way that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and removed from the heterogeneous cell population.

In certain embodiments, the separation (isolation) or isolation (isolation) is performed using a system, device, or apparatus that performs one or more of the isolation, cell preparation, isolation, processing, incubation, culturing, and/or formulation steps of the methods. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, for example to minimize error, user contact, and/or contamination. In one example, the system is the system described in international patent application, publication No. WO2009/072003 or US 20110003380.

In some embodiments, the system or device in an integrated or self-contained system and/or in an automated or programmable manner to perform one or more, such as all, separation, processing, engineering and preparation steps. In some aspects, the system or appliance includes a computer and/or computer program in communication with the system or appliance that allows a user to program, control, evaluate, and/or adjust the results of the processing, separating, engineering, and formulating steps.

In some aspects, the isolation and/or other steps are performed using a CliniMACS system (miltenyi biotec), e.g., for automated cell isolation at a clinical scale level in a closed and sterile system. The assembly may include an integrated microcomputer, magnetic separation unit, peristaltic pump and various pinch valves. In certain aspects, the integrated computer controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. In certain aspects, the magnetic separation unit includes a movable permanent magnet and a support for the selection post. The peristaltic pump controls the flow rate through the tubing set and, together with the pinch valve, ensures a controlled flow of buffer through the system and continuous suspension of the cells.

In certain aspects, the CliniMACS system uses antibody-bound magnetizable particles provided in a sterile, pyrogen-free solution. In some embodiments, after labeling the cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tubing set which in turn is connected to a buffer filled bag and a cell collection bag. The tubing set consists of a pre-assembled sterile tubing, including a pre-column and a separation column, for single use only. After the separation procedure is initiated, the system will automatically apply the cell sample to the separation column. The labeled cells remain within the column, while the unlabeled cells are removed by a series of washing steps. In some embodiments, the population of cells used in the methods described herein is unlabeled and not retained in the column. In some embodiments, the cell population used in the methods described herein is labeled and retained in a column. In some embodiments, the cell population used in the methods described herein is eluted from the column after removal of the magnetic field and collected in a cell collection bag.

In certain embodiments, the separation and/or other steps are performed using the CliniMACS Prodigy system (miltenyi biotec). In certain aspects, the CliniMACS Prodigy system is equipped with a cell processing device that allows for automated washing and separation of cells by centrifugation. The CliniMACS progress system may also include an onboard camera and image recognition software to determine the optimal cell fractionation endpoint by discriminating macroscopic layers of the source cell product. For example, peripheral blood is automatically divided into red blood cells, white blood cells, and plasma layers. The CliniMACS Prodigy system may also include integrated cell culture chambers that perform cell culture protocols such as cell differentiation and expansion, antigen loading, and long-term cell culture. The input port may allow for sterile removal and replenishment of culture media, while the cells may be monitored using an integrated microscope.

In some embodiments, the population of cells described herein is collected and enriched (or depleted) by flow cytometry, wherein cells stained for a plurality of cell surface markers are carried in a fluid stream. In some embodiments, the population of cells described herein is collected and enriched (or depleted) by preparative scale (FACS) -sorting. In certain embodiments, the cell populations described herein are collected and enriched (or depleted) by using a micro-electro-mechanical systems (MEMS) Chip in conjunction with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al (2010) Lab Chip 10, 1567-. In both cases, the cells can be labeled with a variety of markers, allowing the isolation of well-defined high-purity subpopulations of T cells.

In some embodiments, the antibody or binding partner is labeled with one or more detectable labels to facilitate separation of positive and/or negative selections. For example, the separation may be based on binding to a fluorescently labeled antibody. In some examples, cell separation is performed in a fluid stream, such as by Fluorescence Activated Cell Sorting (FACS), based on binding of antibodies or other binding partners specific for one or more cell surface markers, including preparation scale (FACS) and/or micro-electro-mechanical system (MEMS) chips, such as in conjunction with a flow cytometry detection system. Such methods allow for both positive and negative selection based on multiple markers simultaneously.

NK cell processing and obtaining T cells sensitive or resistant to NK cell killing

The methods described herein include contacting a T cell library described herein (e.g., Cas 9) ⁺ B2M ^- sgRNA T cell library, Cas9 ⁺ B2M ^- sgRNA ^iBAR T cell library, Cas9 ⁺ sgRNA T cell library or Cas9 ⁺ sgRNA ^iBAR T cell library) undergoes natural killingTreatment of wounded (NK) cells and obtaining T cells from the T cell bank that are sensitive or resistant to NK cell killing. In some embodiments, treating the T cell library with NK cells comprises culturing the T cell library in the presence of NK cells.

In some embodiments, the treatment with NK cells (hereinafter also referred to as "NK cell treatment step", "NK cell treatment step b)" or "step b)") comprises: i) an initial treatment step comprising contacting the T cell library with NK cells ("initial treatment step"); ii) an optional first enrichment step comprising sorting the mixture of treated cells to obtain a first subpopulation of T cells sensitive or resistant to NK cell killing ("first enrichment step"); iii) an optional first recovery step comprising culturing a first subpopulation of T cells ("first recovery step"); and iv) optionally a second treatment step comprising contacting the first subpopulation of T cells with NK cells ("second treatment step"). In some embodiments, the treating with NK cells step b) comprises a single (e.g., initial) treatment step comprising contacting the T cell library with NK cells. In some embodiments, the treating with NK cells step b) comprises: i) a single (e.g., initial) processing step comprising contacting a library of T cells with NK cells; and ii) a first recovery step comprising culturing the mixture of treated cells. In some embodiments, the treating step b) with NK cells comprises: i) an initial processing step comprising contacting a library of T cells with NK cells; ii) a first recovery step comprising culturing the mixture of treated cells; and iii) a second treatment step comprising contacting the mixture of restored treated cells with NK cells. In some embodiments, the treating with NK cells step b) comprises: i) a single (e.g., initial) processing step comprising contacting a library of T cells with NK cells; ii) a first enrichment step comprising sorting the mixture of treated cells to obtain a first subpopulation of T cells resistant to NK cell killing; and iii) a first recovery step comprising culturing a first subpopulation of T cells. In some embodiments, treating step b) with NK cells comprises: i) an initial processing step comprising contacting a library of T cells with NK cells; ii) a first enrichment step comprising sorting the mixture of treated cells to obtain a first subpopulation of T cells resistant to NK cell killing; iii) a first recovery step comprising culturing a first subpopulation of T cells; and iv) a second treatment step comprising contacting the first subpopulation of T cells with NK cells.

In some embodiments, obtaining T cells from a library of T cells that are sensitive or resistant to NK cell killing (hereinafter also referred to as "T cell obtaining step", "T cell obtaining step c", or "step c)") comprises: i) a sorting step comprising sorting the cells obtained from "NK cell treatment step b)" to obtain a second T cell subpopulation sensitive or resistant to NK cell killing ("harvest sorting step"); and ii) optionally a second recovery step comprising culturing a second subpopulation of T cells prior to harvesting the cells ("second recovery step"). In some embodiments, the T cell obtaining step c) comprises: a sorting step comprising sorting the cells obtained from "NK cell treatment step b)" to obtain a second T cell subpopulation that is sensitive or resistant to NK cell killing. In some embodiments, the T cell obtaining step c) comprises: i) a sorting step comprising sorting the cells obtained from "NK cell treatment step b)" to obtain a second T cell subpopulation resistant to NK cell killing; and ii) a second recovery step comprising culturing a second subpopulation of T cells prior to harvesting the cells.

In some embodiments, the NK cell processing step b) and the T cell obtaining step c) comprise: i) an initial processing step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 0.5:1 for about 72 hours; ii) an enrichment step comprising sorting a mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a first T cell subpopulation that is resistant to NK cell killing; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; iv) a second treatment step comprising contacting the restored first T cell subpopulation with NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3: 1; and v) a sorting step comprising sorting the final mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a second T cell subpopulation that is resistant to NK cell killing.

In some embodiments, the NK cell processing step b) and the T cell obtaining step c) comprise: i) a treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 0.5:1 for about 10 days; and ii) a sorting step comprising sorting a mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing.

In some embodiments, the NK cell processing step b) and the T cell obtaining step c) comprise: i) a treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 1:1 for about 48 hours; ii) a sorting step comprising sorting a mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing; and iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours prior to harvesting the cells.

In some embodiments, the NK cell processing step b) and the T cell obtaining step c) comprise: i) a treatment step comprising contacting the T cell library with NK cells at a ratio of NK cells to T cells of 1:1 for about 48 hours; ii) an enrichment step comprising sorting a mixture of B2M negative (or defective) and viable treated cells, thereby obtaining a first T cell subpopulation that is resistant to NK cell killing; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and iv) a sorting step comprising sorting the recovered first subpopulation of T cells that are B2M negative (or defective) and survive, thereby obtaining a second subpopulation of T cells that are resistant to NK cell killing.

Initial processing step

In some embodiments, the initial treatment step comprises contacting the T cell library with NK cells (e.g., culturing the T cell library in the presence of NK cells) for at least about 48 hours, such as at least any one of about 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, or longer. In some embodiments, the initial processing step comprises contacting the T cell library with NK cells for at least about 48 hours. In some embodiments, the initial processing step comprises contacting the T cell library with NK cells for at least about 72 hours. In some embodiments, the initial treatment step comprises contacting the T cell library with NK cells for at least about 5 days. In some embodiments, the initial treatment step comprises contacting the T cell library with NK cells for at least about 10 days.

In some embodiments, the ratio of NK cells to T cells in the T cell library in the initial processing step is any one of about 0.1:1 to about 100:1, such as about 0.1:1 to about 1:1, about 0.3:1 to about 1:1, about 0.1:1 to about 0.5:1, about 0.5:1 to about 1:1, about 1:1 to about 5:1, about 1:1 to about 10:1, about 5:1 to about 10:1, about 1:1 to about 50:1, about 1:1 to about 20:1, about 10:1 to about 100:1, about 0.1:1 to about 20:1, about 0.5:1 to about 20:1, about 0.1:1 to about 10:1, or about 0.2:1 to about 2: 1. In some embodiments, the ratio of NK cells to T cells in the T cell library in the initial processing step is about 0.5: 1. In some embodiments, the ratio of NK cells to T cells in the T cell library in the initial processing step is about 1: 1.

The longer the NK cell contact time and/or the higher the ratio of NK cells to T cells, the more severe the treatment conditions.

First enrichment step

In some embodiments, the method comprises a first enrichment step, after the initial treatment step, comprising sorting a mixture of treated cells (including NK cells and a treated T cell library) to obtain a first T cell subpopulation that is sensitive or resistant to NK cell killing. In some embodiments, the first enrichment step comprises sorting a mixture of treated cells that are T cells (or non-NK cells) and survive, thereby obtaining a first subpopulation of T cells that are resistant to NK cell killing (also referred to herein as "first live enrichment"). In some embodiments, the first enrichment step comprises sorting the mixture of treated cells that are T cells (or non-NK cells) and die, thereby obtaining a first subpopulation of T cells that are sensitive to killing by NK cells (also referred to herein as "enrichment as the first death").

In some embodiments, the first enrichment step further comprises staining the mixture of treated cells with an antibody that specifically recognizes a T cell-specific marker or an NK cell-specific marker to distinguish between T cells and NK cells prior to sorting. For example, in some embodiments, the first enrichment step comprises staining a mixture of treated cells with anti-CD 3 antibodies and/or anti-CD 56 antibodies and sorting as CD3 ⁺ And/or CD56 ^- (i.e., T cells).

In some embodiments, the first enrichment step further comprises staining the mixture of treated cells with a cell viability marker (e.g., a dye) prior to sorting. Methods and reagents for assessing cell viability are well known in the art, e.g. based on fluorescence or based on colorimetric (enzymatic) methods. For example, detection based on membrane permeability, such as staining with DAPI, Propidium Iodide (PI), 7-AAD, or amine reactive dyes indicates dead cells; whereas acridine orange stains living cells more efficiently. Carboxyfluorescein diacetate (CFDA) is a non-fluorescent, cell-penetrating dye that can be hydrolyzed by non-specific intracellular esterases present only in living cells to form the fluorescent molecule carboxyfluorescein. CFDA-SE is a derivative of CFDA that is better retained after hydrolysis in living cells. Tetramethyl rhodamine ethyl ester (TMRE) and tetramethyl rhodamine methyl ester (TMRM) localize to the mitochondria of healthy cells and the cytoplasm of dying cells. JC-1 is a commonly used potentiometric dye. In healthy cells, JC-1 localizes to mitochondria, where red fluorescent aggregates are formed. After mitochondrial membrane potential elimination, JC-1 spreads throughout the cell and exists as a green fluorescent monomer. Incorporation of BrdU into newly synthesized DNA is indicative of viable cells.

In some embodiments, the first enrichment step further comprises staining the mixture of treated cells with Propidium Iodide (PI) prior to sorting, wherein PI staining indicates cell death. Thus, in some embodiments, the first enrichment step comprises sorting as T cells (e.g., CD 3) ⁺ And/or CD56 ^- ) And PI negative (no PI staining) treated cells, thereby obtaining a first T cell subpopulation that is resistant to NK cell killing. In some embodiments, the first enrichment step comprises sorting as T cells (e.g., CD 3) ⁺ And/or CD56 ^- ) And PI positive (PI staining indicates cell death), thereby obtaining a first T cell subpopulation that is sensitive to NK cell killing.

In some embodiments, a T cell library described herein comprises a B2M mutation (e.g., comprises a B2M sgRNA construct), such as an inactivated B2M mutation. Thus, in some embodiments, the first enrichment step comprises sorting B2M negative or defective (i.e., T cells) and viable (e.g., PI) ^- ) The mixture of treated cells, thereby obtaining a first T cell subpopulation that is resistant to NK cell killing ("first live enrichment"). In some embodiments, the first enrichment step comprises sorting B2M negative or defective (i.e., T cells) and dead (e.g., PI) ⁺ ) The mixture of cells was treated, thereby obtaining a first subpopulation of T cells sensitive to NK cell killing ("enrichment of first death"). The presence or absence of the B2M mutation (e.g., the inactivating B2M mutation) can be assessed by: staining with an anti-B2M antibody, assessing the presence of a B2M sgRNA construct (e.g., a sgRNA vector backbone or B2M sgRNA), assessing the presence of a sgRNA construct targeting another gene that affects B2M expression and/or function, or detecting a B2M mutation, such as by PCR or sequencing (e.g., PCR or sequencing of the B2M locus, or PCR or sequencing of another gene that affects B2M expression and/or function). In some embodiments, the first enrichment step further comprises staining the mixture of treated cells with an anti-B2M antibody prior to sorting. Thus, in some embodiments, the first enrichment step comprises staining the mixture of treated cells with an anti-B2M antibody and PI and sorting the stained mixture of treated cells by: i) B2M ^- (or less B2M expression) and PI ^- Thereby obtaining a first subpopulation of T cells that are resistant to NK cell killing; or ii) B2M ^- (or less B2M expression) and PI ⁺ Thereby obtaining a first subpopulation of T cells sensitive to NK cell killing.

Any cell sorting method may be used herein, such as FACS, MACS, microfluidic cell sorting, Buoyancy Activated Cell Sorting (BACS), and the like. The mixture of treated cells can be sorted by cell type and viability in one sorting step or in separate sorting steps. For example, T cells (live and dead) can be sorted from a mixture of treated cells, and then live T cells (or dead T cells) can be sorted from the mixture of T cells; alternatively, live (or dead) cells (a mixture of T cells and NK cells) may be first sorted from the mixture of treated cells, and then live (or dead) T cells may be sorted from the mixture of T cells and NK cells.

First recovery step

In some embodiments, the method comprises: a first recovery step comprising culturing a mixture of treated cells (NK cells and a library of treated T cells) after an initial treatment step of contacting a library of T cells with NK cells. In some embodiments, the method comprises: a first recovery step comprising culturing a first subpopulation of T cells (i.e., live T cells) after a first enrichment step comprising sorting a mixture of treated cells to obtain the first subpopulation of T cells that are resistant to NK cell killing. In some embodiments, the first recovery step comprises culturing the mixture of treated cells (NK cells and treated T cell library) or the first T cell subpopulation for at least about 24 hours, such as at least any one of at least about 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, or longer. In some embodiments, the first recovery step comprises culturing the mixture of treated cells (NK cells and treated T cell library) or the first T cell subpopulation for about 48 hours.

The culture conditions are suitable for T cell growth and/or proliferation. In some embodiments, the culture conditions do not induce T cells to a particular phenotype during expansion. Such culture conditions are well known in the art. For example, 5% CO at 37 ℃ ₂ An incubator. See also Master et al ("T Cell Media")A Comprehensive Guide to Key Components, "2018). In some embodiments, the medium is a T cell complete medium. In some embodiments, the culture conditions are the same as those of the T cell library prior to NK cell treatment. In some embodiments, the culture conditions are suitable for adoptive T cell therapy, such as CAR-T cells (e.g., allogeneic CAR-T cells). The type of medium successfully cultured may vary from subset of T cells. For T cells, interleukin-2 (IL-2) is a potent cytokine that regulates proliferation and differentiation into effector and memory T cells. The culture conditions may be further modified to polarize the T cells to a particular phenotype during the expansion process. For example, IL-4, IL-7 and IL-15 have been reported to be critical for the induction, survival or renewal of memory T cells, respectively. The most widely used medium for culturing T cells in research laboratories is RPMI1640 supplemented with FBS, whereas "complete" formulations such as X-VIVO 15(Lonza, Inc) and CTS optizer (Thermofisher, Inc) supplemented with human serum are more common for the bio-fabrication of T cells for adoptive cell therapy. In some embodiments, the medium is further supplemented with reagents for a selectable marker, e.g., to select for T cells that do not lose the transgene or mutation during proliferation.

Second treatment step

In some embodiments, the method comprises: a second treatment step comprising contacting the mixture of treated cells (NK cells and treated T cell library) with NK cells after the initial treatment step comprising contacting the T cell library with NK cells (with or without further culturing in the recovery step). In some embodiments, the method comprises: a second processing step comprising contacting the first subpopulation of T cells with NK cells after the first enrichment step (with or without further culture in the recovery step), wherein the first subpopulation of T cells is resistant to NK cell killing in the initial processing step. In some embodiments, contacting the T cell with an NK cell comprises culturing the T cell in the presence of the NK cell.

In some embodiments, the second treatment step comprises contacting the mixture of treated cells (NK cells and treated T cell library) or the first T cell subset that is resistant to NK cell killing with NK cells for at least about 48 hours, such as at least any one of at least about 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, or longer, during the initial treatment step (with or without further culture in the recovery step). In some embodiments, the second treatment step comprises contacting the NK cells for the same or similar (e.g., up to about 30 minutes or so) amount of time as compared to the initial treatment step. In some embodiments, the second treatment step comprises a shorter time of contact with the NK cells than the initial treatment step, such as any of about 35 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days or less than the initial treatment step. In some embodiments, the second treatment step comprises contacting the NK cells for a greater time than the initial treatment step, such as any of about 35 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days or more than the initial treatment step. In some embodiments, the second treatment step comprises, during the initial treatment step (with or without further culturing in the recovery step), contacting the mixture of treated cells (NK cells and treated T cell library) or the first T cell subset that is resistant to NK cell killing with NK cells for about 96 hours.

In some embodiments, in the second NK cell treatment step, the ratio of NK cells to T cells in the first T cell subpopulation that are resistant to NK cell killing during the initial treatment step, or the ratio of NK cells to T cells from the first T cell subpopulation after the recovery step, is from about 0.1:1 to about 100:1, such as from about 0.1:1 to about 1:1, from about 0.3:1 to about 1:1, from about 0.1:1 to about 0.5:1, from about 0.5:1 to about 1:1, from about 1:1 to about 5:1, from about 1:1 to about 10:1, from about 1:1 to about 50:1, from about 1:1 to about 20:1, from about 10:1 to about 100:1, from about 0.1:1 to about 20:1, from about 0.5:1 to about 20:1, from about 0.1:1 to about 10:1, from about 5:1 to about 2:1, or from any of about 2: 1. In some embodiments, the second treatment step comprises contacting the mixture of treated cells (NK cells and treated T cell library), the first T cell subset that is resistant to NK cell killing during the initial treatment step, or T cells from the first T cell subset after the recovery step, at the same NK cell to T cell ratio. In some embodiments, the second treatment step comprises contacting the mixture of treated cells (NK cells and the treated T cell library), the first T cell subpopulation that is resistant to NK cell killing during the initial treatment step, or T cells from the first T cell subpopulation after the recovery step, at a higher ratio of NK cells to T cells, such as any of the NK cell to T cell ratios at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold higher compared to the initial treatment step. In some embodiments, the second treatment step comprises contacting the mixture of treated cells (NK cells and the treated T cell library), the first T cell subset that is resistant to NK cell killing during the initial treatment step, or T cells from the first T cell subset after the recovery step, at a lower NK cell to T cell ratio, such as at least any one of about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold lower NK cell to T cell ratio as compared to the initial treatment step. In some embodiments, in the second NK cell treatment step, the ratio of NK cells to T cells in the first T cell subpopulation that are resistant to NK cell killing during the initial treatment step, or the ratio of NK cells to T cells from the first T cell subpopulation after the recovery step is about 0.3: 1.

Optional additional recovery step

In some embodiments, the method further comprises: an additional recovery step comprising culturing a mixture of treated cells (NK cells and the treated first T cell subpopulation) after the second treatment step. In some embodiments, the additional recovery step has the same culture conditions as in the first recovery step. In some embodiments, the additional recovery step has different culture conditions than in the first recovery step. In some embodiments, the additional recovery step is longer than the first recovery step, such as at least about any of 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days longer than the first recovery step. In some embodiments, the additional recovery step is shorter than the first recovery step, such as at least about any of 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days shorter than the first recovery step.

Harvesting and sorting step

In some embodiments, obtaining T cells sensitive or resistant to NK cell killing from a T cell library ("T cell obtaining step c)") comprises: a sorting step comprising sorting the cells obtained from "NK cell treatment step b)" to obtain a second T cell subpopulation sensitive or resistant to NK cell killing ("harvesting sorting step").

In some embodiments, the cells obtained from "NK cell treatment step b)" are a mixture of treated cells (NK cells and a treated T cell library) after the initial treatment step. In some embodiments, the cells obtained from "NK cell treatment step b)" are a mixture of treated cells (NK cells and treated T cell library) after the initial treatment step, and after a first recovery step comprising culturing the mixture of treated cells. In some embodiments, the cells obtained from "NK cell treatment step b)" are a mixture of treated cells (NK cells and the treated first T cell subpopulation) after the second treatment step. In some embodiments, the cells obtained from "NK cell treatment step b)" are a mixture of treated cells (NK cells and treated T cell library) after the second treatment step, and after an additional recovery step comprising culturing the mixture of treated cells. For such embodiments, the harvest sort step is the same as or similar to the first enrichment step described above.

For example, in some embodiments, the harvesting sorting step comprises sorting a mixture of treated cells obtained from "NK cell treatment step b)" that are T cells (or non-NK cells) and that are viable (with or without further culture in the recovery step), thereby obtaining a second subpopulation of T cells that are resistant to NK cell killing (also referred to herein as "harvest live sorting"). In some embodiments, the harvesting and sorting step comprises sorting a mixture of treated cells obtained from "NK cell treatment step b)" that are T cells (or non-NK cells) and die (with or without further culturing in the recovery step), thereby obtaining a second subpopulation of T cells that are sensitive to NK cell killing (also referred to herein as "harvest-dead sorting").

In some embodiments, the harvesting and sorting step further comprises staining the mixture of treated cells obtained from "NK cell treatment step b" (with or without further culturing in the recovery step) with an antibody that specifically recognizes a T cell-specific marker or an NK cell-specific marker prior to sorting to distinguish between T cells and NK cells. For example, in some embodiments, the harvest sorting step comprises staining a mixture of treated cells obtained from "NK cell treatment step b)" with an anti-CD 3 antibody and/or an anti-CD 56 antibody and sorting as CD3 ⁺ And/or CD56 ^- (i.e., T cells).

In some embodiments, the harvesting and sorting step further comprises staining the mixture of treated cells obtained from "NK cell treatment step b" (with or without further culturing in the recovery step) with a cell viability marker (e.g., a dye) prior to sorting. Any of the reagents and/or methods described above in the section "first enrichment step" can be used herein.

In some embodiments, the harvesting sorting step further comprises staining a mixture of treated cells obtained from "NK cell treatment step b)" (with or without further culturing in the recovery step) with PI prior to sorting, wherein PI staining indicates cell death. Thus, in some embodiments, the harvesting and sorting step comprises sorting the T cells obtained from "NK cell processing step b)" (e.g. CD 3) ⁺ And/or CD56 ^- ) And PI negative (no PI staining) treated cells (with or without further culture in the recovery step), thereby obtaining a second T cell subpopulation that is resistant to NK cell killing. In some embodiments, the harvesting and sorting step comprises sorting the T cells obtained from "NK cell processing step b)" (e.g., CD 3) ⁺ And/or CD56 ^- ) And PI positive (PI staining indicates cell death) with or without further culture in the recovery step, thereby obtaining a second T cell subpopulation that is sensitive to NK cell killing.

In some embodiments, a T cell library described herein comprises a B2M mutation (e.g., comprises a B2M sgRNA construct), such as an inactivated B2M mutation. Thus, in some embodiments, the harvest sort step comprises sorting a mixture of B2M negative or defective (i.e. T cells) and viable (e.g. PI-) treated cells obtained from the "NK cell treatment step B)" to obtain a second subpopulation of T cells that are resistant to NK cell killing ("harvest live sort"). In some embodiments, the harvest sorting step comprises sorting B2M negative or defective (i.e., T cells) and dead (e.g., PI) obtained from "NK cell processing step B)" ⁺ ) A mixture of treated cells (with or without further culture in a recovery step) to obtain a second T cell subpopulation susceptible to NK cell killing ("harvest-dead sorting"). The B2M mutation (e.g., the inactivating B2M mutation)Variation), can be evaluated by: anti-B2M antibody staining, assessing the presence of a B2M sgRNA construct (e.g., a sgRNA vector backbone or B2M sgRNA), assessing the presence of a sgRNA construct targeting another gene that affects B2M expression and/or function, or detecting a B2M mutation, such as by PCR or sequencing (e.g., PCR or sequencing of the B2M locus, or PCR or sequencing of another gene that affects B2M expression and/or function). In some embodiments, the harvesting sorting step further comprises staining the mixture of treated cells obtained from "NK cell treatment step B" (with or without further culture in the recovery step) with an anti-B2M antibody prior to sorting. Thus, in some embodiments, the harvest sorting step comprises staining a mixture of treated cells (with or without further culturing in the recovery step) obtained from "NK cell treatment step B)" with an anti-B2M antibody and PI and sorting the stained mixture of treated cells, which are: i) B2M ^- (or less B2M expression) and PI ^- Thereby obtaining a second subpopulation of T cells that are resistant to NK cell killing; or ii) B2M ^- (or less B2M expression) and PI ⁺ Thereby obtaining a second subpopulation of T cells sensitive to NK cell killing.

The mixture of treated cells (with or without further culturing in the recovery step) obtained from "NK cell treatment step b)" can be sorted by cell type and viability in one sorting step or in a separate sorting step. For example, T cells (live and dead) can be sorted from a mixture of treated cells (with or without further culturing in a recovery step) obtained in "NK cell treatment step b)", and then live T cells (or dead T cells) can be sorted from the mixture of T cells; alternatively, live (or dead) cells (a mixture of T cells and NK cells) may be first sorted from the mixture of treated cells obtained in "NK cell treatment step b)" (with or without further culturing in the recovery step), and then live (or dead) T cells may be sorted from the mixture of T cells and NK cells.

Second recovery step

In some embodiments, the method comprises a second recovery step after the harvest sort step. Thus, in some embodiments, the "T cell obtaining step c)" comprises: i) a sorting step comprising sorting the cells obtained from "NK cell treatment step b)" to obtain a second T cell subpopulation resistant to NK cell killing; ii) a second recovery step comprising culturing a second subpopulation of T cells prior to harvesting the cells.

In some embodiments, the second recovery step is the only recovery step in the methods described herein. In some embodiments, the second recovery step comprises culturing the second T cell subpopulation that is resistant to NK cell killing for at least about 24 hours, such as any one of at least about 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, or longer. In some embodiments, the second recovery step comprises culturing the second subpopulation of T cells that are resistant to NK cell killing for about 48 hours.

The culture conditions are suitable for T cell growth and/or proliferation. Any of the culture conditions and/or methods described in the section "first recovery step" above may be used herein.

In some embodiments, the second recovery step has the same culture conditions as in the first recovery step (and/or optional additional recovery steps). In some embodiments, the second recovery step has different culture conditions than the first recovery step (and/or optional additional recovery steps). In some embodiments, the second recovery step is longer than the first recovery step (and/or optional additional recovery step), such as at least about any of 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days longer than the first recovery step (and/or optional additional recovery step). In some embodiments, the second recovery step is shorter in time than the first recovery step (and/or optional additional recovery step), such as at least about any of 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days shorter than the first recovery step (and/or optional additional recovery step).

T cell harvesting procedure

In some embodiments, the obtained T cells (live or dead) are harvested after obtaining a second T cell subpopulation from the harvest sorting step that is sensitive or resistant to NK cell killing, or after obtaining the second T cell subpopulation and culturing in a second recovery step. In some embodiments, the T cell harvesting step comprises collecting the T cells into a container (e.g., a Falcon tube, EP tube, or centrifuge tube) for storage or for later experimentation. In some embodiments, the T cell harvesting step comprises washing the obtained T cells such that the T cells are in conditions suitable for storage (e.g., 4 ℃, -20 ℃, or-80 ℃) or later experiments (e.g., cell lysis and PCR or sequencing).

NK cell and preparation method

NK cells are lymphocytes involved in the immune response. They have the function of killing tumor cells, cells undergoing oncogenic transformation, and other abnormal cells in vivo, and are an important component of the innate immune surveillance mechanism. NK cells have mechanisms that distinguish between "foreign" or potential target cells and healthy "self" cells through a variety of inhibitory and activating receptors that bind to MHC class I molecules, MHC class I-like molecules, and MHC independent molecules (Caliguri, Blood 2008,112: 461-69). Cells (e.g., T cells, such as allogeneic T cells) with reduced or absent expression of HLAI are targeted by NK cells as "foreign" resulting in rejection (Liu et al.

NK cells express characteristic NK cell surface receptors and lack TCR rearrangement and T cells, B cells, monocytes and/or macrophagesA surface marker. NK cells exhibit cytotoxicity by releasing small cytoplasmic granules of proteins (perforin and granzyme) that cause death of target cells by apoptosis. Killing is triggered in a non-phagocytic process that is contact-dependent, which does not require prior sensitization to the antigen. Human NK cells are characterized by the presence of cell surface markers CD16 and CD56, but by the absence of T cell receptor (CD 3). Human bone marrow-derived NK cells also known as CD2 ⁺ CD16 ⁺ CD56 ⁺ CD3 ^- The phenotype is characterised and comprises a T cell receptor zeta (zeta) chain [ zeta (Q-TCR)]) Typically characterized by NKp46, NKp30 or NKp 44. Inhibitory NK cell receptors include: HLA-E (CD94/NKG 2A); HLA-C (group 1 or 2), KIR2 DL; KIR3DL (HLA-B Bw4) and HLA-A3 or A4+ peptides. Activating NK cell receptors including HLA-E (CD94/NKG 2C); KIR2DS (HLA-C) and KIR3DS (HLA-Bw 4). Other receptors include NK cell receptor protein-1 (called NK1.1 in mice) and the low affinity receptor for the Fc portion of IgG (Fc γ RIII; CD 16).

Methods for the Isolation, culture, induction, expansion and enrichment of NK Cells are well known in the art, for example, US9,938,498 or Magee et al ("Chapter Nine- -Isolation, culture and propagation of Natural Killer Cells," Natural Killer Cells, Basic Science and Clinical Application,2010, P125-135). See also the "T cells and methods of preparation" section above, which methods are applicable to the preparation of NK cells. For example, FACS with antibodies to NK cell-specific markers can be used for the isolation and/or enrichment of NK cells.

The NK cells of the present invention may be derived from any source comprising such cells. NK cells are present in many tissues, for example, from lymph nodes, spleen, liver, lung, intestine, decidua, as well as from iPS cells or Embryonic Stem Cells (ESC). Typically, cord blood, peripheral blood, mobilized peripheral blood, and bone marrow contain heterogeneous lymphocyte populations for providing large numbers of NK cells for research and clinical use. In some embodiments, the method comprises culturing a population of NK cells derived from one of umbilical cord blood, peripheral blood, or bone marrow. In some embodiments, the cells are derived from cells comprising NK cells, CD3 ^- Cells and CD3 ⁺ The heterogeneous population of cells cultures the NK cells.In one embodiment, CD3 ⁺ Partially larger than CD3 ^- NK cell fraction, which is a characteristic feature of bone marrow, umbilical cord blood or peripheral blood. In some embodiments, the population of NK cells is selected or enriched for NK cells. In some embodiments, the NK cells may be propagated from a fresh cell population, while other embodiments propagate NK cells from a stored cell population (e.g., cryopreserved and thawed cells) or a previously cultured cell population. In some embodiments, the NK cells are from a cell line, e.g.

In some embodiments, the NK cells are a homogenous NK cell population (i.e., express the same cell surface marker). In some embodiments, the NK cells are a heterogeneous NK cell population. In some embodiments, a population of cells comprising NK cells is used to process a library of T cells described herein. In some embodiments, the NK cell is a selected NK cell population, e.g., CD56 ⁺ CD3 ^- NK cells, CD56 ⁺ CD16 ⁺ CD3 ^- NK cells or CD56 ⁺ CD16 ^- CD3 ^- NK cells. Methods for selecting NK cells based on phenotype are well known in the art, such as immunodetection or FACS analysis.

Methods for enriching and isolating lymphocytes are well known in the art, and a suitable method may be selected depending on the desired population. For example, in one method, source material of lymphocytes is enriched by removing red blood cells. In its simplest form, removal of red blood cells may involve centrifugation of uncoagulated whole blood or bone marrow. Red blood cells are separated from lymphocytes and other cells according to density. The lymphocyte-rich fraction can then be selectively recovered. Lymphocytes and their progenitors can also be enriched by centrifugation using separation media such as standard Lymphocyte Separation Media (LSM) available from various commercial sources. Alternatively, various affinity-based procedures can be used to enrich for lymphocytes/progenitor cells. Many antibody-mediated affinity preparation methods are known in the art, such as antibody-conjugated magnetic beads. Lymphocyte enrichment can also be performed using commercially available preparations, either negative Sexually selecting unwanted cells, e.g. FICOLL-HYPAQUE ^TM And other density gradient media for enrichment of intact lymphocytes, T cells, or NK cells.

Hit gene identification

The methods described herein include identifying a library of T cells (e.g., Cas 9) ⁺ B2M ^- sgRNA T cell library, Cas9 ⁺ B2M ^- sgRNA ^iBAR T cell library, Cas9 ⁺ sgRNA T cell library or Cas9 ⁺ sgRNA ^iBAR T cell library) obtained from T cells sensitive or resistant to NK cell killing ("hit gene identification step"). In some embodiments, hits identified in T cells sensitive or resistant to NK cell killing obtained from a T cell bank (or a population of treated T cells) are considered target genes whose mutation renders the T cell sensitive or resistant to NK cell killing, respectively.

In some embodiments, the hit gene identification step comprises: i) identifying a sequence comprising a hit gene mutation (e.g., an inactivating mutation) in a T cell obtained from the "T cell obtaining step c)" (or the treated T cell population); and ii) identifying a hit corresponding to a sequence comprising a hit mutation (e.g., an inactivating mutation). In some embodiments, sequences comprising a hit gene mutation (e.g., an inactivating mutation) are identified by sequencing, such as PCR sequencing (e.g., Sanger sequencing) or genomic sequencing (or DNA-seq, such as next generation sequencing or "NGS"). For example, in some embodiments, sequences of T cells (nucleic acid fragments, PCR fragments, or whole genomes) obtained from a T cell library (or treated T cell population) that are sensitive or resistant to NK cell killing are identified by sequencing, and sequences comprising a hit gene mutation (e.g., an inactivating mutation) can be identified and classified as a hit gene by comparison to the wild-type genomic sequence, or by comparison to the genomic sequence of the initial T cell population. In some embodiments, the hit gene identification step further comprises isolating genomic DNA or RNA from T cells obtained in the "T cell obtaining step c" (or the treated T cell population). In some embodiments, the hit gene identification step further comprises PCR amplification of a nucleic acid sequence comprising a hit gene mutation (e.g., an inactivating mutation).

In some embodiments, a T cell library described herein comprises sgRNA constructs or sgrnas directed against the hit genes described herein ^iBAR Constructs. Thus, in some embodiments, the hit gene identification step comprises: i) identifying sgRNA sequences or sgRNAs in T cells obtained from "T cell obtaining step c)" (or treated T cell population) ^iBAR A sequence; and ii) identification of sgRNA or sgRNA ^iBAR The guide sequence of (c) corresponds to (targets) the hit gene. In some embodiments, the sgRNA sequence or sgRNA ^iBAR Sequences are identified by RNA sequencing (RNA-seq) such as RNAGS. In some embodiments, the hit gene identification step comprises: i) identifying sgRNA or sgRNA encoded in T cells obtained from "T cell obtaining step c)" (or treated T cell population) ^iBAR The nucleic acid sequence of (a); and ii) identifying the hit gene corresponding to the leader sequence encoded by the nucleic acid sequence. In some embodiments, the sgRNA or sgRNA is encoded ^iBAR The nucleic acid sequence of (a) is identified by sequencing, for example PCR sequencing (e.g. Sanger sequencing) or genomic sequencing (DNA-seq), for example NGS. In some embodiments, iBAR sequences can be used to identify sgrnas ^iBAR Sequences or encoding sgRNAs ^iBAR The nucleic acid sequence of (1). In some embodiments, the hit gene identification step further comprises isolating genomic DNA or RNA from the T cells obtained in "T cell obtaining step c" (or the treated T cell population). In some embodiments, the hit gene identification step further comprises encoding the sgRNA or sgRNA ^iBAR PCR amplification of the nucleic acid sequence of (1).

Methods of DNA-seq, RNA-seq, PCR-sequencing (e.g., Sanger sequencing), DNA/RNA extraction, cDNA preparation, and data analysis are well known in the art and can be used herein as appropriate to identify hits in T cells from a T cell library (or treated T cell population) that are sensitive or resistant to NK cell killing. Sequencing data can be analyzed and aligned to the genome using any method known in the art.

Target Gene identification

In some embodiments, a hit identified in a T cell from a T cell library (or treated T cell population) that is sensitive or resistant to NK cell killing is considered a target gene in the T cell that modulates T cell activity. For example, in some embodiments, the hit identified in a T cell from a T cell library that is sensitive to NK cell killing (i.e., a dead T cell subpopulation) is a target gene whose mutation (e.g., inactivation) renders the T cell sensitive to NK cell killing. In some embodiments, the hit identified in a T cell from a T cell library that is resistant to NK cell killing (i.e., a subpopulation of live T cells) is a target gene whose mutation (e.g., inactivation) renders the T cell resistant to NK cell killing.

In some embodiments, hits identified in T cells from the T cell library (or treated T cell population) that are sensitive or resistant to NK cell killing are further compared to a control, and/or further ranked and/or filtered with a predetermined threshold level. In some embodiments, identifying the target gene comprises: i) Obtaining a sequence comprising a hit gene mutation (e.g., an inactivating mutation) in the final T cell subpopulation obtained from "T cell obtaining step c"); ii) ordering sequences comprising hit gene mutations (e.g., inactivating mutations) based on sequence count; and iii) identifying hit genes corresponding to sequences comprising hit gene mutations (e.g., inactivating mutations) ranked above a predetermined threshold level. In some embodiments, the ordering step comprises adjusting the ordering of each sequence comprising a hit gene mutation (e.g., an inactivating mutation) based on data consistency between all sequences comprising a hit gene mutation (e.g., an inactivating mutation) corresponding to the same hit gene (or the same target site of the same hit gene). For example, data inconsistencies (e.g., fold changes in different directions relative to a control) will increase the variance of sequences that contain hit gene mutations (e.g., inactivating mutations) corresponding to the same hit genes and decrease the ordering of such hit genes. In some embodiments, the hit is identified as corresponding to a sequence comprising a hit mutation (e.g., an inactivating mutation) whose ordering is consistently better than expected for the aligned sequence under the null hypothesis based on the RRA or alpha-RRA algorithm. In some embodiments, the predetermined threshold level is the value "X" of FDR (e.g., 0.15 or 0.05), and the corresponding to the FDR ≦ "X" hit gene mutations (e.g., inactivating mutations) of the sequence identified as the target gene. In some embodiments, the predetermined threshold level is a value "X" times (e.g., about 2 times) enrichment or depletion, and the hit gene corresponding to the sequence comprising the hit gene mutation having ≧ "X" times enrichment or depletion (e.g., an inactivating mutation) is identified as the target gene. In some embodiments, sequences comprising a hit gene mutation (e.g., an inactivating mutation) are identified by sequencing, e.g., Sanger sequencing or genomic sequencing (or DNA-seq, such as NGS).

In some embodiments, a T cell library described herein comprises sgRNA constructs or sgrnas directed against the hit genes described herein ^iBAR Constructs. Thus, in some embodiments, identifying the target gene comprises: i) obtaining sgRNA sequences or sgRNAs in the final T-cell subpopulation obtained from "T-cell obtaining step c)" ^iBAR A sequence; ii) sequencing of the sgRNA sequence or sgRNA on the basis of sequence counts ^iBAR Ordering the corresponding leader sequences of the sequences; and iii) identifying the hit genes corresponding to the leader sequences that are ranked above a predetermined threshold level. In some embodiments, the ordering comprises adjusting the sgRNA sequence or the sgRNA based on data consistency between all guide sequences corresponding to the same hit gene (or the same target site of the same hit gene) ^iBAR The ordering of each boot sequence of the sequence. For example, data inconsistencies (e.g., different fold changes relative to the control) can increase the variance of the leader sequence corresponding to the same hit, decreasing the rank of the hit. In some embodiments, the hit genes are identified as corresponding to leader sequences whose ordering is consistently better than expected for aligned sequences under the null hypothesis based on the RRA or alpha-RRA algorithm. In some embodiments, the predetermined threshold level is an FDR of value "X" (e.g., 0.15 or 0.05), and a hit gene corresponding to a leader sequence having FDR ≦ X "is identified as the target gene. In some embodiments, the method comprises The thresholding level is a value "X" times (e.g., about 2 times) enriched or depleted, and hits corresponding to leader sequences having ≧ X "times enriched or depleted are identified as target genes. In some embodiments, the sgRNA sequence or sgRNA ^iBAR Sequences are identified by RNA-seq, e.g., RNA NGS. In some embodiments, the sgRNA or sgRNA is encoded ^iBAR The nucleic acid sequence of (a) is identified by genomic sequencing (DNA-seq), e.g., NGS.

In some embodiments, a T cell library described herein comprises sgrnas directed to the hit genes described herein ^iBAR Constructs. In some embodiments, identifying the target gene comprises: i) obtaining sgrnas in the final T cell subpopulation obtained from "T cell obtaining step c)" ^iBAR A sequence; ii) sequence count based on sgRNA ^iBAR Ordering respective ones of the sequences, wherein the ordering includes ordering based on the sgRNA corresponding to the guide sequence ^iBAR Data consistency between iBAR sequences in the sequence adjusts the ordering of each pilot sequence; and iii) identifying the hit genes corresponding to the leader sequences ranked above a predetermined threshold level. In some embodiments, the hits are identified as corresponding to leader sequences whose ordering is consistently better than expected for the aligned sequences under the null hypothesis based on the RRA or alpha-RRA algorithm. In some embodiments, the predetermined threshold level is an FDR of value "X" (e.g., 0.15 or 0.05) and corresponds to having an FDR <The hit gene of the leader sequence of "X" is identified as the target gene. In some embodiments, the predetermined threshold level is at least about 2-fold enrichment or depletion.

In some embodiments, the sequence count of sequences comprising a hit gene mutation (e.g., an inactivating mutation) or a guide RNA is determined by statistical analysis. In some embodiments, the sequence count of the guide RNA and the corresponding iBAR sequence are determined by statistical analysis. An exemplary target gene identification workflow is shown in figure 5. Statistical methods can be used to determine hit gene mutations (e.g., inactivating mutations), sgRNA molecules, or sgrnas that comprise enrichment or depletion in a final T cell subpopulation ^iBAR The identity of the sequence of the molecule. In some embodiments, the NK cell is transfected with a peptideThe treated T cell library is subjected to more than one (e.g., 2, 3, or more) biological or technical replicate. In some embodiments, more than one (e.g., 2, 3, or more) biological or technical replicate is performed on a library of control T cells or a subpopulation of control T cells. In some embodiments, sequences comprising hit gene mutations (e.g., inactivating mutations) or guide RNAs from two or more (e.g., 2, 3, 4, or more) repeats of an NK cell treatment group (or control group) are combined to calculate the mean and variance between repeats of the NK cell treatment group (or control group). Exemplary statistical methods include, but are not limited to: linear regression, generalized linear regression, and hierarchical regression. In some embodiments, the sequence counts are subjected to a normalization method, such as total count normalization or median ratio normalization. In some embodiments, for example, for positive screening, median ratio normalization is preferred. In some embodiments, for example, for sequence counts that follow a normal distribution, the sequence counts are median ratio normalized, followed by mean-variance modeling. In some embodiments, MAGeCK (Li, w.et al. MAGeCK enables robust identification of essential genes from genome CRISPR/Cas9 knock-out probes genome Biol 15,554(2014)) is used to rank sequences comprising hit gene mutations (e.g., inactivating mutations) or sequences of guide RNA sequences, and/or to identify target genes. In some embodiments, the MAGECK is administered in the presence of a pharmaceutically acceptable carrier ^iBAR (Zhu et al, Genome biol.2019; 20:20) for sequencing sequences comprising hit gene mutations (e.g., inactivating mutations) or guide RNA sequences, and/or identifying target genes.

In some embodiments, the sgRNA (or sgRNA) is based on the sgRNA in T cells (or treated T cell population) from the T cell library obtained from step c) that are susceptible or resistant to NK cell killing and in control T cells (or control T cell population) ^iBAR ) Or differences between features of the hit gene mutation, identifying the target gene whose mutation renders the T cell susceptible or resistant to NK cell killing. In some embodiments, the T cells (or treated T cell population) that are sensitive or resistant to NK cell killing based on the T cell library obtained from step c) andthe target gene is identified by the difference between features of the hit gene mutation in the T cell (or control T cell population). In some embodiments, the sgRNA (or sgRNA) in the control T cell (or control T cell population) and the T cell sensitive to or resistant to NK cell killing (or treated T cell population) based on the T cell from the T cell library obtained from step c) are isolated from the control T cell (or control T cell population) and the sgRNA ^iBAR ) The target gene is identified by the difference between the characteristics of (a). In some embodiments, the control T cell population is obtained from the same T cell library cultured under the same conditions without treatment with NK cells, and optionally subjected to the same obtaining method in step c). In some embodiments, the sgRNA (or sgRNA) in the T cells (or treated T cell population) and the control T cells (or control T cell population) that are sensitive or resistant to NK cell killing obtained from step c) from the T cell library ^iBAR ) Or hit the characteristics of a gene mutation, identified by Next Generation Sequencing (NGS), such as DNA-seq or RNA-seq. In some embodiments, the sgRNA (or sgRNA) ^iBAR ) Includes sgRNA (or sgRNA) ^iBAR ) Sequence count of (a), or sgRNA (or sgRNA) ^iBAR ) The sequence count of the corresponding boot sequence. In some embodiments, the sgRNA (or sgRNA) ^iBAR ) Includes encoding sgRNA (or sgRNA) ^iBAR ) Or encodes a corresponding sgRNA (or sgRNA) ^iBAR ) Sequence count of the nucleic acid of the leader sequence of (a). In some embodiments, the characteristics of the hit gene mutation comprise a sequence count of the sequence comprising the hit gene mutation. In some embodiments, the methods described herein further comprise culturing the same T cell library under the same conditions without NK cell treatment, and optionally performing the same obtaining method in step c).

In some embodiments, the sequences obtained from the final T cell subpopulation (or treated T cell population) obtained in "T cell obtaining step c)" are counted (sgrnas or sgrnas) ^iBAR Or a leader sequence thereof, encoding sgRNA or sgRNA ^iBAR Or a nucleic acid sequence of a leader sequence thereof, or a sequence comprising a sequence of a hit gene mutation) is compared to a corresponding sequence count obtained from a control T cell subpopulation or a control T cell library, examples E.g., to provide fold changes (e.g., actual fold changes, or derivatives of fold changes, such as log2 or log10 fold changes), for significance testing (e.g., FDR, p-value), for distribution statistics, and/or to provide gene or sequence ordering by scoring and/or derivation. In some embodiments, the control T cell subpopulation (or control T cell population) is obtained from the same T cell library cultured under the same conditions without undergoing NK cell treatment, e.g., from the start of the test to the final sample harvest, cultured under the same culture conditions as the test group for the same amount of time (treated with NK cells) (see fig. 2). In some embodiments, the control T cell subpopulation is a whole T cell library cultured under identical conditions, which has not undergone NK cell treatment, nor any selection or acquisition method in "T cell acquisition step c)", hereinafter also referred to as "control T cell library". In some embodiments, the control T cell subpopulation is obtained from the same T cell library cultured under the same conditions, which has not undergone NK cell treatment, and employs the same acquisition method as in "T cell acquisition step c)".

In some embodiments, the methods described herein further comprise culturing the same T cell library under the same conditions without NK cell treatment, and optionally performing the same obtaining method in step c) to obtain a control T cell population. In some embodiments, the method further comprises identifying a population of control T cells or a library of control T cells that comprises a hit gene mutation (e.g., an inactivating mutation) or a sgRNA or sgRNA ^iBAR The sequence of the leader sequence of (1). In some embodiments, a gene comprising a hit mutation (e.g., an inactivating mutation) or a sgRNA or sgRNA is identified as compared to a gene from a control population of T cells or a control T cell library ^iBAR Is not present, and identifying the presence in the T cells (or treated T cell population) obtained from step c) that are sensitive or resistant to NK cell killing, identifying the hit as a target gene. In some embodiments, a gene comprising a hit mutation (e.g., an inactivating mutation) or a sgRNA or sgRNA is identified as compared to a gene from a control T cell population or a control T cell library ^iBAR Of the leader sequence ofIdentifying the presence of the hit gene in the target gene if it is not present in the T cells (or treated T cell population) sensitive or resistant to NK cell killing obtained from step c). For example, for a T cell library comprising mutations A, B and C in individual T cells, if only mutation a is identified from a treated (e.g., surviving) population of T cells, while the absence of mutations B and C is identified in the treated (e.g., surviving) population of T cells, it is indicated that the hits B and C are target genes, e.g., conferring sensitivity to NK cell killing when mutated. For another example, if only mutation a is identified from a treated population of T cells (e.g., dead), and the absence of mutations B and C is identified in the treated population of T cells (e.g., dead), it is an indication that the hit genes B and C are target genes, e.g., conferring resistance to NK cell killing when mutated.

In some embodiments, the obtained treated T cell population is a live T cell that is resistant to NK cell killing. In some embodiments, identifying the target gene comprises: sgRNA (or sgRNA) obtained from the treated T cell population ^iBAR Or a guide sequence thereof, or encodes a sgRNA or sgRNA ^iBAR Or a guide sequence thereof) with sgrnas (or sgrnas) obtained from a control T cell population ^iBAR Or a guide sequence, or encodes a sgRNA or sgRNA ^iBAR Or a nucleic acid of a leader sequence thereof) is compared, wherein: i) in at least one NK cell treatment of FDR ≦ 0.05 (e.g., any of FDR ≦ 0.04, 0.03, 0.02, 0.01, 0.001, or less), or in at least two separate different NK cell treatments of FDR ≦ 0.15 (e.g., any of FDR ≦ 0.1, 0.05, 0.01, 0.001, or less), their corresponding sgRNAs (or sgRNAs) ^iBAR ) The leader sequence is identified as a hit gene that is enriched (and/or has at least about a 2-fold enrichment, such as any of at least about a 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more fold enrichment) in the treated T cell population (e.g., viable, resistant to NK cell killing) compared to a control T cell population, as a target gene whose mutation renders the T cell resistant to NK cell killing; and/or ii) at FDR ≦ 0.01 (e.g., FDR ≦ 0.009, 0.0 07. 0.005, 0.001, 0.0005 or less), or in at least two separate different NK cell treatments of FDR ≦ 0.05 (e.g., any of FDR ≦ 0.04, 0.03, 0.02, 0.01, 0.001 or less), their respective sgrnas (or sgrnas ^iBAR ) The leader sequence is identified as a hit that is depleted (and/or has at least about 2-fold depletion, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more depletion) in the treated population of T cells (e.g., viable, resistant to NK cell killing) compared to a control population of T cells, and is identified as a target gene whose mutation sensitizes the T cells to NK cell killing. In some embodiments, the sgrnas (or sgrnas) ^iBAR Or a leader sequence thereof, or encodes sgRNA or sgRNA ^iBAR Or the nucleic acid of its leader sequence) were normalized for median ratio and then modeled for mean-variance. In some embodiments, identifying the target gene comprises: comparing the sequence count of the hit gene mutations obtained from the treated T cell population to the sequence count of the hit gene mutations obtained from the control T cell population, wherein: i) in at least one NK cell treatment of FDR ≦ 0.05 (e.g., any of FDR ≦ 0.04, 0.03, 0.02, 0.01, 0.001, or less), or in at least two separate different NK cell treatments of FDR ≦ 0.15 (e.g., any of FDR ≦ 0.1, 0.05, 0.01, 0.001, or less), whose corresponding hit mutant sequences were identified as having an enrichment (and/or having at least about 2-fold enrichment, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) in a treated T cell population (e.g., surviving, resistant to NK cell killing) compared to a control T cell population, are identified as target genes whose mutation renders the T cells resistant to NK cell killing; and/or ii) in at least one NK cell treatment of FDR ≦ 0.01 (e.g., any of FDR ≦ 0.009, 0.007, 0.005, 0.001, 0.0005 or less), or in at least two separate, different NK cell treatments of FDR ≦ 0.05 (e.g., any of FDR ≦ 0.04, 0.03, 0.02, 0.01, 0.001 or less), whose corresponding hit gene mutation sequences were identified as being at treatment as compared to a control T cell population Hit genes that are depleted (and/or have at least about 2-fold depletion, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more depletion) in a subsequent population of T cells (e.g., viable, resistant to NK cell killing) are identified as target genes whose mutation sensitizes the T cells to NK cell killing. In some embodiments, the sequence counts of hit gene mutations are median ratio normalized, followed by mean-variance modeling.

In some embodiments, the obtained population of treated T cells is dead T cells susceptible to NK cell killing. In some embodiments, identifying the target gene comprises: sgRNA (or sgRNA) obtained from the treated T cell population ^iBAR Or a guide sequence thereof, or encodes a sgRNA or sgRNA ^iBAR Or a guide sequence thereof) with sgrnas (or sgrnas) obtained from a control T cell population ^iBAR Or a guide sequence, or encodes a sgRNA or sgRNA ^iBAR Or a leader sequence thereof) is compared, wherein: i) in at least one NK cell treatment of FDR ≦ 0.01 (e.g., any of FDR ≦ 0.009, 0.007, 0.005, 0.001, 0.0005, or less), or in at least two separate, different NK cell treatments of FDR ≦ 0.05 (e.g., any of FDR ≦ 0.04, 0.03, 0.02, 0.01, 0.001, or less), their corresponding sgRNAs (or sgRNAs) ^iBAR ) The leader sequence is identified as a hit gene that is enriched in (and/or has at least about a 2-fold enrichment, such as any of at least about a 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more fold enrichment) the treated T cell population (e.g., dead, susceptible to NK cell killing) compared to a control T cell population, as a target gene whose mutation sensitizes the T cell to NK cell killing; and/or ii) in at least one NK cell treatment of FDR ≦ 0.05 (e.g., any of FDR ≦ 0.04, 0.03, 0.02, 0.01, 0.001, or less), or in at least two separate, different NK cell treatments of FDR ≦ 0.15 (e.g., any of FDR ≦ 0.1, 0.05, 0.01, 0.001, or less), its corresponding sgRNA (or sgRNA ^iBAR ) The leader sequence is identified as depleted in the treated T cell population (e.g., dead, sensitive to NK cell killing) as compared to the control T cell populationAn exhausted (and/or at least about 2-fold exhausted, such as at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more exhausted any of) hit gene is identified as a target gene whose mutation renders the T cell resistant to NK cell killing. In some embodiments, the sgRNA (or sgRNA) is administered to a subject ^iBAR Or a guide sequence thereof, or encodes a sgRNA or sgRNA ^iBAR Or the nucleic acid of its leader sequence) is subjected to median ratio normalization and then to mean-variance modeling. In some embodiments, identifying the target gene comprises: comparing the sequence count of the hit gene mutations obtained from the treated T cell population to the sequence count of the hit gene mutations obtained from the control T cell population, wherein: i) in at least one NK cell treatment of FDR ≦ 0.01 (e.g., FDR ≦ 0.009, 0.007, 0.005, 0.001, 0.0005, or less), or in at least two separate different NK cell treatments of FDR ≦ 0.05 (e.g., FDR ≦ 0.04, 0.03, 0.02, 0.01, 0.001, or less), whose corresponding hit gene mutation sequences were identified as enriched (and/or having at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more fold enrichment) in a treated T cell population (e.g., dead, sensitive to NK cell killing) compared to a control T cell population, as a target gene whose mutation renders the T cell sensitive to NK cell killing; and/or ii) in at least one NK cell treatment of FDR ≦ 0.05 (e.g., any of FDR ≦ 0.04, 0.03, 0.02, 0.01, 0.001, or less), or in at least two separate, different NK cell treatments of FDR ≦ 0.15 (e.g., any of FDR ≦ 0.1, 0.05, 0.01, 0.001, or less), whose corresponding hit gene mutation sequences were identified as hits that are depleted (and/or have at least about 2-fold depletion, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more-fold depletion) in a treated T cell population (e.g., dead, NK cell-sensitive) as compared to a control T cell population, are identified as target genes whose mutation renders the T cells resistant to NK cell killing. In some embodiments, the sequence counts for hit gene mutations are median ratio normalized, followed by mean-variance normalization And (6) modeling. In some embodiments, the sgRNA library is a sgRNA ^iBAR A library. In some embodiments, the sgRNA corresponding to the guide sequence is based on ^iBAR Data consistency between iBAR sequences in the sequence adjusts the variance of each pilot sequence. In some embodiments, the variance of each leader sequence or sequence comprising a hit gene mutation (e.g., an inactivating mutation) is adjusted based on data consistency between the same genes. As used herein, "data identity" refers to the identity of the sequencing results (e.g., sequence counts, normalized sequence counts, ordering, or fold changes) of the same leader sequence corresponding to different iBAR sequences in a screening experiment; or identity of the sequencing results of different hit gene mutations, such as inactivating mutations (e.g., at different targets of the same hit gene) or different sgRNA sequences corresponding to the same gene. Theoretically, the true hits of the screen should have biologically relevant similarity in performance, such as sgrnas with the same leader sequence but different ibars ^iBAR Similar normalized sequence counts, orderings, and/or fold changes corresponding to the constructs; and/or similar normalized sequence counts, orderings, and/or fold changes corresponding to the same gene but different hit gene mutant sequences, such as inactivating mutant sequences (e.g., at different target sites of the hit gene) or different sgRNA sequences. How to model mean-variance and how to base on sgRNA corresponding to the guide sequence ^iBAR Data consistency between iBAR sequences in the sequence adjusts the variance of each pilot sequence, see also WO 2020125762.

In some embodiments, the sgrnas corresponding to each guide sequence are determined based on the direction of fold change of each iBAR sequence ^iBAR Data consistency between iBAR sequences in a sequence, wherein the variance of the leader sequence increases if the fold changes of the iBAR sequences are in different directions relative to each other (e.g., increasing versus decreasing, increasing versus invariant, or decreasing versus invariant, all considered to be different directions). In some embodiments, data consistency between different hit mutation (e.g., inactivating mutation) sequences or different sgRNA sequences corresponding to the same gene is based on each hit mutation(e.g., inactivating mutation) sequence or the direction of fold change for each sgRNA sequence, wherein the variance of the hit (e.g., inactivating mutation) sequence or guide sequence increases if the fold change for different hit (e.g., inactivating mutation) sequences or different sgRNA sequences is in a different direction relative to each other. This data inconsistency leads to increased variance, which helps to rule out rare but significantly changing hit mutations (e.g., inactivating mutations)/sgrnas in positive screening at high MOI ^iBAR And (4) sequencing. For example, for the iBAR system, due to the high MOI during library construction, "sloshing" of false positive sgrnas associated with sgrnas directed against true positive hit genes may occur. As used herein, a "knock-in car" refers to a sgRNA that targets an unrelated sequence (e.g., an unrelated hit) that is mis-associated with a sgRNA that targets a true positive hit to enter the same T cell. In some embodiments, the sgRNA ^iBAR Is based on a set of sgRNAs ^iBAR The direction of enrichment for different ibars for each leader sequence in the construct was corrected. If a group of sgRNAs ^iBAR All ibars of the construct (i.e. all ibars corresponding to the same leader sequence) exhibit the same direction of fold change, i.e. are all larger or smaller than that of the control group, then the sgrnas of this group ^iBAR The variance of the construct (or the variance of the leader sequence) will remain unchanged. If a group of sgRNAs ^iBAR The ibars of the constructs (or ibars corresponding to the same leader sequence) show non-uniform direction of fold change relative to the control, and the corresponding leader sequence is penalized by increasing its variance. In some embodiments, the non-uniform sgRNA ^iBAR The final adjusted variance of (a) is the model estimated variance (e.g., by mean-variance modeling) plus the experimental variance calculated from NK cell treated samples and controls. In some embodiments, the hit gene comprises two or more (e.g., 2, 3, 4, 5, or more, such as 2) hit gene mutations (e.g., inactivating mutations), or the hit gene is targeted at different target sites by two or more (e.g., 2, 3, 4, 5, or more, such as 2) different guide sequences (e.g., two or more different sgrnas, or two different sgrnas) Group or more sgRNAs ^iBAR Constructs, each comprising a targeting sequence to a different target site). In some embodiments, the sgRNA corresponding to each guide sequence and to the same hit gene ^iBAR Data consistency between iBAR sequences in a sequence is determined based on the direction of fold change of each iBAR sequence, wherein the variance of a leader sequence increases if the fold changes of the corresponding iBAR sequences are in different directions relative to each other, and the variance of a leader sequence (or the variance of a hit) further increases if two or more (e.g., 2, 3, 4, 5 or more, such as 2) different leader sequences targeting the same hit have fold changes in different directions relative to each other. For example, for sgRNA a and sgRNA targeting different target sites of the same hit gene X, the variance of each guide sequence or hit gene is unchanged if the guide sequences of both sgRNA a and sgRNA are enriched or depleted compared to a control; if the guide sequence of the sgrna is enriched compared to the control, while the guide sequence of the sgrna is depleted, the variance per guide sequence or hit increases. In some embodiments, sgrnas corresponding to the same hit gene are determined based on the direction of fold change for each iBAR sequence ^iBAR Data consistency between iBAR sequences in a sequence, wherein if fold changes of iBAR sequences corresponding to the same hit gene are in different directions relative to each other, the variance of each leader sequence targeting the same hit gene increases, and the variance of each leader sequence targeting the same hit gene (or the variance of hit genes) increases. For example, if 2 sgRNAs ^iBAR (4 sgRNAs per group ^iBAR ) Targeting 2 different target sites of the same hit gene, the variance of these two leader sequences remains unchanged if all 8 iBAR sequences are identified as enriched compared to the control; if some iBAR sequences are identified as enriched and others as unchanged or depleted compared to the control, the variance of the two leader sequences increases.

In some embodiments, fold changes between their corresponding target sites show inclusion of hit gene mutations at different target sites of the same hit gene in different directionsA sequence that is altered (e.g., an inactivating mutation) in a fold change between its corresponding target sites shows sgrnas or sgrnas that target different target sites of the same hit gene in different directions ^iBAR Or fold change between the corresponding ibars, show that sgrnas in different directions can be penalized by increased variance, resulting in lower scores and ranks for certain hit genes. For example, if 2 sgRNAs ^iBAR (4 sgRNAs per group ^iBAR ) Targeting 2 different target sites of the same hit gene, if all 8 iBAR sequences are identified as enriched compared to the control, the hit gene has low variance and therefore higher rank and/or score (e.g., genes sensitive to NK cell killing are ranked high, have a high sensitivity score); if certain iBAR sequences are identified as enriched compared to controls, while others are identified as unchanged or depleted, the hit genes have high variance and are therefore ranked and/or scored lower (e.g., genes that are resistant to NK cell killing are ranked lower, have a low resistance score).

In a group of sgRNAs ^iBAR In the construct, the ordering of the leader sequences can be adjusted according to the identity of the predetermined threshold number m of enrichment directions of the different iBAR sequences in the set, where m is an integer between 1 and n. For example, if sgRNA ^iBAR At least m iBAR sequences of the set exhibit the same fold change direction, i.e. are all greater or less than the fold change of a control T cell subpopulation, then the ordering (or variance) of the leader sequence is unchanged. However, if more than n-m different iBAR sequences reveal inconsistent fold change directions, then sgRNA ^iBAR Sets will be penalized by decreasing their rank (e.g., by increasing their variance). In some embodiments, the ordering of sequences or leader sequences containing a hit mutation (e.g., inactivating mutation) may be adjusted (or further adjusted) according to the consistency of the enrichment directions of a predetermined threshold number m of different hit mutations (e.g., inactivating mutations) or different leader sequences corresponding to the same hit, where m is an integer between 1 and n. For example, if at least m hit gene mutations (e.g., inactivating mutations) or m leader sequences corresponding to the same hit gene exhibit the same fold The direction of change, i.e., both greater or less than the fold change of the control T cell subpopulation, then the ordering (or variance) is unchanged. However, if more than n-m different hit gene mutations (e.g., inactivating mutations) or more than n-m different leader sequences show inconsistent directions of fold change, then sequences or leader sequences comprising hit gene mutations (e.g., inactivating mutations) will be penalized by decreasing their ordering (e.g., by increasing their variance).

In some embodiments, the P value, or sgRNA, of each sequence comprising a hit gene mutation (e.g., an inactivating mutation) is calculated using the mean and variance of the treatment group (e.g., experimental variance, model estimated variance, or corrected variance based on data inconsistency) as compared to the control group ^iBAR P value for each pilot sequence.

Robust ranking Aggregation (RRA; Kolde R et al. bioinformatics.2012; 28: 573-580) or modified RRA (e.g., α -RRA in MAGECK; Li W et al. genome biol.2014; 15:554) are one of the statistical and ranking tools available in the art that can detect that ranking is consistently better than the expected gene under the zero hypothesis of uncorrelated input, assign a significance score to each gene, and combine the ranked list into a single ranking. It assumes that all information normalized ordering comes from a distribution strongly biased towards zero and detects these distributions by computing binomial probabilities from the assumed uniformly ordered distribution. And the P value assigned to each element in the aggregated list is used to rank the genes and describe how well it ranks than expected, making randomly ranked genes less important. The underlying probabilistic model frees the RRA algorithm parameters and is robust against outliers, noise and errors. The significance score also provides a rigorous approach to keep only statistically relevant genes in the final list. These attributes make this approach robust and attractive in many settings. Briefly, in RRA and a-RRA, for each sequence corresponding to a hit that includes a hit mutation (e.g., an inactivating mutation), each sgRNA guide sequence, or each sgRNA ^iBAR Leader sequence (hereinafter also referred to as "CommandMiddle gene mutation (e.g., inactivating mutation)/sgRNA guide sequence/sgRNA ^iBAR Guide sequence ") (e.g., when there are two sgRNAs targeting the same hit), the algorithm looks at all hits mutations (e.g., inactivating mutations) obtained from a T cell library (NK cell-treated T cell library, or control T cell/control T cell library) for such sequences/sgRNA guide sequence/sgRNA ^iBAR How it is located in the normalized ordered list of guide sequences and compare it to all hit mutations (e.g., inactivating mutations)/sgRNA guide sequences/sgrnas ^iBAR The baseline cases of random scrambling of the boot sequence ("permuted sequence") were compared. Thus, for all hit mutations (e.g., inactivating mutations) corresponding to their hits/sgRNA guide sequence/sgRNA ^iBAR The leader sequence is assigned a P value showing how well its position in the sorted list is than expected by chance. This P value is used to reorder hit mutations (e.g., inactivating mutations)/sgRNA guide sequences/sgrnas corresponding to the hits ^iBAR The sequences are guided and their significance is determined. Those skilled in the art will appreciate that other tools may be used for such statistics and ordering. In some embodiments, the final score for each gene hit is calculated using RRA or a-RRA to obtain an ordering of the gene hits based on the mean and variance (e.g., modified variance) of each gene hit.

In some embodiments, a hit gene mutation (e.g., an inactivating mutation), sgRNA guide sequence, or sgRNA is included ^iBAR Guide sequence (hit gene mutation (e.g., inactivating mutation)/sgRNA guide sequence/sgRNA ^iBAR Guide sequence), based on P-values calculated using the mean and variance of a Negative Binomial (NB) distribution model (e.g., modified variance adjusted for data disagreement) to estimate each hit gene mutation (e.g., inactivating mutation)/sgRNA guide sequence/sgRNA across biological/experimental replicates and treatment and control groups ^iBAR Probability of leader sequence, then applying RRA or a-RRA algorithm to identify hit gene mutation (e.g., inactivating mutation)/sgRNA leader with highest rank (e.g., highest a%, such as highest 5%) (ii)Leader sequence/sgRNA ^iBAR The targeting sequence of (a) corresponds to the hit gene of the positive selection or the negative selection. Lower RRA scores correspond to stronger hit gene enrichment. In some embodiments, the selection is below a threshold (e.g., P value)<0.25) such top-ranked hit gene mutations (e.g., inactivating mutations)/sgRNA guide sequences/sgrnas ^iBAR The P value of the leader sequence and the corresponding hit was identified as the target gene. In some embodiments, such an earlier-ordered hit mutation (e.g., inactivating mutation)/sgRNA guide sequence/sgRNA below a threshold (e.g., FDR ≦ 0.05) is selected ^iBAR The FDR of the sequence was guided and the corresponding hit was identified as the target gene. In some embodiments, when multiple hit mutations (e.g., inactivating mutations)/sgRNA guide sequences/sgrnas are designed for the same hit ^iBAR Top hit mutation (e.g., inactivating mutation)/sgRNA guide sequence/sgRNA when the sequences are guided ^iBAR Only one gene in the leader sequence is considered in the RRA or alpha-RRA calculation. RRA or a-RRA hypothesizes that if the hit has no effect on T cell sensitivity/resistance against NK cell treatment, the hit mutation (e.g., inactivating mutation)/sgRNA guide sequence/sgRNA corresponding to the hit ^iBAR Guide sequence, all hit mutations obtained from a T cell library (e.g., inactivating mutations)/sgRNA guide sequence/sgRNA ^iBAR The boot sequence should be evenly distributed in the sorted list. In some embodiments, all hit mutations (e.g., inactivating mutations)/sgRNA guide sequences/sgrnas ^iBAR Leader sequences, sorted and compared between treatment and control groups by RRA or alpha-RRA according to their relative ordering within each group and different distribution among groups. By mutating hit genes (e.g., inactivating mutation)/sgRNA guide sequence/sgRNA ^iBAR The beta-bias of the leader sequence was compared to a consistent null hypothesis model, ranking all hit genes covered by the T cell library, and their corresponding hit gene mutations (e.g., inactivating mutations)/sgRNA leader sequence/sgRNA ^iBAR The guide sequence ordering is always higher than the expected hit gene, and the guide sequence ordering adopts the Benjie-HohbergThe (Benjamini-Hochberg) program was statistically significant (P-value) by ranking tests and/or acceptable FDRs, giving preference to treatment in RRA or alpha-RRA (lower RRA score). Such RRA or a-RRA analysis can significantly reduce or eliminate false positives due to perturbations in the experiment or sampling. In some embodiments, the mutation (e.g., inactivating mutation)/sgRNA guide sequence/sgRNA based on the corresponding hit gene obtained by median ratio normalization followed by mean-variance modeling ^iBAR The ranking score of the leader sequence ranks the hit genes. In some embodiments, multiple hit mutations (e.g., inactivating mutations)/sgRNA guide sequence/sgRNA are contemplated for the same hit ^iBAR Leader sequence, further ordering of hits by RRA or α -RRA.

In some embodiments, the predetermined threshold level is from all hit gene mutations (e.g., inactivating mutations)/sgRNA guide sequence/sgRNA obtained from the experiment (treatment or control) ^iBAR FDR value for permutation test of the leader sequence. In some embodiments, FDR values are determined by considering the largest potential true target gene in a particular screen (e.g., involving a particular pathway in response to NK cell processing). In some embodiments, the threshold is the top β% of the sequence counts (normalized or unnormalized) obtained from the T cell library, and the corresponding hit genes are identified as target genes.

Any target identification method known in the art can be used herein. For example, empirical Bayesian methods (by likelihood identification of targets) or algorithms based thereon, such as CasTLE (cas9 high throughput maximum likelihood estimator) use an empirical Bayesian framework to account for multiple sources of variability, including reagent efficacy and off-target effects on large scale genome perturbation screening assays, and provide CasTLE scores for ranking and thresholding cut-off (Morgans, D.W.et al. (2016) Nat Biotechnol 34, 634-. In some embodiments, log2 ratio differences and p-values from t-tests can be used to identify target genes. For example, RIGER (Luo, J.et al. (2009). Cell 137,835-848) sorts shRNA according to their differential effects between two types of samples, and then identifies the genes targeted by the shRNA at the front of the sorted list, from which they were identified But genes that are critical for the differences between classes. The LFC and P values may be used for sorting and threshold cutoff. In some embodiments, the probability mass function of the binomial distribution (or an algorithm based thereon) may be used for target gene identification. For example, STARS (Doench, J.G., et al. (2016) Nat Biotechnol 34, 184-191), where the STAR score can be used for sorting and thresholding. In some embodiments, a negative binomial model and a-RRA algorithm based can be used for target gene identification, such as MAGeCK (Li, w.et al, (2014) Genome Biol 15,554), and RRA scores can be used for ranking and threshold cutoff. In some embodiments, algorithms based on beta-binomial modeling can be used for target gene identification, such as CRISPRBetaBinomial (CB) ² ) (Jeong, H.H.et al. (2019). Genome Res 29, 999-. In some embodiments, the sgRNA or sgRNA, e.g., during a stringent positive screen ^iBAR Raw read count ordering, normalized read count ordering, and/or log 2-fold change between treatment and control groups can be used for target gene identification, e.g., the hits corresponding to the first X% of the read counts are identified as target genes.

In some embodiments, target gene identification is a positive screen, i.e., by identifying hit gene mutation (e.g., inactivating mutation) sequences or leader sequences that are enriched in the final T cell subpopulation. In some embodiments, target gene identification is a negative screen, i.e., by identifying a hit gene mutation (e.g., inactivating mutation) sequence or leader sequence that is depleted in the final T cell subpopulation. Hit gene mutation (e.g., inactivating mutation) sequences or leader sequences enriched in the final T cell subpopulation are higher in order based on sequence count or fold change; while hit gene mutation (e.g., inactivating mutation) sequences or leader sequences that are depleted in the final T cell subpopulation are lower in rank based on sequence count or fold change. In some embodiments, enrichment or depletion is related to the total sequence count obtained from the final T cell subpopulation. In some embodiments, the enrichment or depletion is associated with a control T cell subpopulation or a corresponding sequence count in a control T cell library, such as a T cell subpopulation obtained from the same T cell library not treated with NK cells. In some embodiments, enrichment or depletion is calculated based on the RRA or alpha-RRA algorithm.

In some embodiments, the method comprises: subjecting the T cell library to at least two (e.g., at least 3, 4, 5, 6, 7, 8, 10 or more) separate distinct treatments with NK cells in step b), and obtaining NK cell killing-sensitive or resistant T cells for each treatment in step c) for target gene identification. In some embodiments, the method comprises identifying one or more hit genes in the treated population of T cells from step c) obtained from each treatment, and i) obtaining one or more hit genes identified from all treatments whose mutation renders the T cell susceptible to NK cell killing, thereby identifying a target gene whose mutation renders the T cell susceptible to NK cell killing in the T cell; or ii) obtaining one or more hits identified from all treatments whose mutation renders the T cell resistant to NK cell killing, thereby identifying a target gene whose mutation renders said T cell resistant to NK cell killing in the T cell. In some embodiments, the method comprises identifying one or more hit genes in the treated T cell population of step c) obtained from each treatment, and i) combining the one or more hit genes identified from all treatments whose mutation renders the T cell susceptible to NK cell killing, thereby identifying a target gene whose mutation renders the T cell susceptible to NK cell killing in the T cell; or ii) combining one or more hits identified from all treatments whose mutation renders the T cell resistant to NK cell killing, thereby identifying a target gene whose mutation renders said T cell resistant to NK cell killing in the T cell. In some embodiments, identifying the target gene comprises: using NK cells to identify hits in T cells obtained from at least two (e.g., at least 3, 4, 5, 6, 7, 8, 10 or more) separate distinct treatments, wherein: i) hits identified as depleted from (and/or having at least about 2-fold depletion, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more-fold depletion) the final T cell subpopulation that is resistant to NK cell killing in at least one NK cell treatment of FDR ≦ 0.01 (e.g., any of FDR ≦ 0.009, 0.007, 0.005, 0.001, 0.0005 or less), or in at least two separate different NK cell treatments of FDR ≦ 0.05 (e.g., any of FDR ≦ 0.04, 0.03, 0.02, 0.01, 0.005, 0.001, or less), identified as having a target gene whose mutation (e.g., such as, inactivated) renders T cells susceptible to NK cell killing; ii) hits identified as being enriched from (and/or having at least about 2-fold enrichment, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more enrichment) a final subset of T cells that are resistant to killing by NK cells in at least one NK cell treatment of FDR ≦ 0.05 (e.g., any of FDR ≦ 0.04, 0.03, 0.02, 0.01, 0.005, 0.001, or less), or at least two separate different NK cell treatments of FDR ≦ 0.15 (e.g., any of FDR ≦ 0.14, 0.13, 0.12, 0.11, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, or less), identified as target genes whose mutation (e.g., inactivation) renders the T cells resistant to killing by NK cells; iii) hits identified as depleted (and/or having at least about 2-fold depletion, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more depletion) from a final subpopulation of T cells that are sensitive to killing of NK cells, in at least one NK cell treatment of FDR ≦ 0.05 (e.g., any of FDR ≦ 0.04, 0.03, 0.02, 0.01, or less), or at least two separate different NK cell treatments of FDR ≦ 0.15 (e.g., any of FDR ≦ 0.14, 0.13, 0.12, 0.11, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, or less), identified as a target gene whose mutation (e.g., inactivated) renders the T cells resistant to killing; and/or iv) hits identified as enriched from (and/or having at least about 2-fold enrichment, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more enrichment) a final subset of T cells that are susceptible to NK cell killing in at least one NK cell treatment of FDR ≦ 0.01 (e.g., any of FDR ≦ 0.009, 0.007, 0.005, 0.001, 0.0005 or less), or at least two separate distinct NK cell treatments of FDR ≦ 0.05 (e.g., any of FDR ≦ 0.04, 0.03, 0.02, 0.01, 0.005, 0.001, or less), are mutated (e.g., mutated) target genes whose mutation (e.g., mutation) renders the T cells susceptible to NK cell killing.

In some embodiments, the methods described herein comprise subjecting a library of T cells to at least two of 4 separate assays for target gene identification: (I) test I: i) an initial processing step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 0.5:1 for about 72 hours; ii) an enrichment step comprising sorting as T cells (e.g., B2M negative or defective, or CD3 ⁺ ) And a mixture of viable treated cells, thereby obtaining a first T cell subpopulation that is resistant to NK cell killing; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; iv) a second treatment step comprising contacting the restored first T cell subpopulation with NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3: 1; and v) a sorting step comprising sorting as T cells (e.g., B2M negative or defective, or CD3 ⁺ ) And a final mixture of viable treated cells, thereby obtaining a second subpopulation of T cells resistant to NK cell killing; (II) run II: i) a treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 0.5:1 for about 10 days; and ii) a sorting step comprising sorting as T cells (e.g., B2M negative or defective, or CD 3) ⁺ ) And a mixture of viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing; (III) test III: i) a treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 1:1 for about 48 hours; ii) a sorting step comprising sorting as T cells (e.g., B2M negative or defective, or CD3 ⁺ ) And a mixture of viable treated cells, thereby obtaining a subpopulation of T cells that are resistant to NK cell killing; and iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours prior to harvesting the cells; and (IV) test IV: i) a treatment step comprising contacting a T cell library with NK cells at a ratio of NK cells to T cells of about 1:1 for about 48 hours; ii) an enrichment step comprising sorting as T cells (e.g., B2M negative or defective, or CD3 ⁺ ) And of viable treated cells(ii) the mixture, thereby obtaining a first subpopulation of T cells that are resistant to NK cell killing; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and iv) a sorting step comprising sorting recovered T cells (e.g., B2M negative or defective, or CD 3) ⁺ ) And surviving the first T cell subpopulation, thereby obtaining a second T cell subpopulation NK cells that are resistant to T cell killing. In some embodiments, identifying the target gene comprises identifying hits from at least two of 4 separate trials, wherein: i) hits identified as depleted (and/or having at least about 2-fold depletion, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more-fold depletion) from the final T cell subpopulation in at least one assay for FDR ≦ 0.01, or in at least two assays for FDR ≦ 0.05 (e.g., any of FDR ≦ 0.04, 0.03, 0.02, 0.01, 0.005, 0.001, or less), are identified as target genes whose mutation renders T cells susceptible to NK cell killing; and/or ii) hits identified as being enriched from the final T cell subpopulation (and/or having at least about 2-fold enrichment, such as any of at least about 3-, 4-, 5-, 10-, 20-, 50-, 100-fold or more enrichment) in at least one assay wherein FDR ≦ 0.05 or at least two assays wherein FDR ≦ 0.15 (e.g., any of FDR ≦ 0.14, 0.13, 0.12, 0.11, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, or less), are identified as target genes whose mutation renders the T cells resistant to NK cell killing.

In some embodiments, the method further comprises ranking the identified target genes, wherein target gene ranking is based on sgrnas or sgrnas in the treated T cell population (T cells obtained from step c) compared to a control T cell population ^iBAR The guide sequence or the hit gene mutation enrichment or depletion degree (e.g., enrichment fold, depletion fold, enrichment FDR, or depletion FDR). In some embodiments, the target gene ordering is further adjusted based on data consistency between all sequences comprising a hit gene mutation (e.g., an inactivating mutation) corresponding to the same target gene. In some embodiments, the sgRNA library is a sgRNA ^iBAR Library based on priming with target genesGuide sequence corresponding sgRNA ^iBAR Data consistency between iBAR sequences in the sequence, and/or data consistency between all guide sequences corresponding to the same target gene (e.g., the same or different target sites), further adjusts target gene ordering. In some embodiments, the RRA or alpha-RRA algorithm is used to rank the identified target genes. In some embodiments, the ordering of the identified target genes is: i) based on data consistency between all sequences comprising hit gene mutations (e.g., inactivating mutations) corresponding to the same target gene; or ii) based on sgRNA corresponding to the guide sequence of the target gene ^iBAR Data consistency between iBAR sequences in a sequence; and/or iii) based on sgrnas or sgrnas corresponding to the same target gene (e.g., the same or different target sites) ^iBAR Data consistency between all boot sequences; wherein the identified target genes are ranked from high to low based on the degree of data consistency from high to low. In some embodiments, the treated T cell population (T cells obtained from step c) is a viable population, i.e., resistant to NK cell killing. In some embodiments, the methods further comprise assigning a sensitivity score or a resistance score to the identified target gene, wherein the sgRNA or sgRNA in the treated T cell population (e.g., live, resistant to NK cell killing) is based on comparison to the control T cell population ^iBAR The guide sequence or hit gene mutation enrichment times (or based on enrichment FDR-FDR smaller, ranking higher, or based on data consistency degree-data consistency degree higher, ranking higher), its mutation makes T cell to NK cell killing resistant target genes from high to low ranking, and each target gene is assigned a high to low resistance score; and/or wherein the sgRNA or sgRNA in the treated T cell population (e.g., viable, resistant to NK cell killing) is based on a comparison to a control T cell population ^iBAR The targeting sequence or hit gene mutation exhaustion multiple (based on exhaustion FDR-FDR smaller, ranking higher, or based on data consistency degree-data consistency degree higher, ranking higher), its mutation makes T cell sensitive to NK cell killing target gene from high to low ranking, and each target gene is correspondinglySensitivity scores were assigned from high to low. In some embodiments, the treated population of T cells (T cells obtained from step c) is a dead population, i.e., sensitive to NK cell killing. In some embodiments, the methods further comprise assigning a sensitivity score or a resistance score to the identified target gene, wherein the sgRNA or sgRNA in the treated T cell population (e.g., dead, sensitive to NK cell killing) is based on comparison to a control T cell population ^iBAR The guide sequence or the hit gene mutation is enriched in multiples (or ranked higher based on the smaller the enriched FDR-FDR; or ranked higher based on the degree of data consistency-the higher the degree of data consistency), the mutation ranks the target genes whose T cells are sensitive to NK cell killing from high to low, and each target gene is assigned a sensitivity score from high to low accordingly; and/or wherein the sgRNA or sgRNA in a treated T cell population (e.g., dead, sensitive to NK cell killing) is based on comparison to a control T cell population ^iBAR The targeting sequences or hits are mutated to a multiple of depletion (either based on the smaller the depleted FDR-FDR, the higher the ranking; or based on the degree of data consistency-the higher the degree of data consistency, the higher the ranking), their mutations are ranked from high to low for target genes that are resistant to NK cell killing by T cells, and each target gene is assigned a resistance score from high to low accordingly. In some embodiments, the method further comprises validating the identified target gene by: a) modifying a T cell by making a mutation (e.g., an inactivating mutation) in a target gene of the T cell; b) determining the sensitivity or resistance of the modified T cell to NK cell killing. In some embodiments, the method comprises subjecting the modified T cell to any NK cell treatment step b) described herein and optionally any T cell obtaining step c). Any cell viability assay known in the art and described herein can be used to determine the sensitivity or resistance of modified T cells to NK cell killing. When the modified T cell is a homogeneous population (i.e., contains the same mutation, such as a loss of viability mutation), more cell viability assays can be used, such as assays based on metabolic activity, e.g., resazurin (oxidation-reduction (redox reaction) indicator), tetrazolium salts MTT and XTT, dihydrorhodamine, -calcein or-fluorescein, luminescence ATP determination. In some embodiments, the validation method further comprises generating a mutation (e.g., an inactivating mutation) in B2M in the T cell or the target gene modified T cell. The generation of mutations (e.g., inactivating mutations) in the target gene and B2M can be performed simultaneously or sequentially, using the same or different mutagenesis methods (e.g., both using CRISPR/Cas-mediated gene editing). Mutations (e.g., inactivating mutations) in the target gene and/or B2M can be generated by any method known in the art and described herein, such as by a mutagen, or TALEN-, ZFN-, or CRISPR/Cas-mediated gene editing (e.g., using Cas, sgrnas directed against the target gene, and/or B2M sgrnas). In some embodiments, the T cell prior to generating the mutation (e.g., inactivating mutation) in the target gene comprises a mutation (e.g., inactivating mutation) in B2M. In some embodiments, the method comprises: a) modifying a T cell by making a mutation (e.g., an inactivating mutation) in a target gene of the T cell; b) optionally an enrichment step of the target gene modified T cells; and c) modifying the target gene modified T cell by making a mutation (e.g., an inactivating mutation) in B2M. In some embodiments, the method comprises: a) modifying T cells by making mutations (e.g., inactivating mutations) in B2M; b) an optional enrichment step of B2M modified T cells; and c) modifying the B2M modified T cell by making a mutation (e.g., an inactivating mutation) in the target gene.

Methods of producing modified T cells

One aspect of the invention provides methods of generating modified T cells, such as modified T cells with increased resistance to NK cell killing. In some embodiments, a method of producing a modified T cell comprises: inactivating one or more target genes identified by any of the screening methods described herein in a host T cell (e.g., an allogeneic T cell, a precursor T cell, a PBMC-derived T cell, or a CAR-T cell (e.g., an allogeneic CAR-T cell)). In some embodiments, the host T cell further comprises a mutation (e.g., an inactivating mutation) in B2M. In some embodiments, the method further comprises generating one or more mutations (e.g., inactivating mutations) in B2M of the host T cell or modified T cell. In some embodiments, the host T cell expresses a CAR. In some embodiments, the method further comprises introducing a nucleic acid or vector encoding a CAR into the host T cell or modified T cell. Also provided are modified T cells produced by any of the methods described herein.

In some embodiments, a method of producing a modified T cell comprises: one or more mutations (e.g., inactivating mutations) are generated at one or more target genes identified by any of the screening methods described herein. In some embodiments, the method comprises: contacting a host T cell (e.g., an allogeneic T cell, a precursor T cell, a PBMC-derived T cell, or a CAR-T cell (e.g., an allogeneic CAR-T cell)) with a mutagen, and selecting a modified T cell that comprises one or more mutations (e.g., inactivating mutations) on one or more target genes identified herein. Methods for detecting such mutations are well known in the art, such as by PCR. In some embodiments, the method comprises: one or more mutations (e.g., inactivating mutations) are generated in one or more target genes identified herein in a host T cell (e.g., an allogeneic T cell, a precursor T cell, a PBMC-derived T cell, or a CAR-T cell (e.g., an allogeneic CAR-T cell)) by gene editing, as any gene editing method known in the art or described herein. For example, nonhomologous end joining (NHEJ) -or homologous recombination-mediated gene disruption, or ZFN-, TALEN-, or CRISPR/Cas-mediated gene disruption. In some embodiments, a method of producing a modified T cell comprises: introducing a sgRNA construct into a host T cell (e.g., an allogeneic T cell, a precursor T cell, a PBMC-derived T cell, or a CAR-T cell (e.g., an allogeneic CAR-T cell)), wherein the sgRNA construct comprises or encodes a sgRNA (e.g., a sgRNA, or a vector (e.g., a viral vector, such as a lentiviral vector) carrying a nucleic acid encoding the sgRNA), wherein the sgRNA comprises a guide sequence that is complementary (e.g., at least any one of about 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site in a target gene identified herein. In some embodiments, a method of generating a modified T cell is provided, comprising introducing a sgRNA library into a host T cell (e.g., an allogeneic T cell, a precursor T cell, a PBMC-derived T cell, or a CAR-T cell (as an allogeneic CAR-T cell)), wherein the sgRNA library comprises one or more sgRNA constructs, wherein each sgRNA construct comprises or encodes a sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary to a target site in a target gene (e.g., at least about any one of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary), the target gene selected from the group consisting of: TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34 and PACS 2. In some embodiments, the method further comprises: introducing a vector (e.g., a viral vector, such as a lentiviral vector) carrying a nucleic acid encoding a Cas protein (e.g., Cas9) or a Cas (e.g., Cas9) mRNA into a host T cell or a host T cell comprising the sgRNA construct. In some embodiments, the method further comprises: introducing a B2M sgRNA construct into a host T cell or a host T cell comprising a sgRNA construct directed to a target gene, wherein the B2M sgRNA construct comprises or encodes a B2M sgRNA (e.g., a B2M sgRNA, or a vector (e.g., a viral vector, such as a lentiviral vector) carrying a nucleic acid encoding a B2M sgRNA, wherein the B2M sgRNA comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site in B2M in some embodiments the host T cell comprises a B2M mutation (e.g., an inactivated B2M mutation) in some embodiments, a Cas component directed to the sgRNA construct, the B2M sgRNA construct, and/or a mRNA component comprising a Cas protein or a nucleic acid encoding a Cas protein (e.g., a vector or a mRNA), (and/or nucleic acids encoding a chimeric receptor such as a CAR or an engineered TCR, e.g., for use in generating CAR-T or TCR-T cells) are introduced into a host T cell at the same time. In some embodiments, the nucleic acid encoding the target gene sgRNA, the nucleic acid encoding the B2M sgRNA, and/or the nucleic acid encoding the Cas protein, (and/or the nucleic acid encoding the chimeric receptor such as a CAR or an engineered TCR, e.g., for generating CAR-T or TCR-T cells) are on the same vector, under the control of the same promoter, or under the control of different promoters. In some embodiments, a nucleic acid encoding a target gene sgRNA, a nucleic acid encoding B2M sgRNA, and/or a nucleic acid encoding a Cas protein, (and/or a nucleic acid encoding a chimeric receptor such as a CAR or an engineered TCR, e.g., for generating a CAR-T or TCR-T cell) are linked by one or more IRES linking sequences and under the control of the same promoter. In some embodiments, the nucleic acid encoding the target gene sgRNA, the nucleic acid encoding the B2M sgRNA, and/or the nucleic acid encoding the Cas protein, (and/or the nucleic acid encoding the chimeric receptor such as a CAR or an engineered TCR, e.g., for generating a CAR-T or TCR-T cell) are on different vectors. In some embodiments, the host T cell comprises a B2M mutation (e.g., an inactivated B2M mutation). In some embodiments, a sgRNA construct directed against a target gene, a B2M sgRNA construct, and/or a Cas component comprising a Cas protein or a nucleic acid encoding a Cas protein (e.g., a vector or mRNA), (and/or a nucleic acid encoding a chimeric receptor such as a CAR or an engineered TCR, e.g., for generating a CAR-T or TCR-T cell) are introduced into a host T cell sequentially.

In some embodiments, when a population of host T cells (or an initial population of T cells) is used to produce modified T cells described herein, the method further comprises one or more isolation and/or enrichment steps, e.g., isolating and/or enriching T cells comprising one or more mutations (e.g., inactivating mutations) in the target gene and/or B2M, the target gene sgRNA construct, the B2M sgRNA construct, or the Cas component from a population of T cells contacted with any of the modifying agents described herein. In some embodiments, the methods further comprise isolating and/or enriching for T cells expressing a chimeric receptor, such as a CAR or an engineered TCR. Such isolation and/or enrichment steps can be performed using any known technique in the art and described herein, such as FACS or Magnetic Activated Cell Sorting (MACS). See also the methods described in the "isolation and enrichment of modified T cells", "first enrichment step" and "harvest sorting step" sections above.

In some embodiments, the host T cell is derived from: blood, bone marrow, lymph or lymphatic organs. In some aspects, the host T cell is a human T cell. In some embodiments, the host T cell is derived from a T cell line. In some embodiments, the host T cell is obtained from a heterologous source, e.g., from: mouse, rat, non-human primate, or pig. In some embodiments, the host T cell is an engineered T cell, such as an engineered T cell comprising a mutation (e.g., a B2M mutation), a CAR-T cell (e.g., an allogeneic CAR-T cell), a T cell with an endogenous TCR knockout, or a T cell expressing an exogenous Nef protein.

In some embodiments, the nucleic acid (DNA or RNA) or a vector encoding the same (e.g., a non-viral vector, or a viral vector such as a lentiviral vector), or a virus comprising a nucleic acid encoding the same (e.g., a lentivirus) is transduced/transfected. In some embodiments, the cells are passed through a microfluidic system such as by inserting the protein into the cell membrane while passing the cells through the membrane

(see, e.g., U.S. patent application publication US20140287509), a Cas component (e.g., a Cas9 protein) is introduced into a host T cell.

Methods of introducing vectors (e.g., viral vectors) or isolated nucleic acids into mammalian cells are known in the art. The nucleic acids or vectors described herein can be transferred into T cells by physical, chemical, or biological means.

Physical methods for introducing vectors (e.g., viral vectors) into T cells include calcium phosphate precipitation, lipofection, microprojectile bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al (2001) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, the vector (e.g., viral vector) is introduced into the T cell by electroporation.

Biological methods for introducing vectors into T cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian (e.g., human) cells.

Chemical methods for introducing vectors (e.g., viral vectors) into T cells include colloidally dispersed systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

In some embodiments, an RNA molecule (e.g., a sgRNA or an mRNA encoding Cas 9) can be prepared by conventional methods (e.g., in vitro transcription) and then introduced into a T cell by known methods such as mRNA electroporation. See, e.g., Rabinovich et al, Human Gene Therapy 17: 1027-.

In some embodiments, the sgRNA encoding any of the target genes, described herein, will be included ^iBAR A viral vector (lentiviral vector) or a virus (e.g., lentivirus) of a nucleic acid of the B2M sgRNA and/or Cas protein is contacted with a host T cell (or initial T cell population), e.g., at an MOI of at least about 1, such as at least about any of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9 or 10. In some embodiments, the sgRNA encoding any target gene, or both described herein is included ^iBAR B2M sgRNA and/or Cas protein at an MOI of about 3 to host T cells (or initial T cell population).

In some embodiments, the transduced/transfected T cells are propagated ex vivo following introduction of the vector or isolated nucleic acid. In some embodiments, the transduced/transfected T cells are cultured to proliferate for at least any of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. In some embodiments, the transduced/transfected T cells are further evaluated or screened to select for desired modified T cells as described herein.

Reporter genes can be used to identify potentially transfected/transduced cells and to evaluate the function of regulatory sequences. Generally, a reporter gene is a gene that is not present or expressed by the recipient organism or tissue and encodes a polypeptide whose expression is evidenced by some easily detectable property, such as enzymatic activity. After the DNA/RNA is introduced into the recipient cells, the expression of the reporter gene is determined at an appropriate time. Suitable reporter genes may include: genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or Green Fluorescent Protein (GFP) (e.g., Ui-Tei et al, FEBS Letters 479: 79-82 (2000)). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Antibiotic selection markers can also be used to identify potential transfected/transduced cells.

Other methods of confirming the presence of any of the nucleic acids described herein (e.g., sgRNA constructs) or mutations (e.g., inactivating mutations) in the target gene of a modified T cell include, for example, molecular biological assays well known to those of skill in the art, such as Southern and Northern blots, RT-PCR, DNA-seq or RNA-seq; biochemical analysis, such as detecting the presence or absence of a particular peptide, for example by immunological methods (e.g. ELISA and western blot), Fluorescence Activated Cell Sorting (FACS) or Magnetic Activated Cell Sorting (MACS).

In some embodiments, the method further comprises: the modified T cells (e.g., modified T cells that are more resistant to NK cell killing) are formulated with at least one pharmaceutically acceptable carrier. In some embodiments, the method further comprises: administering to an individual (e.g., a human) an effective amount of a modified T cell (e.g., a modified CAR-T cell that is more resistant to NK cell killing (such as an allogeneic CAR-T cell)), or an effective amount of a pharmaceutical formulation thereof. In some embodiments, the individual has cancer. In some embodiments, the subject is not histocompatible with the donor of the host T cell from which the modified T cell is derived.

Pharmaceutical compositions comprising modified T cells

The present application also provides a pharmaceutical composition comprising: any of the modified T cells (e.g., modified T cells that are more resistant to NK cell killing) comprising one or more mutations (e.g., inactivating mutations) in one or more target genes identified herein, and optionally a pharmaceutically acceptable carrier. Also provided are methods of treating a disease (e.g., cancer, an immune disease such as infection, etc.) in an individual (e.g., a human) using a modified T cell (e.g., a modified CAR-T cell, or a modified allogeneic CAR-T cell) or a pharmaceutical composition thereof (e.g., resistant to NK cell killing) described herein, comprising administering to the individual an effective amount of the modified T cell or a pharmaceutical composition thereof. In some embodiments, the modified T cell (e.g., resistant to NK cell killing) is a CAR-T cell. In some embodiments, the CAR specifically recognizes an antigen, such as a cancer/tumor antigen, an antigen of an infectious agent (e.g., a virus, bacterium, fungus, parasite, etc.).

In some embodiments, a modified T cell (e.g., an allogeneic T cell or CAR-T (e.g., an allogeneic CAR-T cell)) is provided that comprises one or more mutations (e.g., inactivating mutations, such as a knock-out) in one or more target genes, wherein the target genes are selected from the group consisting of: TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34 and PACS 2. In some embodiments, a modified T cell (e.g., an allogeneic T cell or CAR-T (e.g., an allogeneic CAR-T cell)) is provided that comprises one or more mutations (e.g., inactivating mutations, such as knockouts) in PSCS 2. In some embodiments, there is provided a pharmaceutical composition comprising: i) one or more modified T cells (e.g., allogeneic T cells or CAR-T (e.g., allogeneic CAR-T cells)) comprising one or more mutations (e.g., inactivating mutations, such as a knock-out) in one or more target genes, wherein the target genes are selected from the group consisting of: TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34 and PACS 2; and ii) optionally a pharmaceutically acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising: i) one or more modified T cells (e.g., allogeneic T cells or CAR-T (e.g., allogeneic CAR-T cells)) comprising one or more mutations (e.g., an inactivating mutation, such as a knock-out) in PSCS 2; and ii) optionally a pharmaceutically acceptable carrier. In some embodiments, the modified T cell comprises a mutation (e.g., an inactivating mutation, such as a knock-out) in all target genes. In some embodiments, the modified T cell further comprises a mutation (e.g., an inactivating mutation) in B2M. In some embodiments, the modified T cell has a higher resistance to NK cell killing in a tissue-incompatible individual than a primary T cell isolated from a host T cell donor from which the modified T cell is derived. In some embodiments, the modified T cell has at least about 1.2-fold higher resistance to NK cell killing (e.g., at least about any of 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or more) in a tissue-incompatible individual as compared to a primary T cell isolated from a host T cell donor from which the modified T cell is derived. In some embodiments, the amount of modified T cells killed by NK cells in a tissue incompatible individual is at least about 10% lower (e.g., at least about any of 15%, 20%, 30%, 40%, 50%, 60%, 80%, 90%, or 95% lower) than primary T cells isolated from a host T cell donor from which the modified T cells are derived. In some embodiments, up to about 70% (such as up to any of about 60%, 50%, 40%, 30%, 20%, 10%, or 5%) of the modified T cells in tissue incompatible individuals are killed by NK cells.

Pharmaceutical compositions can be prepared by mixing the modified T cell populations described herein with optional pharmaceutically acceptable carriers, excipients, or stabilizers in the form of an aqueous solution (Remington's Pharmaceutical Sciences 16th edition, Osol, a.ed. (1980)). In some embodiments, the population of modified T cells is homogeneous (i.e., comprises the same mutation, such as an inactivating mutation). In some embodiments, the modified T cell population is heterogeneous (i.e., comprises at least one different mutation, such as an inactivating mutation). In some embodiments, at least about 70% (e.g., any of at least about 75%, 80%, 85%, 90%, or 95%) of the population of modified T cells comprises the same mutation, such as an inactivating mutation.

Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include: the buffer and antioxidant include ascorbic acid, methionine, vitamin E, and sodium pyrosulfite; preservatives, isotonicity agents, stabilizers, metal complexes (e.g., zinc-protein complexes); chelating agents such as EDTA and/or nonionic surfactants.

The buffer is used to control the pH within a range that optimizes the therapeutic effect, especially where stability is pH dependent. Buffers suitable for use in the present invention include organic and inorganic acids and salts thereof. For example citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. In addition, the buffer may comprise histidine and trimethylamine salts such as Tris.

Preservatives are added to prevent microbial growth and are typically present in the range of 0.2% to 1.0% (w/v). Preservatives suitable for use in the present invention include: octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; tea phenol; resorcinol; cyclohexanol, 3-pentanol and m-cresol.

Other excipients may include agents that prevent adhesion to the walls of the container.

Nonionic surfactants or detergents (also known as "wetting agents") may also be present. Suitable nonionic surfactants include: polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.),

A polyhydric alcohol,

Polyoxyethylene sorbitan monoether (

Etc.), lauryl alcohol 400, polyethylene glycol 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. Anionic detergents which may be used include: sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

In order for pharmaceutical compositions to be administered in vivo, they must be sterile. The pharmaceutical composition may be rendered sterile by filtration through sterile filtration membranes. The pharmaceutical compositions herein are typically placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is according to known and accepted methods, such as by single or multiple bolus injections or by prolonged infusion in a suitable manner, for example by injection or infusion by subcutaneous, intravenous, intratumoral, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, or by sustained or extended release means. Suitable examples of sustained release formulations include: semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained release matrices include: polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRONDEPOT ^TM (injectable microsphere composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D- (-) -3-hydroxybutyric acid.

The active ingredient may also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-and poly (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition.

The pharmaceutical compositions described herein may also contain more than one active compound or agent as required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressive agent or growth inhibitory agent. Such molecules are present in appropriate combinations in amounts effective for the intended purpose.

V. kits and articles of manufacture

The present application also provides kits and articles of manufacture for any embodiment of a method for identifying a target gene in a T cell described herein, such as using a sgRNA library or sgrnas described herein ^iBAR A library. Kits and articles of manufacture for generating modified T cells with increased resistance to NK cell killing are also provided.

In some embodiments, a kit for identifying a target gene in a T cell that modulates T cell activity (e.g., sensitivity or resistance to NK cell treatment) is provided, comprising any of the sgRNA libraries or sgrnas described herein ^iBAR Any of the libraries. In some embodiments, the kit further comprises a Cas protein or a nucleic acid encoding a Cas protein. In some embodiments, the kit further comprises a sgRNA construct comprising or encoding a sgRNA whose guide sequence is complementary (e.g., at least any one of about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site in B2M (e.g., a viral vector encoding B2M sgRNA). In some embodiments, the kit further comprises a sgRNA ^iBAR One or more positive and/or negative control groups of the construct, or one or more positive and/or negative controls of the sgRNA construct. In some embodiments, the kit further comprises NK cells and/or a population of naive T cells, such as allogeneic T cells, PBMC-derived T cells, precursor T cells, CAR-T cells (such as allogeneic CAR-T cells), or T cells comprising a B2M mutation (e.g., an inactivated B2M mutation). In some embodiments, the kit further comprises data analysis software. In some embodiments The kit comprises instructions for performing any of the methods described herein.

In some embodiments, a kit is provided for identifying a target gene that modulates T cell activity (e.g., sensitivity or resistance to NK cell treatment) in a T cell, comprising any of the T cell libraries described herein, such as a T cell library comprising mutations in some or all of the hits in the genome (e.g., inactivating mutations), or comprising any of the sgRNA libraries or sgrnas described herein ^iBAR T cell library of library. In some embodiments, the kit further comprises a Cas protein or a nucleic acid encoding a Cas protein. In some embodiments, the T cell library further comprises a mutation (e.g., an inactivating mutation) in B2M. In some embodiments, the T cell library further comprises sgRNA constructs comprising or encoding sgrnas whose guide sequences are complementary (e.g., at least any one of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to the target site in B2M. In some embodiments, the kit further comprises NK cells. In some embodiments, the kit further comprises a control T cell library, such as one or more mutations (e.g., inactivating mutations) in a nongenic region in the genome, or one or more positive and/or negative controls comprising a sgRNA construct, or a sgRNA ^iBAR One or more positive and/or negative control groups of constructs. In some embodiments, the kit further comprises data analysis software. In some embodiments, the kit comprises instructions for performing any one of the methods described herein.

In some embodiments, kits are provided for generating modified T cells with increased resistance to NK cell killing, comprising a sgRNA library (or sgrnas) containing one or more sgRNA constructs ^iBAR Library), wherein each sgRNA construct comprises or encodes a sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary (e.g., at least any one of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site in a target gene that is targeted for expression in a subjectSelected from the group consisting of: TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34 and PACS 2. In some embodiments, the kit further comprises a sgRNA construct comprising or encoding a sgRNA whose guide sequence is complementary (e.g., any of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site in B2M. In some embodiments, the kit further comprises a Cas protein or a nucleic acid encoding a Cas protein. In some embodiments, the kit further comprises an initial population of T cells, such as allogeneic T cells, PBMC-derived T cells, precursor T cells, CAR-T cells (such as allogeneic CAR-T cells), or T cells comprising a B2M mutation (e.g., an inactivated B2M mutation). In some embodiments, the kit comprises instructions for performing the modified T cell production method.

In some embodiments, a kit is provided comprising a modified T cell or a pharmaceutical composition thereof, wherein the modified T cell comprises one or more mutations (e.g., inactivating mutations, such as knockouts) in one or more target genes selected from the group consisting of: TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34 and PACS 2. In some embodiments, kits are provided comprising a modified T cell or a pharmaceutical composition thereof, wherein the modified T cell comprises one or more mutations (e.g., inactivating mutations, such as knockouts) in PSCS 2. In some embodiments, the modified T cell further comprises a mutation in B2M (e.g., an inactivating mutation). In some embodiments, the modified T cell is more resistant to NK cell killing. In some embodiments, the kit further comprises instructions for use. In some embodiments, the kit comprises a homogeneous population of modified T cells. In some embodiments, the kit comprises a heterogeneous population of modified T cells. In some embodiments, the modified T cell further comprises a CAR.

The kit may comprise additional components, such as containers, reagents, media, primers, buffers, enzymes, and the like, to facilitate performance of any of the screening methods described herein. In some embodiments, the kit comprises a library for combining sgrnas or sgrnas ^iBAR Reagents, buffers and vectors for introducing the library and the Cas protein or Cas protein-encoding nucleic acid into T cells. In some embodiments, the kit comprises primers, reagents, and enzymes (e.g., polymerases) for preparing a sequencing library of sequences comprising hit gene mutations (e.g., inactivating mutations), sgRNA sequences, or sgrnas extracted from a selected T cell subpopulation ^iBAR And (4) sequencing.

The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to: vials, bottles, jars, flexible packaging (e.g., sealed mylar or plastic bags), and the like. The kit may optionally provide additional components such as buffers and explanatory information. The present application thus also provides articles of manufacture including vials (e.g., sealed vials), bottles, jars, flexible packages, and the like.

The article of manufacture may comprise a container and a label or package insert on or associated with the container. Suitable containers include: such as bottles, vials, syringes, etc. The container may be formed from a variety of materials, such as glass or plastic. Typically, the container contains the composition (e.g., modified T cells with higher resistance to NK cell killing) and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some embodiments, the label or package insert indicates that the composition is used to treat a particular condition or enhance an immune response in an individual. The label or package insert will further comprise instructions for administering the composition to an individual. A package insert refers to instructions typically contained in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings regarding the use of such therapeutic products. In addition, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may also include other desirable materials from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

The kit or article of manufacture may include a plurality of unit doses of the pharmaceutical composition and instructions for use, packaged in amounts sufficient for storage and use in pharmacies, such as hospital pharmacies and compound pharmacies.

Examples

The following examples and exemplary embodiments are intended to be purely exemplary of the invention and therefore should not be considered as limiting the invention in any way. The following examples and detailed description are provided by way of illustration and not limitation.

Example 1: identification of T cell regulatory genes

This example provides exemplary methods for identifying T cell regulatory genes. Briefly, sgrnas carrying targeting each human gene were constructed for Cas 9-mediated gene knock-out (KO) ^iBAR And a T cell library targeting the sgRNA of B2M. B2M is a component of MHC class I molecules. T cells with B2M KO will be killed by NK cells. B2M constructed by detection ^- sgRNA ^iBAR NK cell killing efficacy of the T cell library, genes conferring a resistant or sensitive phenotype to NK cell killing after KO can be identified. Fig. 1 shows a workflow.

Isolation and culture of T cells

After blood collection, PBMCs are isolated from the donor blood sample. T cells were isolated from PBMC using immunomagnetic beads and then incubated at 37 ℃ with 5% CO ₂ In an incubator containing 10% FBS, 1% GlutaMAX and 0.1% recombinant human IL-2 ^TM 15 medium (hereinafter referred to as "T cell complete medium").

T cell activation and expansion

Mixing with 800 μ L

Human T-activator CD3/CD28 was split into 4 1.5mL portionsEppendorf tubes, 200. mu.L/tube. Add 1mL PBS per tube, pipette and resuspend

The 1.5mL tube was placed on a magnetic rack and allowed to stand for 1 minute, and then the supernatant was removed. This washing step was repeated twice. 1mLT cell complete medium was then added to each tube and the washed tubes were resuspended by gentle pipetting

Mixing 3.2X 10 ⁷ The cultured T cells were transferred to a T150 cell culture flask and then 4mL of resuspended

Add to T cells and mix gently. The mixture was heated at 37 ℃ with 5% CO ₂ Cultured in an incubator to activate and expand T cells. The activated T cells are expanded to a sufficient amount to construct a T cell library.

3. Human genome-scale CRISPR sgRNA ^iBAR Construction of libraries and T cell libraries

Human genome-scale CRISPR sgRNA ^iBAR Library design and construction is similar to that described in WO2020125762 and Zhu et al (Zhu et al, "Guide RNAs with embedded barcodes boost CRISPR-porous cultures," Genome biol.2019; 20:20), the contents of each of which are incorporated herein by reference in their entirety. Briefly, 19,114 annotated protein-encoding genes were retrieved from the UCSC human genome. The DeepRank algorithm (see Zhu et al.) was used to design sgRNAs targeting each gene and 4 6-bp IBARs (iBARs) were used ₆ ) Random assignment to each sgRNA ("sgRNA ^iBAR "). The internal barcode sequence was designed to be placed in four loops of the gRNA scaffold outside of the Cas9-sgRNA ribonucleoprotein complex, which did not affect the activity of its upstream guide sequence. The encoded sgRNA was designed and synthesized in large quantities ^iBAR The DNA oligonucleotide of (1), and then PCR amplification is performed. The PCR product was cloned into an internally modified lentiviral sgRNA based on pLenti-sgRNA-Lib (adddge #53121) ^iBAR In the expression of the framework,to obtain sgRNA ^iBAR Library plasmids encoding 156,848 sgRNAs ^iBAR Covering 19,114 human genes (group 2 sgRNA) ^iBAR Each gene is directed against 2 different target sites; each group of sgRNAs ^iBAR Comprises 4 sgRNAs ^iBAR ). sgRNA was then obtained using standard protocols ^iBAR A library lentivirus.

Addition of sgRNA ^iBAR Library lentiviruses to activate T cells at MOI 3 and mix gently. The cell mixture was incubated at 37 ℃ with 5% CO ₂ Incubate overnight in an incubator for infection. The supernatant was discarded the next day, and the same amount of T cell complete medium supplemented with puromycin was added at 37 ℃ with 5% CO ₂ Culturing in an incubator overnight. Unsuccessfully infected T cells were then removed, yielding sgrnas ^iBAR A T cell library carrying sgrnas targeting each of the 19,114 annotated functional genes.

Cas9+ beta-2-microglobulin KO (B2M-) sgRNA ^iBAR Construction of T cell libraries

The sgRNA was synthesized ^iBAR Library lentivirus infected T cells were transferred to a 50mL centrifuge tube and placed on a magnetic rack for 10 minutes. The supernatant was then transferred to a new 50mL centrifuge tube and placed on a magnetic rack and allowed to stand for 5 minutes to remove as much as possible

The supernatant containing the T cells was transferred to a clean 50mL centrifuge tube, centrifuged at 400g for 5 minutes, resuspended with 20mL DPBS, washed twice, and then centrifuged at 400g for 5 minutes. The supernatant was discarded and 600. mu.L of the solution was used

The T cells were resuspended in reduced serum medium and cell counted (6.60X 10) ⁷ Individual T cells). These T cells were isolated in three 1.5mL Eppendorf tubes and placed on ice. Mu.g of Cas9 mRNA and 16. mu.g of sgRNA specifically targeting B2M (internal design and manufacture) were added to each tube and mixed gently, and the cell mixture was then transferred separately to 4mm BTX electroporation cuvettes for electrotransformation. Transfer of the electroporated T cells to T150 cellsThe cell density was adjusted to 1X 10 by adding the complete T cell medium to the flask ⁶ cell/mL, then 5% CO at 37 ℃ ₂ Culturing in an incubator. Cell passages were performed every two days. 96 hours after electrotransformation, target gene (sgRNA) ^iBAR Targeted human genes) and B2M were considered to be effectively knocked-out (KO efficiency of approximately 91% as detected for B2M) yielding Cas9 ⁺ B2M ^- sgRNA ^iBAR T cell libraries for screening.

5. Cas9 treated with NK cells ⁺ B2M ^- sgRNA ^iBAR Screening of T cell libraries

Addition of NK cells to Cas9 ⁺ B2M ^- sgRNA ^iBAR T cell library to check NK cell killing efficacy. The killing efficacy depends on the NK cells and B2M ^- Proportion of T cell library and total incubation time. Thus, 4 test groups were set up with different treatment intensities and screening protocols (trials 3-6; see FIG. 2). Setting control group without NK cell treatment, Cas9 ⁺ B2M ^- sgRNA ^iBAR T cells were cultured in T cell complete medium with passages every two days. Two biological replicates were set up per group. To ensure sgRNA in T cell libraries ^iBAR Abundance of (2), 3.56X 10 per repeat use ⁷ T cells (each sgRNA) ^iBAR On average about 100 fold coverage, or on average about 800 fold coverage per hit gene).

In the test set, Cas9 ⁺ B2M ^- sgRNA ^iBAR After incubation of the T cell library with NK cells for a period of time, all cells were harvested, stained with Propidium Iodide (PI) dye (indicating dead cells) and anti-B2M antibody, and then FACS was used to sort PI-negative and B2M-negative or defective cells (i.e., live Cas 9) ⁺ B2M ^- sgRNA ^iBAR T cells). For CRISPR screening, multiple rounds of NK cell treatment helped to enrich target sgrnas ^iBAR T cells and increase the signal-to-noise ratio. Since activated T cells can only be cultured in vitro for a limited time, if the live Cas9 sorted after the first round of NK cell treatment ⁺ B2M ^- sgRNA ^iBAR T cells in appropriate conditions, then can be performed for the second round of NK cell processing, then for PI negative and B2M negative or defective cells were re-stained and FACS sorted (i.e., enriched live Cas 9) ⁺ B2M ^- sgRNA ^iBAR A T cell; see tests 3 and 6 in figure 2). Common targets under different screening conditions can be identified.

5.1 NK cell treatment

Addition of appropriate amount of NK cells to Cas9 according to different screening protocols (fig. 2) ⁺ B2M ^- sgRNA ^iBAR T cell library, then 5% CO at 37 ℃ ₂ Co-culturing in an incubator.

⁺ ^- ^iBAR 5.2 FACS sorting of Cas9B2MsgRNA target cells

The test group cells were collected, centrifuged at 300g for 10 minutes, and the supernatant was discarded. Resuspend cells in 500. mu.L PBS buffer and add PE anti-human beta 2-microglobulin antibody (per 1X 107 cells) ^An Cell 5. mu.L antibody), left at room temperature for 15 minutes in the dark. Then 2ml of PBS was added to the cell mixture and centrifuged at 400g for 5 minutes. The supernatant was removed and the cells were resuspended in 1.5mL of buffer (PBS + 1% FBS +10 XPS). Add 150 μ Ι PI dye to cell suspension and mix gently, then sort cells into PI-negative and PE-negative cells by FACS (i.e. live Cas 9) ⁺ B2M ^- sgRNA ^iBAR T cells).

6. Target gene analysis

FACS-sorted PI-negative and B2M-negative or defective cells (i.e., live Cas 9) ⁺ B2M ^- sgRNA ^iBAR T cells) were used for genome extraction. sgRNA ^iBAR The encoded fragment, amplified from the extracted genome, purified and prepared for NGS sequencing. MAGECK ^iBAR Algorithms are used for sequencing data analysis (see Zhu et al, "Guide RNAs with embedded barcodes boost CRISPR-porous cultures," Genome biol.2019; 20: 20; the contents of which are incorporated herein by reference in their entirety), comprising three major components: analytical preparation, statistical tests and rank aggregation. Briefly, each sgRNA was enriched or depleted according to the extent of each gene between the test group and the control group ^iBAR The targeted gene is scored and ranked to identify the geneWhether the candidate gene is a candidate gene with high confidence. See fig. 5, workflow for target gene identification. Top ranked candidates (dark grey dots above the horizontal dashed line) from each trial are shown in fig. 3A-3B, where candidate genes whose deletion results in a phenotype sensitive to NK cell killing were identified from the negative screen, while candidate genes whose deletion results in a phenotype resistant to NK cell killing were identified from the positive screen. The top ranked candidates with FDR ≦ 0.15 for each trial are plotted. These top ranked candidate genes were found to be involved in autoimmune responses (e.g., TAAC2, HES1, LILRB4, KLHL24, ARNTL, LRRC69, PACS2, CSK, and MYB, etc.), tumor malignancy transformation (e.g., CJD2, FANCB, TPM3, TFG, SMAD6, PTPN14, or MEF12, etc.), or tumor metastasis (e.g., STON1, PLS1, SIX1, PIK3R6, PDE4C, SRRM3, SSPO, TLN1, PIH1D2, or SLC35C2, etc.). FIG. 4 shows Venn plots of top ranked candidates in various experiments with FDR ≦ 0.15.

7. Results

Candidate genes that appeared in the negative screen of at least two experiments with FDR ≦ 0.05 were classified as T cell regulatory genes, the deletion of which resulted in a sensitive phenotype to NK cell killing (Table 1). Candidate genes that appeared in the positive screen for any trial with an FDR ≦ 0.05, or at least two trials with an FDR ≦ 0.15 were classified as T cell regulatory genes, the deletion of which resulted in a resistant phenotype to NK cell killing (Table 2). Among these, phosphofurin acidic amino acid cluster sortilin 2 (PACS-2) was identified as a T cell regulatory gene that confers resistance to NK cell killing upon deletion. This is consistent with the pro-apoptotic effector role of PACS-2 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) -mediated apoptosis (Werneburg et al, J Biol chem.2012; 287(29):24427 24437). Another example is PTEN, identified as a T cell regulatory gene, which confers sensitivity to NK cell killing upon deletion. This is consistent with the role of PTEN in cell proliferation, transcriptional regulation, and ubiquitination.

The results obtained here, in particular the finding that the gene whose deletion confers T cell resistance to NK cell killing, demonstrate a valuable target for avoiding host rejection in allogeneic T cell therapy.

TABLE 1T cell regulatory genes sensitive to NK cell killing after deletion

Table 2: t cell regulatory genes resistant to NK cell killing after deletion

Claims

1. A method of identifying a target gene in a T cell that regulates the activity of said T cell, comprising:

a) providing a T cell library comprising a plurality of T cells, wherein each of the plurality of T cells has a mutation at a hit gene in the genome ("hit gene mutation"), wherein at least two of the plurality of T cells have mutations in the genome The hit genes are different from each other;

b) treating the T cell library with NK cells;

c) obtaining T cells sensitive or resistant to NK cell killing from the T cell library; and

d) Identifying hit genes in the T cells obtained in step c), thereby identifying target genes in T cells that regulate the activity of said T cells.

2. The method of claim 1, wherein the T cell library is generated by genome-wide gene editing of a naive T cell population.

3. The method of claim 1 or 2, wherein the T cell library is generated by contacting an initial population of T cells with: i) a single-stranded guide RNA ("sgRNA") comprising a plurality of sgRNA constructs ") a library, wherein each sgRNA construct comprises or encodes an sgRNA, and wherein each sgRNA comprises a guide sequence complementary to the target site of the hit gene in the genome; and optionally, ii) after allowing the sgRNA construct to be combined with any The selected Cas component is introduced into the naive T cell population under conditions that comprise a Cas protein or a Cas component of a nucleic acid encoding said Cas protein.

4. The method of claim 3, wherein the Cas protein is Cas9.

5. The method of claim 4, wherein each sgRNA comprises a leader sequence fused to a second sequence, wherein the second sequence comprises a repeat-anti-repeat stem-loop that interacts with Cas9.

6. The method of claim 5, wherein the second sequence of each sgRNA further comprises stem loop 1, stem loop 2 and/or stem loop 3.

7. The method of any one of claims 3-6, wherein each sgRNA further comprises an internal barcode (iBAR) sequence ("sgRNA ^iBAR "), wherein each sgRNA ^iBAR is operable with the Cas protein to The hit gene is modified.

8. The method of claim 7, wherein the iBAR sequence of each sgRNA ^iBAR is inserted into the loop region of the repeat-trans-repeat stem-loop.

9. The method of claim 7, wherein each sgRNA ^iBAR comprises a first stem sequence and a second stem sequence in the 5' to 3' direction, wherein the first stem sequence hybridizes with the second stem sequence to form a Cas protein interacting double-stranded RNA (dsRNA) region, and wherein the iBAR sequence is located between the 3' end of the first stem sequence and the 5' end of the second stem sequence.

10. The method of any one of claims 3-9, wherein each leader sequence comprises from about 17 to about 23 nucleotides.

11. The method of any one of claims 7-10, wherein each iBAR sequence comprises from about 1 to about 50 nucleotides.

12. The method of any one of claims 7-11, wherein the sgRNA library ("sgRNA ^iBAR library") comprising multiple sgRNA ^iBAR constructs comprises multiple groups of sgRNA ^iBAR constructs, wherein each group of sgRNA ^iBAR constructs Comprising three or more sgRNA ^iBAR constructs, each of which comprises or encodes an sgRNA ^iBAR , wherein the guide sequences of the three or more sgRNA ^iBAR constructs are identical, wherein each of the three or more sgRNA ^iBAR constructs The iBAR sequences of the sgRNAs differ from each other, and where the guide sequences of each set of sgRNA ^iBAR constructs are complementary to different target sites in the genome.

13. The method of claim 12, wherein each set of sgRNA ^iBAR constructs comprises 4 sgRNA ^iBAR constructs, and wherein the iBAR sequence of each of the 4 sgRNA ^iBAR constructs is different from each other.

14. The method of claim 12 or 13, wherein the sgRNA ^iBAR library comprises at least about 100 sets of sgRNA ^iBAR constructs.

15. The method of any one of claims 12-14, wherein the iBAR sequences of at least two sets of sgRNA ^iBAR constructs are identical.