WO2025076306A1 - Prime editors having improved prime editing efficiency - Google Patents

Abstract

Prime editing systems having improved prime editing efficiency, and methods of their use, are described.

Description

Prime Editors having Improved Prime Editing Efficiency

Cross-Reference to Related Applications

[0001] The present application claims priority to U.S. Provisional Patent Application Serial No. 63/588,435 filed October 6, 2023, and U.S. Provisional Patent Application Serial No. 63/643,634 filed May 7, 2024. The foregoing applications are hereby incorporated herein by reference in their entirety.

Government Rights in Invention

[0002] This invention was made with government support under TR002668 and HL 150669 awarded by the National Institutes of Health. The government has certain rights in the invention.

Technical Field

[0003] The present invention relates to improved prime editing systems, and more particularly to prime editing systems having prime editors with improved solubility and dNTP affinity.

Background Art

[0004] Prime editing has the potential for broad therapeutic application for a variety of human diseases due to its versatility in performing targeted base conversion, deletions and insertions¹. A prime editor (PE) has 3 core components: Cas9 nickase, M-MLV reverse transcriptase (MMLV-RT), and prime editing guide RNA (pegRNA)^{1, 2}. Prime editing efficiency is enhanced by using a sgRNA to direct a nick to the target strand (PE3 system), and a high level of precision can be maintained by directing the nicking guide to recognize sequence changes encoded by the pegRNA (PE3b system)^{1, 3}. These prime editing components have undergone optimization to improve prime editing outcomes^{1, 2}’ ^4-7 The original PE2 system¹ and the improved PEmax system² utilize a pentamutant MMLV-RT that increases thermostability, affinity for the RNA-DNA template and processivity. Subsequent studies have evaluated additional MMLV mutations⁸, alternate reverse transcriptases (RTs)^{5, 9}'¹¹, DNA-dependent polymerases^{12, 13}, split prime editor systems^{3, 9, 10}, or evolved RTs¹¹ to enhance the efficiency of prime editing with notable improvements in precise editing outcomes. Despite these advances, precise editing rates remain modest in some primary cell types and in vivo in some tissues^{14, 15}. Summary of the Embodiments

[0005] In accordance with one embodiment of the invention, a prime editor protein, the prime editor protein comprising a Cas9 nickase fused to a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase domain, wherein the M-MLV reverse transcriptase domain comprises a V223M and a L435K mutation.

[0006] In accordance with another embodiment of the invention, an isolated nucleic acid encoding the prime editor protein.

[0007] In accordance with one embodiment of the invention, a programmable prime editing system for modification of a double-stranded target DNA sequence comprising a target strand and a complementary non-target strand, the system comprising the prime editor protein; and a prime editing guide RNA (pegRNA) comprising a spacer sequence having a region of complementarity to the target strand of the double-stranded target DNA sequence.

[0008] In some embodiments, the programmable prime editing system further comprises an inhibitor of SAMHDl. The inhibitor of SAMHD1 may be Vpx, Vpx (S13E), EBV BGLF4, HHV-6/7 U69, HCMV UL97, or KSHV ORF36.

[0009] In some embodiments the programmable prime editing system comprises a nicking sgRNA.

[0010] In accordance with a further embodiment of the invention, a method of modifying a double-stranded target DNA sequence, the method comprising contacting the double-stranded target DNA sequence with the prime editor protein and the pegRNA.

[0011] In accordance with an additional embodiment of the invention, a method of modifying a double-stranded target DNA sequence in a cell, the method comprising: sequentially introducing into the cell, in the following order: (1) a programmable prime editing system, the programmable prime editing system comprising (i) a prime editor protein comprising a Cas9 nickase fused to a M-MLV reverse transcriptase domain and (ii) a pegRNA comprising a spacer sequence having a region of complementarity to a target strand of the double-stranded target DNA sequence; and (2) a nicking sgRNA. In some embodiments, the M-MLV reverse transcriptase domain comprises a V223M and a L435K mutation. In some embodiments, the programmable prime editing system comprises an inhibitor of SAMHDL The inhibitor of SAMHDl may be Vpx, Vpx (S13E), EBV BGLF4, HHV-6/7 U69, HCMV UL97, or KSHV ORF36. [0012] In some embodiments, the nicking sgRNA is introduced into the cell 2-36 hours after introducing the programmable prime editing system into the cell. In other embodiments, the nicking sgRNA is introduced into the cell 6-24 hours after introducing the programmable prime editing system into the cell.

[0013] In some embodiments, the programmable prime editing system is introduced into the cell by a first method selected from the group consisting of electroporation, transfection, viral delivery, virus-like particle delivery, and lipid nanoparticle delivery.

[0014] In some embodiments, the nicking sgRNA is introduced into the cell by a second method selected from the group consisting of electroporation, transfection, viral delivery, virus-like particle delivery, and lipid nanoparticle delivery.

Brief Description of the Drawings

[0015] The foregoing features of embodiments will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawings, in which:

[0016] Fig. 1A is a plot of fold-change in PE2 and Twin-PE editing efficiency of prime editor variants compared to PEmax in HEK293T cells. All individual values of n = 3 independent replicates across multiple target sites are shown. One-way ANOVA was used to compare multiple prime editor architectures. * indicates P <0.05, ** indicates P <0.01, ** indicates P <0.0001, ns indicates no significance.

[0017] Fig. IB is a plot showing in vitro biochemical analysis of PEmax and PEmax** RNP activity under different dNTP concentrations by assessing 20 nt tag incorporation at the HEK4 target site in purified gDNA (qPCR, n=3 independent replicates). Two-way ANOVA was used to compare PEmax and PEmax** for each condition; * indicates P <0.05, ** indicates P <0.01.

[0018] Fig. 1C is a chart showing PEmax, PEmax* (V223M) and PEmax** (V223M & L435K) protein and mRNA mediated prime editing efficiency with synthetic pegRNA/sgRNA delivered by electroporation in iMyoblasts and primary T cells. PE3 editing at HEK4 (+4+5 GG to CC) in iMyoblasts, and at FANCF (+5 G to T) in iMyoblasts and in activated primary T cells, respectively (n=3 independent replicates). PAM sequence is boxed, protospacer sequence is underlined, and edited bases are indicated in lowercase letters. Unpaired two-sample t-test and one-way ANOVA was used to compare the intended edit across all the groups for each graph; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P< 0.001, **** indicates P< 0.0001. Figure 1C discloses SEQ ID NOS 2-5, respectively, from left to right and top to bottom.

[0019] Fig. ID is a chart showing PEmax** protein improves PE2 editing rates in zebrafish. Comparison of PEmax and PEmax** RNP editing efficiency at different doses when introducing R841W at the tek locus in zebrafish embryos (n=3 independent replicates). Codons with synonymous or non-synonymous mutations are indicated by bold underline. One-way ANOVA was used to compare the intended edit across all the groups for each graph; **** indicates P < 0.0001. Figure ID discloses SEQ ID NOS 6-7, respectively, in order of appearance.

[0020] Fig. IE shows violin plots of fold-change in PE2 or Twin-PE editing efficiency of prime editor variants compared to PEmax in HEK293T cells under different conditions (standard, co-delivery of VPX or supplementation of dNs). Horizontal white lines indicate quartiles. Medians are indicated by the black horizontal lines. Two-way ANOVA was used to compare the intended edit from multiple prime editor architectures under different conditions, and standard PEmax editing was used as control for multiple comparisons. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P< 0.001, **** indicates P< 0.0001, ns indicates not significant.

[0021] Fig. IF is a chart showing co-delivery of VPX mRNA significantly improves prime editing efficiency in primary T cells and SC-derived islet cells. PEmax, PE6d and PEmax** mediated PE3 editing and Cas9-NG-PEmax and Cas9-NG-PEmax** mediated Twin-PE editing to install the CCR5^delta32 HIV-1 resistance mutation in activated human primary T cells (triplicates from n=2 different donors). Co-delivery of VPX mRNA with PEmax mRNA increases prime editing at FANCF with PE3b system in SC-Derived islet cells. Two-way ANOVA was used to compare the intended edit from multiple prime editor architectures under different conditions. * indicates P < 0.05, *** indicates P< 0.001, **** indicates P< 0.0001, ns indicates not significant. Figure IF discloses SEQ ID NOS 8-11, respectively, from left to right and top to bottom.

[0022] Fig. 2A is a schematic overview of simultaneous and sequential delivery of PE3b Nicking sgRNA for PE3b prime editing. [0023] Fig. 2B is a chart showing PE3b editing mediated by PEmax, PE6d and PEmax** at FANCF (+5 G to T) in fibroblasts, human bronchial epithelial (HBE) cells and EbvB cells using simultaneous or sequential delivery of PE3b nicking sgRNA with or without co-delivery of VPX mRNA (n=3 independent replicates). PAM sequences are boxed, protospacer regions are underlined, and edited sequences are indicated as lowercase letters. The protospacer for PE3b nicking sgRNAs is indicated on bottom strand. Figure 2B discloses SEQ ID NOS 9 and 11, respectively, in order of appearance.

[0024] Fig. 2C is a chart showing PEmax, PE6d and PEmax** mRNA-mediated PE3b editing at MECP2 (+2+4+5 CTG to TCC) to revert the T158M mutation in patient derived fibroblasts with co-delivery of synthetic pegRNA with or without VPX mRNA using simultaneous or sequential delivery of PE3b nicking sgRNA (n=3 independent replicates). Dark bars indicate PE3b editing without Vpx. Relatively lighter gray bars indicate PE3b editing with co-delivery of Vpx. Figure 2C discloses SEQ ID NOS 12-13, respectively, in order of appearance.

[0025] Fig. 2D is a chart showing PEmax, PE6d and PEmax** mRNA-mediated PE3b editing at CFTR (insert CTT) to correct the AF508 mutation in HBE cells with codelivery of synthetic pegRNA with or without VPX mRNA using simultaneous or sequential delivery of PE3b nicking sgRNA (n=3 independent replicates). Dark bars indicate PE3b editing without Vpx. Relatively lighter gray bars indicate PE3b editing with co-delivery of Vpx. Figure 2D discloses SEQ ID NOS 14-15, respectively, in order of appearance.

[0026] Fig. 2E is a chart showing PEmax, PE6d and PEmax** mRNA-mediated PE3b editing at HEX4^127S+TATC (delete TATC) to correct the microduplication in EbvB cells (e) with co-delivery of synthetic pegRNA with or without VPX mRNA using simultaneous or sequential delivery of PE3b nicking sgRNA (n=3 independent replicates). Dark bars indicate PE3b editing without Vpx. Relatively lighter gray bars indicate PE3b editing with co-delivery of Vpx. Figure 2E discloses SEQ ID NOS 16-17, respectively, in order of appearance.

[0027] Fig. 2F is a violin plot showing a comparison of precise prime editing between simultaneous and sequential delivery of PE3b nicking sgRNA across multiple endogenous genomic loci and human cell types, with (+VPX) and without (-VPX) co- delivery of Vpx. White lines indicate quartiles. Medians are indicated by black horizontal lines.

[0028] Fig. 2G is a schematic overview of a combination of methods used to boost prime editing efficiency.

Detailed Description of Specific Embodiments

[0029] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0030] The terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0031] The term “Cas9” or “Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 domain, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). A “Cas9 domain” as used herein, is a protein fragment comprising an active or inactive cleavage domain of Cas9 and/or the gRNA binding domain of Cas9. A “Cas9 protein” is a full length Cas9 protein. A Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a transencoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 domain. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3 '-5' exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of which are hereby incorporated by reference. Cas9 recognizes a short motif absent from the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an Ml strain of Streptococcus pyogenes ' Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W ., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor Rnase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471 :602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, .S’. pyogenes, Staphylococcus lugdunensis, Neisseria meningitidis and S. thermophilus . Cas9 and Cas9 nickases derived from the aforementioned species, as well as variants thereof, are also contemplated as part of the present disclosure. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on U.S. Patent No.

11,447,770, which is hereby incorporated by reference for its disclosure of Cas9 nucleases, and such Cas9 nucleases and Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease comprises one or more mutations that partially impair or inactivate the DNA cleavage domain. U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of Cas9.

[0032] The term “nickase,” “Cas9 nickase,” and “nCas9” refers to a Cas9 with one of its two nuclease domains inactivated. This enzyme is capable of cleaving only one strand of a target DNA. U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of Cas9 nickases.

[0033] As used herein, the terms “DNA synthesis template,” “reverse transcriptase template,” and “RTT” are used interchangeably to refer to the region or portion of the extension arm of a pegRNA that is utilized as a template strand by a polymerase, such as reverse transcriptase, of a prime editor to encode a 3' replacement DNA flap that contains a desired edit and which then, through the mechanism of prime editing, replaces the corresponding endogenous strand of DNA at the target site. US Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of DNA synthesis templates, the mechanisms of prime editing, components of prime editing systems, and methods of using prime editors and prime editing systems.

[0034] As used herein, the terms “upstream” and “downstream” are terms of relativity that define the linear position of at least two elements located in a nucleic acid molecule (whether single or double-stranded) that is orientated in a 5'-to-3' direction. In particular, a first element is upstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 5' to the second element. For example, a SNP is upstream of a Cas9-induced nick site if the SNP is on the 5' side of the nick site. Conversely, a first element is downstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 3' to the second element. For example, a SNP is downstream of a Cas9-induced nick site if the SNP is on the 3' side of the nick site. The nucleic acid molecule can be a DNA (double or single stranded). RNA (double or single stranded), or a hybrid of DNA and RNA. The analysis is the same for single strand nucleic acid molecule and a double strand molecule since the terms upstream and downstream are in reference to only a single strand of a nucleic acid molecule, except that one needs to select which strand of the double stranded molecule is being considered. Often, the strand of a double stranded DNA which can be used to determine the positional relativity of at least two elements is the “sense” or “coding” strand. In genetics, a “sense” strand is the segment within double-stranded DNA that runs from 5' to 3', and which is complementary to the antisense strand of DNA, or template strand, which runs from 3' to 5'. Thus, as an example, a SNP nucleobase is “downstream” of a promoter sequence in a genomic DNA (which is double- stranded) if the SNP nucleobase is on the 3' side of the promoter on the sense or coding strand.

[0035] The term “extension arm” refers to a nucleotide sequence component of a pegRNA which provides several functions, including a primer binding site (PBS) and a DNA synthesis template (also referred to as an “reverse transcriptase template” or “RTT”) for reverse transcriptase. In some embodiments the extension arm is located at the 3' end of the guide RNA. In various embodiments, the extension arm comprises the following components in a 5' to 3' direction: the DNA synthesis template and the primer binding site. Since polymerization activity of the reverse transcriptase is in the 5' to 3' direction, the preferred arrangement of the DNA synthesis template and primer binding site is in the 5' to 3' direction such that the reverse transcriptase, once primed by an annealed primer sequence, polymerizes a single strand of DNA using the DNA synthesis template as a complementary template strand.

[0036] The extension arm may also be described as comprising generally two regions: a primer binding site (PBS) and a DNA synthesis template. The primer binding site binds to a primer sequence that is formed from the endogenous DNA strand of the target site when it becomes nicked by the prime editor complex, thereby exposing a 3' end on the endogenous nicked strand. The binding of the primer sequence to the primer binding site on the extension arm of the pegRNA creates a duplex region with an exposed 3' end (i.e., the 3' of the primer sequence), which then provides a substrate for reverse transcriptase to begin polymerizing a single strand of DNA from the exposed 3' end along the length of the DNA synthesis template. The sequence of the single strand DNA product is the complement of the DNA synthesis template. Polymerization continues towards the 5' of the DNA synthesis template (or extension arm) until polymerization terminates. Thus, the DNA synthesis template represents the portion of the extension arm that is encoded into a single strand DNA product (i.e., the 3' single strand DNA flap containing the desired genetic edit information) by the polymerase of the prime editor complex and which ultimately replaces the corresponding endogenous DNA strand of the target site that sits immediate downstream of the PE-induced nick site. Without being bound by theory, polymerization of the DNA synthesis template continues towards the 5' end of the extension arm until a termination event. Polymerization may terminate in a variety of ways, including, but not limited to (a) reaching a 5' terminus of the pegRNA (e.g., in the case of the 5' extension arm wherein the DNA polymerase simply runs out of template), (b) reaching an impassable RNA secondary structure (e.g., hairpin or stem/loop), or (c) reaching a replication termination signal, e.g., a specific nucleotide sequence that blocks or inhibits the polymerase, or a nucleic acid topological signal, such as, supercoiled DNA or RNA. US Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of extension arms.

[0037] The term “fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy -terminal (C-terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. A protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a nucleic-acid editing protein. For example, a fusion protein may comprise a Cas9 nickase fused to a reverse transcriptase. Such a fusion protein is referred to herein as a “prime editor protein.”

[0038] As used herein, the term “guide RNA” is a particular type of guide nucleic acid which is mostly commonly associated with a Cas protein of a CRISPR-Cas9 and which associates with Cas9, directing the Cas9 protein to a specific sequence in a DNA molecule that includes complementarity to a spacer sequence of the guide RNA. However, this term also embraces the equivalent guide nucleic acid molecules that associate with Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and which otherwise program the Cas9 equivalent to localize to a specific target nucleotide sequence. “Prime editing guide RNA” (or “pegRNA”) is a guide RNA that has been modified and designed for the prime editing methods and systems disclosed herein. A pegRNA associates with a prime editor protein. U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of guide RNA.

[0039] As used herein, the terms “prime editing guide RNA” or “pegRNA” refers to a specialized form of a guide RNA that has been modified to include one or more additional sequences for implementing the prime editing methods and systems described herein. The additional sequences comprise (i) a “DNA synthesis template” which encodes (copied by the polymerase, e.g., reverse transcriptase, of the prime editor) a single-stranded DNA which, in turn, has been designed to be (a) homologous with the endogenous target DNA to be edited, and (b) which comprises at least one desired nucleotide change (e.g., a transition, a transversion, a deletion, or an insertion) to be introduced or integrated into the endogenous target DNA; and (ii) a “primer binding site.” As used herein the “primer binding site” comprises a sequence that hybridizes to a single-strand DNA sequence having a 3' end generated from the nicked DNA of the R-loop. U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of pegRNA.

[0040] As used herein, “PE2” refers to a PE complex comprising a fusion protein comprising a Cas9 nickase and a reverse transcriptase (RT), and a desired pegRNA. U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of PE2.

[0041] As used herein, “PE3” refers to PE2 plus a second-strand nicking guide RNA (Nk sgRNA) that complexes with the PE2 and introduces a nick in the non-edited DNA strand in order to induce preferential replacement of the edited strand. U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of nicking guide RNA, second-strand nicking, and PE3.

[0042] As used herein, the term “polymerase” refers to an enzyme that synthesizes a nucleotide strand and which may be used in connection with the prime editor systems described herein. Reverse transcriptase is a polymerase. U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of polymerases.

[0043] As used herein, the term “prime editing” refers to an approach for gene editing using a nucleic acid programmable fusion protein comprising a Cas9 nickase and a polymerase (e.g., a reverse transcriptase), and specialized guide RNAs that include a DNA synthesis template for encoding desired new genetic information (or deleting genetic information) that is then incorporated into a target DNA sequence. U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of prime editing.

[0044] The term “prime editor protein” refers to fusion constructs comprising a nucleic acid programmable Cas9 nickase and a polymerase (e.g., reverse transcriptase) that is capable of carrying out prime editing on a target nucleotide sequence in the presence of a pegRNA. The term “prime editor” may refer to the fusion protein or to the fusion protein complexed with a pegRNA, or to the fusion protein complexed with a second-strand nicking sgRNA. A nicking sgRNA may have a spacer sequence comprising a region of complementarity to the modified sequence of a non-target strand (PE3b) or to a sequence adjacent to the modified sequence of the non-target strand (PE3). As used herein with respect to nicking sgRNA, a non-target strand is the strand opposite the target strand, the target strand having a sequence complementary to the spacer sequence of a pegRNA, and to which the prime editorpegRNA RNP complex binds. U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of prime editors and prime editor proteins.

[0045] The terms “primer binding site,” “primer binding sequence,” and “PBS” are used interchangeably to refer to the nucleotide sequence located on a pegRNA as component of the extension arm (typically at the 3' end of the extension arm) and serves to bind to the primer sequence that is formed after Cas9 nicking of the target site sequence by the prime editor. U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of primer binding sites.

[0046] The term “reverse transcriptase” describes a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Avian myoblastosis virus (AMV) reverse transcriptase was the first widely used RNA-dependent DNA polymerase (Verma, Biochim. Biophys. Acta 473:1 (1977)). The enzyme has 5 '-3' RNA- directed DNA polymerase activity, 5 '-3' DNA-directed DNA polymerase activity, and RNase H activity. RNase H is a processive 5' and 3' ribonuclease specific for the RNA strand for RNA-DNA hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)). Another reverse transcriptase which is used extensively in molecular biology is reverse transcriptase originating from Moloney murine leukemia virus (M-MLV). See, e.g., Gerard, G. R , DNA 5:271-279 (1986) and Kotewicz, M. L., et al., Gene 35:249-258 (1985). M-MLV reverse transcriptase substantially lacking in RNase H activity has also been described. See, e.g., U.S. Pat. No. 5,244,797. U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of reverse transcriptases.

[0047] An “M-MLV reverse transcriptase domain,” “M-MLV-RT domain ,” and the like, means a protein domain of a prime editor protein having reverse transcriptase activity and at least 95% amino acid sequence identity with a corresponding contiguous sequence of wild-type M-MLV reverse transcriptase, as known to those of ordinary skill in the art, e.g., as disclosed in U.S. Publication No. US 2021/0292769, incorporated by reference herein at least for its disclosure of an M-MLV reference amino acid sequence and numbering convention used to refer to amino acid residues within the reference M-MLV sequence.

[0048] As used herein, amino acid residue numbering for an M-MLV RT domain of a prime editor refers to the amino acid residue numbering of M-MLV reverse transcriptase itself — not the entire prime editor protein — for example, the M-MLV RT amino acid residue numbering disclosed in the reference M-MLV reference amino acid sequence of U.S. Publication No. US 2021/0292769.

[0049] An M-MLV RT domain includes, but is not limited to, various M-MLV-RT domain prime editor architectures, as known to those of ordinary skill in the art. For example, an M-MLV reverse transcriptase domain includes wild-type M-MLV reverse transcriptase, RNaseH minus M-MLV reverse transcriptase (a deletion of the C-terminal RNaseH domain of M-MLV RT), and an N-terminal deletion of an M-MLV-RT.

[0050] The term “target site” refers to a sequence within a nucleic acid molecule that is edited by a prime editor (PE) disclosed herein. The target site further refers to the target sequence within a nucleic acid molecule to which a complex of the prime editor (PE) and gRNA binds.

[0051] As used herein the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant Cas9 is a Cas9 comprising one or more changes in amino acid residues as compared to a wild type Cas9 amino acid sequence. The term “variant” encompasses homologous proteins having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% percent identity with a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence, and which display the same or substantially the same functional activity or activities as the reference sequence.

[0052] “Engineered pegRNA” or “epegRNA” is a pegRNA comprising a 3’ structured RNA pseudoknot (3’ of the PBS of the pegRNA) that protects the 3' extension arm from degradation by exonucleases. epegRNAs are described in Liu et al., International PCT Application PCT/US2021/052097, filed September 24, 2021, published as WO/2022067130 on March 31, 2022, the contents of which are incorporated herein by reference for its disclosure of epegRNA.

[0053] The prime editor proteins disclosed herein form a complex with (e.g., bind or associate with) one or more RNA(s) that is not a target for cleavage. In some embodiments, an RNA-programmable nuclease, such as a prime editor protein, when in a complex with an RNA, may be referred to as a ribonucleoprotein complex, ribonucleoprotein, RNP, PE RNP, or RNP complex. The bound RNA(s) may be, for example, a pegRNA, epegRNA, or sgRNA.

[0054] “Melting temperature” (“Tm”) is the temperature at which one half of the strands of a population of duplexed nucleic acid will dissociate to become single-stranded. In some embodiments, the duplexed nucleic acid is a RNA:DNA duplex. Melting temperature is a function of both the sequence and the length of the duplex. Methods of calculating Tm are well known in the art. See, e.g., Dumousseau et al. (2012) BMC Bioinformatics, 13, 101, hereby incorporated by reference for its disclosure of melting temperature calculation.

[0055] By “hybridizable” (and derivatives thereof) or “complementary” it is meant that a nucleic acid (e.g. RNA) comprises a sequence of nucleotides that enables it to non- covalently bind, i.e. form Watson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. As is known in the art, standard Watson-Crick base-pairing includes; adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) [DNA, RNA], In addition, it is also known in the art that for hybridization between two RNA molecules (e.g., dsRNA), guanine (G) base pairs with uracil (U). For example, G/U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. In the context of this disclosure, a guanine (G) of a protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule is considered complementary to a uracil (U), and vice versa. As such, when a G/U base-pair can be made at a given nucleotide position a protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule, the position is not considered to be non- complementary, but is instead considered to be complementary. U.S. Publication No. US 2019/0010520 is hereby incorporated by reference for its disclosure of complementarity (including “complementary”) and hybridization (including “hybridizable”).

[0056] An “inhibitor of SAMHD1” means a compound that inhibits or reduces the activity of SAMHD1, directly or indirectly (e.g., by increasing degradation of SAMHD1 or inhibiting its enzymatic activity), thereby causing an increase in intracellular dNTP levels. Examples of an inhibitor of SAMHD1 include, but are not limited to, Vpx and variants thereof (Vpx (S13E) described by Miyakawa et al. Nat Commun. 2019 Apr 23; 10(1): 1844) and various protein kinases from beta and gamma herpesvirus, e g., EBV BGLF4, HHV-6/7 U69, HCMV UL97, and KSHV ORF36 (see, e.g., Zhang et al. Cell Rep. 2019 Jul 9;28(2):449-459.e5). The present disclosure contemplates inhibitors of SAMHD1 as known to those of ordinary skill in the art now and in the future.

[0057] In primary cell types, intracellular dNTP levels are tightly regulated in a cell cycle dependent manner. We have discovered that prime editing efficiency is increased by mutations that improve the enzymatic properties of MMLV-reverse transcriptase and treatments that increase intracellular dNTP levels. In addition, sequential delivery of prime editing and second strand nicking reagents improves precise editing rates.

[0058] Prime editing efficiency in primary cells may be hampered by inherent properties of MMLV-RT. For example, most prior optimization of prime editing components has been performed in rapidly dividing mammalian cells^{1, 9}> ⁿ. Since intracellular dNTP concentration can be 100-fold lower in post-mitotic or quiescent cells than cycling transformed cell lines¹⁶'¹⁹, this scarcity may suppress MMLV-RT processivity due to its low affinity for dNTPs^{18, 20}. In addition, we observed MMLV-RT -dependent protein aggregation during purification of the PEmax prime editor protein⁷, which suggests that its solubility may limit prime editing activity. [0059] We have surprisingly discovered that a prime editor protein comprising a Cas9 nickase fused to a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase domain having mutations (1) V223M, which increases dNTP affinity and (2) L435K, which increases prime editor solubility, significantly improves prime editing rates. In some embodiments, a prime editor protein comprising a Cas9 nickase fused to a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase domain, wherein the M-MLV reverse transcriptase domain comprises a V223M and a L435K mutation is disclosed.

[0060] In some embodiments, an isolated nucleic acid encoding the prime editor protein having the V223M and L435K mutations is contemplated as part of the present disclosure.

[0061] The present disclosure also contemplates a programmable prime editing system for modification of a double-stranded target DNA sequence comprising a target strand and a complementary non-target strand, the system comprising a prime editor protein having the V223M and L435K mutations; and a prime editing guide RNA (pegRNA) comprising a spacer sequence having a region of complementarity to the target strand of the double-stranded target DNA sequence. In some embodiments, the prime editing system comprises a nucleic acid encoding the prime editor protein and/or a nucleic acid encoding the pegRNA.

[0062] In some embodiments, the programmable prime editing system further comprises an inhibitor of SAMHD1 known to those of skill in the art, now and in the future. The inhibitor of SAMHD1 may, e.g., be Vpx, Vpx (S13E), EBV BGLF4, HHV-6/7 U69, HCMV UL97, or KSHV ORF36. In some embodiments, the inhibitor of SAMHD1 comprised by the system is a protein. In other embodiments, the system comprises a nucleic acid encoding the inhibitor of SAMHD1.

[0063] A prime editing system of the present disclosure may be a twin prime editing (Twin PE) system. Twin PE systems utilize a prime editor protein and two pegRNAs targeting opposite strands of locus to be modified, allowing for longer sequence edits.

[0064] In some embodiments, the programmable prime editing system comprises a nicking sgRNA. In other embodiments, the prime editing system comprises a nucleic acid encoding the nicking sgRNA. [0065] In accordance with a further embodiment of the invention, a method of modifying a double-stranded target DNA sequence, the method comprising contacting the double-stranded target DNA sequence with the prime editor protein and the pegRNA.

[0066] In accordance with additional embodiments of the invention, a method of modifying a double-stranded target DNA sequence in a cell, the method comprising: sequentially introducing into the cell, in the following order: (1) a programmable prime editing system, the programmable prime editing system comprising (i) a prime editor protein comprising a Cas9 nickase fused to a M-MLV reverse transcriptase domain and (ii) a pegRNA comprising a spacer sequence having a region of complementarity to a target strand of the double-stranded target DNA sequence; and (2) a nicking sgRNA. In some embodiments, the prime editor may be introduced into the cell by introducing a nucleic acid encoding the prime editor into the cell. In some embodiments, the prime editor may be introduced into the cell by introducing the prime editor protein into the cell. In some embodiments, the pegRNA may be introduced into the cell by introducing a nucleic acid encoding the pegRNA into the cell. In some embodiments, the pegRNA may be introduced into the cell by introducing the pegRNA into the cell. In some embodiments, the nicking sgRNA may be introduced into the cell by introducing the nicking sgRNA into the cell. In some embodiments, the nicking sgRNA may be introduced into the cell by introducing a nucleic acid encoding the nicking sgRNA into the cell.

[0067] In some embodiments of the method, the M-MLV reverse transcriptase domain comprises a V223M and a L435K mutation. In some embodiments, the programmable prime editing system further comprises an inhibitor of SAMHD1. The inhibitor of SAMHD1 may be Vpx, Vpx (S13E), EBV BGLF4, HHV-6/7 U69, HCMV UL97, or KSHV ORF36. In some embodiments, the inhibitor of SAMHD1 is a protein introduced into the cell. In other embodiments, the inhibitor of SAMHD1 is introduced into the cell by introducing a nucleic acid encoding the inhibitor of SAMHD1.

[0068] In some embodiments, the nicking sgRNA is introduced into the cell 2-96 hours after introducing the programmable prime editing system into the cell. In some embodiments, the nicking sgRNA is introduced into the cell 2-72 hours after introducing the programmable prime editing system into the cell. In some embodiments, the nicking sgRNA is introduced into the cell 2-48 hours after introducing the programmable prime editing system into the cell. In some embodiments, the nicking sgRNA is introduced into the cell 2- 36 hours after introducing the programmable prime editing system into the cell. In other embodiments, the nicking sgRNA is introduced into the cell 6-24 hours after introducing the programmable prime editing system into the cell.

[0069] In some embodiments, the nicking sgRNA is introduced into the cell 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,

30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,

55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,

80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours after introducing the programmable prime editing system into the cell.

[0070] In some embodiments, the programmable prime editing system is introduced into the cell by a first method selected from the group consisting of electroporation, transfection, viral delivery, virus-like particle delivery, and lipid nanoparticle delivery.

[0071] In some embodiments, the nicking sgRNA is introduced into the cell by a second method selected from the group consisting of electroporation, transfection, viral delivery, virus-like particle delivery, and lipid nanoparticle delivery.

[0072] Example 1: PEmax** (V223M and L435K MMLV-RT mutations)

[0073] Several previously described MMLV-RT sequence variants were screened for their impact on PEmax prime editing activity in transformed cell lines (HEK293T and U2OS cells) and primary fibroblasts. MMLV-RT processivity under limiting dNTP levels has been enhanced by a Q221R mutation²² and a variety of mutations at position 223^22-24. V223M changes the MMLV-RT active site from an onco-retroviral RT to a more efficient lentiviral- like RT, reducing its K_m for dNTPs by 2 to 4-fold^22-24. V223A and V223Y mutations have been previously introduced in MMLV-RT to improve prime editing rates^{11, 23}.

[0074] We evaluated the impact of Q221R and three different V223 mutations (V223A, V223M, and V223Y) individually on prime editing rates at multiple target sites under subsaturating editor levels to discern differences in activity. Among the dNTP affinity mutants, V223M (PEmax*) and V223Y modestly enhanced prime editing rates, with V223M displaying the most substantial improvement in activity (Fig. 1A).

[0075] MMLV-RT solubility has been improved by a 24 amino acid N-terminal truncation or a L435K mutation without substantial impact on its catalytic performance²⁶. In the context of PEmax, the N-terminal MMLV-RT truncation reduced prime editing efficiency, whereas the L435K mutation significantly enhanced prime editing efficiency (Fig. 1A)

[0076] Next, we combined L435K mutant with the panel of dNTP affinity mutants (V223A, V223M, and V223Y) in PEmax and assessed prime editing rates in comparison to a recently described evolved PE6d variant¹¹. The combination of V223M and L435K MMLV- RT mutations (PEmax**) proved the most robust combination, outperforming PEmax by an average of ~2.5-fold, and displaying a ~1.4 fold improvement over PE6d across a panel of target sites (Figure 1A).

[0077] Example 2; PEmax** Protein has Improved Solubility

[0078] We introduced the L435K and V223M mutations in our previously described PEmax protein expression construct⁷, to evaluate their impact on protein solubility and prime editing activity (PEmax**).

[0079] Consistent with our prior results⁷, purified PEmax protein formed aggregates when concentrated above 30 pM. In contrast, purified PEmax** protein could be concentrated to 140 pM without visible aggregates. The improved solubility of the PEmax** prime editor protein resulted in a 7-fold increase in protein yield.

[0080] Example 3; PEmax** has Improved Prime Editing Efficiency Compared to PEmax and PEmax*

[0081] We evaluated the prime editing efficiency of the PEmax and PEmax** proteins in vitro under limiting dNTP concentrations. The incorporation rate of a 20- nucleotide sequence tag²⁷ plateaued at -0.2 pM dNTPs for both proteins (Fig. IB). PEmax** displayed significantly higher enzymatic activity at subsaturating dNTP levels (0.05 to 0.15 pM dNTPs), consistent with the improved dNTP affinity of the V223M mutation²².

[0082] Next, we compared prime editing activity for PEmax and PEmax** in induced myoblasts (iMyoblasts), which are differentiated from iPSCs using a transgene-free myogenic protocol²⁸. Editing rates were evaluated at the FANCF or HEK4 target sites (PE3 system)¹. We observed a 1.3- to 1.7-fold increase in precise editing outcomes when employing PEmax** RNPs in iMyoblasts with maximal editing efficiencies ranging from 15 to 40% (Fig. 1C). Similar improvements in editing efficiency were achieved for the delivery of prime editor mRNA with synthetic pegRNA/sgRNA, where PEmax ^V223M (PEmax*) displayed a more modest increase in editing rates. A comparable enhancement (~2-fold) in precise editing rate was observed in primary human T cells for PEmax** at the FANCF target site (PE3 system) (Fig. 1C).

[0083] We also compared the in vivo prime editing activity of PEmax and PEmax** in zebrafish and mice. We programmed PE RNPs with a synthetic pegRNA (PE2 system) to generate two non-synonymous mutations - R841W or L907F - within the tek locus in zebrafish embryos⁷. We observed a ~2-fold increase in the introduction of the R841W mutation for PEmax** RNPs. Dose-dependent editing was observed, where precise editing rates reached -30% (Fig. ID). A similar improvement in prime editing efficiency for PEmax** was observed for introduction of the L907F mutation.

[0084] We then evaluated the utility of PEmax** for introducing a Q155H mutation into Pcsk9 in the mouse liver¹⁵. We delivered lipid nanoparticles (LNPs) containing PEmax or PEmax** mRNA along with previously described epegRNA and nicking guide RNA¹⁵ by retro-orbital injection in young adult mice. We observed 1.8% precise editing .Pcsk9 in the mouse liver with PEmax**, a 1.7-fold improvement over PEmax. Thus, PEmax** increases precise editing rates in vivo in zebrafish embryos and adult mice in two different delivery formats.

[0085] Example 4; VPX Co-delivery and Deoxynucleoside Supplementation Increase Prime Editing Efficiency

[0086] Our biochemical, in vitro and in vivo prime editing studies are consistent with intracellular dNTP levels representing a bottleneck for MMLV-RT -based prime editing. Cells employ multiple pathways to regulate dNTP levels to limit unplanned DNA synthesis²⁹. SAMHD1 is a deoxynucleotide triphosphohydrolase that maintains low dNTP levels in cells that are not undergoing DNA replication, which restricts infection by retroviruses^{30, 31}. Many viruses subvert SAMHD1 activity to promote infectivity³². HIV-2 packages an accessory protein - VPX - within the virion that targets SAMHD1 for degradation via the cullin 4-based (CRL4) E3 ubiquitin ligase pathway³³' ³⁴. SAMHD1 inactivation increases intracellular dNTP levels, which promotes reverse transcription and successful viral infection in quiescent cells^{31, 35}. An alternate approach for increasing intracellular dNTP levels in vitro is supplementation of deoxynucleosides (dNs) in growth media³⁶’³⁸. [0087] We investigated the impact of co-delivering VPX or supplementing cellular growth media with dNs on prime editing rates in HEK293T cells, U2OS cells and primary fibroblasts. Because excess dNs can negatively impact cell fitness and proliferation, we evaluated the impact of increasing dN concentration on cell viability to find a balance between cell fitness and dN concentration³⁹'⁴². We observed that dN supplementation or VPX co-delivery enhanced prime editing rates, where VPX in combination with PEmax** increased prime editing rates -3.5-fold relative to PEmax alone (Fig. IE). Consistent with intracellular levels of dNTPs playing a critical role in precise editing outcomes, co-delivery of SAMHD1 suppressed precise editing rates and increased imprecise editing outcomes (data not shown).

[0088] Next, we used the EvaGreen system⁴³ to assess the impact of VPX on intracellular dNTP levels in fibroblasts and activated T cells. Delivery of VPX increased intraellular dNTP levels in fibroblasts by more than 2-fold and depleted SAMHD1 protein, whereas VPX^Q76A, which cannot target SAMHD1 for proteasomal degradation³⁴, did not appreciably change intracellular dNTP levels (data not shown). Overexpression of SAMHD1 in fibroblasts depleted intracellular dNTP levels (data not shown), mirroring the decrease in prime editing efficiency observed upon its co-delivery (data not shown).

[0089] Co-delivery of VPX protein or mRNA increased precise editing rates for PEmax, PEmax* or PEmax** at the FANCF locus in resting T cells (data not shown). VPX mRNA co-delivery significantly increased prime editing rates at the CCR5 locus in activated T cells, where a TWIN-PE approach⁴⁴ with Cas9-NG⁴⁵ PEmax** efficiently introduced the CCR5^deIta32 deletion (-40%) that is associated with HIV-1 R5-trophic virus resistance⁴⁶'⁴⁸ (Fig. IF). Similarly, co-delivery of VPX mRNA significantly improved prime editing efficiency with PEmax** in stem cell-derived islet (SC-islet) cells^{49, 50} (Fig. IF). Co-delivery of VPX increased the ratio of precise to imprecise prime editing outcomes, while also modestly increasing the rate of pegRNA scaffold insertions¹¹ (data not shown).

[0090] VPX co-delivery with PEmax** may be used with LNPs or eVLPs⁴⁶ to improve editing outcomes in various organ systems in vivo. DNA polymerase-based prime editing systems may provide an alternate method to overcome low dNTP levels that restrict prime editing in quiescent and post-mitotic cells due to their superior processivity and dNTP affinity relative to RTs^41,42. [0091] Example 5: Sequential Delivery of Nicking sgRNA Increases Prime Editing Rates Compared to Simultaneous Delivery of Nicking sgRNA in PE3b Prime Editing System

[0092] The PE3b prime editing system provides an efficient method to increase prime editing rates while reducing unwanted editing byproducts through delivery of a nicking guide RNA that recognizes the modified sequence introduced by prime editing^{1, 3}. Because the protospacer recognized by the nicking sgRNA contains prime editing directed mutations, nicking of the unmodified genomic strand should occur after MMLV-RT polymerization of the pegRNA-encoded template sequence into the genome^{1, 3}. Since the nicking sgRNA has higher affinity for the prime editor than the pegRNA, prime editing rates can be reduced by competition between the sgRNA and pegRNA for the prime editor⁷.

[0093] We hypothesized that delivery of the prime editor and pegRNA followed by delivery of the nicking sgRNA after a delay to allow the initiation of prime editing would increase precise editing rates by minimizing sgRNA competition (Fig. 2A). We examined the impact of simultaneous and sequential delivery of the PE3b nicking reagents on prime editing in an HEK293T mCherry reporter system⁴ and observed that sequential delivery of the PE3b nicking reagents enhanced prime editing rates relative to simultaneous delivery (data not shown).

[0094] We next examined sequential PE3b editing with PEmax, PE6d or PEmax** at the FANCF locus in three cell types: EBV-transformed B cells, immortalized human bronchial epithelial (HBE) cells and primary fibroblasts. In all cases sequential PE3b editing increased precise editing rates relative to simultaneous delivery, and delivery of VPX further improved precise editing rates (Fig. 2B).

[0095] We then compared simultaneous and sequential PE3b editing for the precise correction of three therapeutically relevant targets: Rett syndrome (A/ECP2^T158M)⁵¹, cystic fibrosis (CF7A^delF508)⁵² and Tay-Sachs (HEXA^1278+TATC)⁵³. For all three target sites with three different prime editors (PEmax, PE6d or PEmax**), sequential PE3b editing outperformed simultaneous PE3b editing, and VPX further improved precise editing rates (Figs. 2C-E).

[0096] Sequential PE3b editing with PEmax** and VPX mRNA co-delivery increases precise editing rates by ~5-fold over standard PE3b editing with PEmax in primary fibroblasts and immortalized cell lines (Fig. 2F-G). Sequential PE3b editing involves the delivery of Cas9^H840A nickase and the nicking sgRNA, where the additional nickase may also help to stimulate precise editing outcomes. Because the nicking sgRNA in PE3b systems is programmed to recognize a precisely edited sequence¹’ ³, the co-delivery of nicking sgRNA with the pegRNA can only initially produce unproductive competition by complexing with the prime editor⁷. Sequential delivery prevents guide RNA competition under conditions of limiting prime editor protein.

[0097] Employing PEmax** with sequential PE3b nicking along with methods to increase intracellular dNTPs may have broad utility for editing quiescent and post-mitotic cells.

[0098] Materials and methods

[0099] General methods and molecular cloning

[0100] Expression plasmids used for pegRNAs have been previously described²⁷. Mammalian expression plasmids for different PEmax variants (N terminal 24aa deletion, Q221R, V223A, V223M, V223Y, L435K, L435K/Q221R, L435K/V223A, L435K/V223M, L435K/V223Y) were generated by site-directed mutagenesis on pCMV-PEmax (pCMV- PEmax and pCMV-PE6d are gifts from David Liu, Addgene plasmid #174820 and #207854). For NG-PEmax mammalian expression vector, the

LI 111R/D1135V/G1218R/E1219F/A1322R/R1335V/T1337R (VRVRFRR) fragment⁴⁵ was synthesized by IDT and Gibson-cloned into the PEmax mammalian expression and IVT plasmids (Addgene plasmid #174820 and #204472) digested with Pmll/Rsr2 enzymes. All plasmids used for transient transfection experiments were purified with an endotoxin removal step (ZymoPURE Plasmid Miniprep Kit from Zymo Research). The PEmax** protein expression vector (pET-21a-PEmax**-6His) was generated by amplifying the fragment of MMLV-RT containing the V223M and L435K mutations from pCMV- PEmax**. This PCR product was digested with BamHI and BsrGI and cloned into pET-2 la- PEmax-6His (Addgene plasmid #204471) digested with the same enzymes. The PEmax* and PEmax** mRNA in vitro transcription (IVT) vector was generated by amplifying the fragment containing the MMLV-RT mutants from different PEmax variants or pCMV-PE6d separately. These PCR products were Gibson-cloned into the PEmax mRNA plasmid (Addgene plasmid #204472) digested with EcoRI enzyme. VPX coding sequence was amplified from pscALPS gag-gfp/deltavpx vector (Addgene #115807) and inserted into PEmax mRNA plasmid (Addgene plasmid #204472) digested by Sall and EcoRI to replace the PEmax coding sequence. The VPX^Q76A vector was generated from the VPX mRNA vector by Gibson cloning. SAMHDl coding sequence was amplified from U2OS cell cDNA library, then Gibson-cloned into the pCMV-PEmax or PEmax IVT mRNA plasmid to replace the PEmax coding sequence.

[0101] pegRNA designs

[0102] We designed the pegRNAs with a primer binding site (PBS) length and composition that reduces the auto-inhibitory interaction between the PBS and spacer sequence within the pegRNA, as described in our previous study⁷, we utilized the MELTING 5 program⁵⁸ to identify a PBS length nearest to the ideal T_m value identified (37°C). The RNA sequence of the PBS was entered in the 5’ to 3’ direction along with the nucleic acid concentration, hybridization type and sodium concentration based on the 10 pL RNP complex reaction conditions (nucleic. acid. concentration = 2e-05, hybridization. type = “rnadna”, Na. cone = 1 and method. nn = “sug95”). Based on the output of predicted T_m values, a PBS sequence length was chosen that has a Tm close to 37°C. The PBS length for all pegRNAs used in this study except the PCSK9 pegRNA were designed using the MELTING 5 program. For epegRNAs design, we used pegLIT (peglit.liugroup.us) to identify a suitable linker sequence by inputting the spacer, PBS, and reverse transcriptase template (RTT) for each specific target site⁴.

[0103] In vitro transcription of mRNA for PEmax, PEmax variants, SAMHDl and VPX

[0104] PEmax, PEmax*, PEmax**, PE6d, SAMHDl, VPX and VPXQ76A IVT mRNA plasmid were linearized using Pmel to cleave after the polyA tail. mRNA was transcribed from 500 ng purified linearized template using the HiScribe T7 High-Yield RNA Synthesis Kit (New England BioLabs) with co-transcriptional capping by CleanCap AG (TriLink Biotechnologies) and full replacement of UTP with Nl-Methylpseudouridine-5’- triphosphate (TriLink Biotechnologies). After 1 hour of in vitro transcription, the DNA template was digested by 1 uL DNasel (Thermo Fisher Scientific) for 15 min. Transcribed mRNAs were purified by RNA Clean & Concentrator-25 kit from Zymo Research, then purified mRNA was dissolved in nuclease-free water. The resulting mRNA was quantified with a NanoDrop One UV-Vis spectrophotometer (Thermo Fisher Scientific) and stored at - 80°C.

[0105] PEmax and PEmax** protein purification

[0106] PEmax protein purification followed our previously described protocol ⁷. Briefly, PEmax and PEmax** protein expression constructs were introduced into E. coli Rosetta2(DE3)pLysS cells (EMD Millipore). Bacteria were grown at 37°C in baffle flasks to an OD600 of ~0.6, then pre-chilled in an ice bath for 10 minutes and shifted to growth at 18°C. At an OD600 of -0.8 the cells were induced for 16 hours with IPTG (0.7 mM final concentration). Following induction, cells were pelleted by centrifugation (3500 g, 20 min) and then resuspended with Nickel-NTA buffer (20 mM TRIS + 1 M NaCl + 20 mM imidazole + 1 mM TCEP, pH 7.5) supplemented with HALT Protease Inhibitor Cocktail, EDTA-Free (100X) [ThermoFisher] and lysed with LM-20 Microfluidizer (Microfluidics) following the manufacturer’s instructions. The lysate was transferred to a centrifuge tube and spun at 20,000 g for 20 minutes. The clarified lysate was then purified with Ni-NTA resin (Qiagen) in batch mode, washed with wash buffer (20 mM TRIS + 1 M NaCl + 20 mM imidazole + 1 mM TCEP, pH 7.5) and eluted with an elution buffer (20 mM TRIS, 500 mM NaCl, 250 mM Imidazole, 10% w/v glycerol, pH 7.5). The eluted proteins were dialyzed overnight at 4°C in 20 mM HEPES, 500 mM NaCl, 1 mM EDTA, 10% w/v (8% v/v) glycerol, pH 7.5. Subsequently, the proteins were step-dialyzed from 500 mMNaCl to 250 mM NaCl to 200 mM NaCl (not exceeding one hour incubation per step; final dialysis buffer: 20 mM HEPES, 200 mM NaCl, 1 mM EDTA, 10% w/v glycerol, pH 7.5). Next, the proteins were purified by anion and cation exchange chromatography [the protein was loaded on a stacked column of 5ml HiTrap-Q HP and 5ml HiTrap-S (Cytiva), Buffer A = 20 mM HEPES pH 7.5 + 1 mM TCEP, Buffer B = 20 mM HEPES pH 7.5 + 1 M NaCl + 1 mM TCEP, Flow rate = 5 ml/min, CV = column volume = 5 ml]. The anion exchange column was removed prior to the elution of the prime editor protein from the cation exchange column. The primary prime editor protein peak fractions were dialyzed into 20 mM HEPES pH 7.5, 300 mM NaCl and then concentrated to ~30pM for PEmax and 140pM for PEmax** using a 100 kDa amicon ultra centrifugal filter (UFC910008, Millipore).

[0107] Culture conditions for immortalized cell lines, Rett patient-derived fibroblasts, 16HBEge CFTR AF508 cells and HEXA^1278+TATC EbvB cells. [0108] HEK293T cells and U2OS cells were purchased from ATCC. Patient-derived fibroblasts containing the T158M mutation were a gift from the Rett Syndrome Research Trust. These cell lines were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS at 37°C and 5% CO2. CFF-16HBEge CFTR A508 (16HBE) cells were a gift of the Cystic Fibrosis Foundation. 16HBE cells were grown at 37 °C/5% CO2 in MEM (Gibco) supplemented with 10% FBS (Gibco) and 1% Penicillin/Streptomycin (Gibco). Plates/flasks for 16HBE cell growth were prepared by incubating a thin layer of coating solution [LHC-8 basal medium (Gibco), 1.34 pl/ml Bovine serum albumin 7.5% (Gibco), 10 pl/ml Bovine collagen solution (Gibco), Type 1 (Advanced BioMatrix), 10 pl/ml Fibronectin from human plasma (Thermo Fisher Scientific)] at 37 °C/5% CO2 for 2-3 h followed by thorough removal of coating solution and storage at 4 °C until use. Cells were dissociated with Trypsin (Gibco) and centrifuged at 120 x g for 5 min before seeding. Medium was changed three times weekly. HEXA^I278+TATC EbvB Cells were purchased from Coriell (GM11852). HEXA EbvB cells were cultured in RPMI 1640 medium (Gibco) with 20% of Fetal Bovine Serum (Gibco), 4 mM L-glutamine (twice the normal culture concentration, Gibco) and 1% Penicillin/Streptomycin at 37 °C/5% CO2. Deoxynucleosides (Sigma-Aldrich) were resuspended in nuclease-free water at 25 mM each, filter-sterilized, aliquoted and stored at -20°C freezer. For deoxynucleoside (dN) supplementation in the growth media, cells were cultured in the presence of a specific amount of dNs 12 hours before transfection or electroporation. For editing experiments, 50 pM of each dN (final concentration) was added to the media for HEK293T cells and U2OS cells, and 30 pM of each dN was added to the media for Fibroblast cells.

[0109] Transfection of HEK293T and U2OS cells

[0110] HEK293T and U2OS cells were plated 40,000 cells per well in a 48-well plate. 24 hours later, the cells were co-transfected with 1000 ng prime editor plasmid and 330 ng pegRNA plasmid using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. 250 ng SAMHD1 or VPX expression plasmid was included for overexpression of these factors. To determine editing rates at endogenous genomic loci, cells were cultured 3 days following transfection, after which the media was removed, the cells were harvested, and genomic DNA was isolated using QIAamp DNA mini kit (QIAGEN) according to the manufacturer’s instructions. The editing rates were then determined by targeted amplicon deep sequencing.

[0111] Electroporation of HEK293T, U2OS cells, iMyoblasts, Fibroblast cells, HBE cells and EbvB cells

[0112] PEmax mRNA mixed with synthetic pegRNA/sgRNA mixtures or RNPs programmed with synthetic pegRNA/sgRNA were delivered by electroporation using the NEON Nucleofection System 10 pL kit (Thermo Fisher Scientific). Simultaneous or sequential delivery are used for some mRNA-based editing experiments in Fibroblast cells, HBE cells and EbvB cells. For simultaneous PE3b, 100k cells were pelleted at 300 g for 5 min and resuspended in 12 pL NEON Buffer R containing 2.5 pg PEmax or NG-PEmax variant mRNA, 180 pmol synthetic pegRNA, 30 pmol synthetic Nicking sgRNA (IDT) and 600 ng of VPX/VPX^Q/oA mRNA (for VPX mRNA co-delivery), then electroporated using cell type specific conditions (HEK293T: 1150v, 20ms, 2 pulses; U2OS: 1200v, 20ms, 2 pulses; Fibroblast cells: 700v, 20ms, 2 pulses; HBE cells: 1400v, 30ms, 2 pulses; and EbvB cells: 1750v, 20ms, 2 pulses). Cells were harvested and genomic DNA was isolated 3 days later. For Sequential PE3b delivery, nicking guide was not included in the initial electroporation. 12 to 16 hours after electroporation, 500 ng SpCas9 H840A nickase or SpCas9-NG H840A nickase mRNA and 50 pmol synthetic sgRNA (IDT, for sequential PE3b approaches; Supplementary Table) were transfected into the cells using Lipofectamine MessengerMAX Reagent. For editing experiments in iMy oblasts, Limb girdle muscular dystrophy 2G (LGMD2G) iMyoblasts were differentiated from LGMD2G iPSCs and maintained as previously described²⁸’ ⁵⁹. For mRNA-based editing experiments, 200,000 iMy oblast cells were resuspended into 10 pl of NEON Buffer R, mixed with Ipg PEmax variant mRNA, 100 pmol synthetic pegRNA (IDT) and 50pmol sgRNA (IDT), and then electroporated using NEON Nucleofection System 10 pL kit (Thermo Fisher Scientific, pulse voltage 1,500 V, pulse width 20 ms, 1 pulse). After electroporation, the iMy oblast cells were plated into a 6-well plate with HMP growth medium. For RNP-based editing experiments in iMyoblasts, 50 pmol of PEmax or PEmax** protein was incubated with 200 pmol of synthetic pegRNA (IDT) and 15 pmol of sgRNA (IDT) in R buffer to a total volume of 10 pL for 15 min at room temperature for complex formation. 200k cells were electroporated with 10 pL of PEmax RNP complex using the same electroporation and culture conditions described above for mRNA delivery. gDNA was isolated 3 days after electroporation from each group and stored at -80°C for Illumina library preparation for targeted amplicon deep sequencing.

[0113] Prime editing experiments in human primary CD4⁺ T cells

[0114] Leukopaks were obtained from anonymous, healthy blood donors (New York Biologies, Southampton, New York). To generate primary CD4+ T cells, peripheral blood mononuclear cells (PBMCs) were isolated from human donor leukopaks by gradient centrifugation on lymphoprep (cat#07861, Stemcell Technologies). Thereafter, PBMCs were depleted of CD14 mononuclear cells using anti-CD14 microbead antibodies (cat#130-050- 201, Miltenyi Biotec) and the flowthrough was enriched for CD4+ T cells by positive selection using anti-CD4 microbead antibodies (cat#130-045-101, Miltenyi Biotec). CD4+ T cell enrichment was confirmed by determining the percentage of CD3+/CD4+ cells via flow cytometry. Isolated CD4+ T cells were cultured in complete RPMI-IL2 media (RPMI-1640 media (cat# 11875093, Thermofisher Scientific) supplemented with 10% heat-inactivated Cosmic Calf Serum (cat#SH30087.03, GE Life Sciences), 25 mM HEPES pH 7.2 (cat#25- 060-CI, Corning), 20 mM GlutaMAX (cat#3505-061, Gibco), 1 mM Sodium pyruvate (cat#25-000-CI, Coming), IX MEM non-essential amino acids (cat#25-025-CI, Corning), 1% penicillin-streptomycin (cat#l 5140-122, Gibco), and 1 :2000 human interleukin-2 (made in-house from IL-2 expressing cell line). 3 days prior to electroporation, primary CD4+ T cells were activated with anti-CD3/CD28 antibodies (cat#10971, Stemcell Technologies). For activated primary CD4+ T cells, 5xl0⁵ cells per sample were pelleted by centrifugation for 5 min at 300 g and resuspended in 11 mL NEON Buffer T (Thermo Fisher Scientific). The cell solution was added to a mix of 1.5 mg PEmax (or the variants) mRNA, 120 pmol synthetic pegRNA (Integrated DNA Technologies), 30 pmol synthetic sgRNA (Synthego), and 0 to 1 mg VPX (or VPX^Q76A) mRNA. Synthetic pegRNAs and sgRNAs were dissolved in TE buffer (10 mM Tris-HCl, pH 8.0; 0.1 mM EDTA). Mock control electroporations were performed with 3 mL NEON Buffer T without any RNA added. Electroporation on the NEON Transfection System (Thermo Fisher Scientific) was carried out using 10 mL NEON tips with the following parameters: 1,400 V, 10 ms, three pulses. Cells were plated in 600 mL fresh T cell media in a 24-well plate. 2 days after electroporation, ImL fresh T cell media was added to cells. 4 days after electroporation, cells were pelleted by centrifugation for 5 min at 300g and genomic DNA was isolated using the Qiagen QiAamp DNA Blood Mini Kit (cat#51104, Qiagen). For resting CD4+ T cell experiments, le⁶ cells were electroporated using the P3 primary cell nucleofector kit and program FI-115 on the Amaxa 4D-Nucleofector immediately after isolation. The cell solution was added to a mix of 1 pg PEmax (or the mutants) mRNA, 100 pmol synthetic pegRNA (Integrated DNA Technologies), 50 pmol synthetic sgRNA (Synthego), and 0 to 500 ng VPX (or VPX^Q76A). CD4+ T cells were allowed to recover in RPMI-IL2 media for 72 hours post nucleofection before genomic extraction using the Qiagen QiAamp DNA Blood Mini Kit (cat#51104, Qiagen). For VPX co-delivery experiments, 0.5 - 4 pg of VPX protein (ab267924, Abeam) or 500 ng of VPX/VPX^Q76A mRNA was added to the electroporation mixture described above.

[0115] Prime editing experiments in HuES8-derived islet cells

[0116] Generation of human SC-islets. HuES8-derived SC-islet cells were maintained and differentiated as described⁶⁰ in protocol version 8, except the medium used for Stage 6 was the Stage 7 medium described by Balboa and colleagues⁶¹, modified to include non-essential amino acids and to omit ZM447439, T3, and NAC. In addition, differentiating cultures were treated throughout Stage 5 and for Days 1-7 of Stage 6 with 1 pM aphi dicolin (A-0781, Sigma).

[0117] In vitro glucose-stimulated insulin secretion (GSIS). SC-islet clusters were washed twice MCDB131 medium (CM134-050, GenDepot) supplemented with glutagro (25- 015-CI, Corning) and 5.6 mM glucose and preincubated at 37° C for 1 h containing MCDB13 1 medium containing glutagro and 2.5 mM glucose (basal). Clusters were then challenged with MCDB131 + glutagro containing basal glucose (2.5 mM), high glucose (20 mM), followed by depolarization with MCDB131 + glutagro containing 2.5 mM glucose + 20 mM KC1. Each treatment lasted 1 h, after which supernatant was collected and human insulin quantified using a human insulin TRF -PINCER® Assay (#IHTR1010, Mediomics).

[0118] Flow cytometric analysis of SC-islets. SC-islet cells were dissociated and stained with the following: BV786 mouse anti-Ki-67 (cat. #563756, BD Biosciences), Alexa Fluor 790 mouse anti-glucagon (cat. #sc514592 AF790, Santa Cruz Biotechnology), Alexa Fluor 647 mouse anti-C-peptide (cat. #565831, BD Biosciences), and PE mouse anti-Sox2 (cat. #560291, BD Biosciences). Flow cytometry data were acquired using a FACSymphony™ Al cytometer (BD Biosciences) and analyzed using FlowJo software (BD Biosciences).

[0119] Electroporation of SC-islets. Differentiated SC-islet cells (stage 6, day 19) were cultured in complete CMRL 1066 media (cat. #15-110-CV, Coming) supplemented with 10% fetal bovine serum (cat. #EF-0500-A, Atlas Biologicals), 1% penicillinstreptomycin solution (cat. #30-002-CI, Coming), and 1% GlutaMAX (cat. #35050-061, Gibco) at 37 °C, 10% CO2 for 24 h prior to electroporation. SC-islet cells were washed once with phosphate buffered saline (cat. #21-040-CM, Corning), pelleted by centrifugation for 3 min at 300 x g, and resuspended in 1 mb of TrypLE Express (cat. #12604, Gibco). SC-islet cells were then incubated for 5-10 min at 37 °C and triturated with a P1000 pipette every 2 min until 80-90% dissociated. Once dissociated, 2 mL of complete CMRL 1066 were added to the cell solution and the cells were pelleted by centrifugation for 3 min at 300 x g. The dissociated cells were resuspended in 5 mL of complete CMRL 1066, passed through 37 mM filters (cat. #27215, STEMCELL Technologies), and counted by trypan blue (cat. #15250- 061, Gibco) staining. Cells were then aliquoted into 1.5 x 10⁶ cells per sample. PEmax mRNA with synthetic pegRNA/sgRNA mixtures were delivered by electroporation using the Lonza™ P3 Primary Cell 4D-Nucleofector™ X Kit L (cat. # V4XP-3024, Lonza Bioscience) and the 4D-Nucleofector™ X Unit (cat. #AAF-1003X, Lonza Bioscience). Samples were pelleted and resuspended in 20 mL of supplemented P3 buffer containing 12 mg PE mRNA, 600 pmol PegRNA, 100 pmol sgRNA, and 0 or 2 mg VPX mRNA. Mock control electroporation samples were resuspended in 20 mL of supplemented P3 buffer. Electroporation on the 4D-Nucleofector™ X Unit was performed on the CA137 setting. Immediately after electroporation, 880 mL of warm RPMI 1640 media (cat. #11875-093, Gibco) was added to each sample and cells were incubated at 37 °C for 10 min.

[0120] Islet reaggregation and DNA extraction. Aggrewell 800 24-well plates (cat. #34811, STEMCELL Technologies) were prepared by adding 500 mL of anti-adherence rinsing solution (cat. #07010, STEMCELL Technologies) to each well. The plate was spun at 1300 x g for 5 minutes and wells were rinsed with 2 mL of warm complete CMRL 1066 media. This media was aspirated and replaced with 1 mL of warm complete CMRL 1066 media. The electroporated samples were then pipetted evenly into each well of the plate and the plate was centrifuged at 100 x g for 3 min. Cells were cultured at 37 °C, 10% CO2 for five days post-electroporation, with media replacement after 24 h with fresh complete CMRL 1066. Genomic DNA was extracted from the pseudo-islets with the DNeasy Tissue kit (cat. #69504, Qiagen).

[0121] Quantification of dNTP tolerance by PE-tag based qPCR

[0122] PE-tag based quantitative PCR (qPCR) was described previously²⁷. Briefly, 100 pmol synthetic pegRNA (IDT) targeting HEK4 was incubated with 50 pmol prime editor protein in 15 ml PBS for 20 min at room temperature to form RNP. Following complex formation, the PE RNP was mixed with the reaction buffer: dNTPs at a desired concentration (0~l mM), 5% glycerol, 100 mM KC1, 10 mM HEPES, pH 7.5, 0.2 mM EDTA, 3 mM MgCh, and 5 mM DTT) and 2 pg purified gDNA in a total volume of 30 pl for 2 h at 37 °C. Next the reaction was treated with 10 pl RNase A (50 pg/ml) to digest the pegRNA, and the gDNA was purified using a DNeasy Blood & Tissue Kit (Qiagen). The tag incorporation rate was quantified by qPCR with a tag-specific primer and a locus-specific primer. A pair of primers located approximately 2,000 bp upstream of the target site served as a qPCR internal control for gDNA normalization.

[0123] Immunoblot for SAMHD1 Protein from fibroblasts and Primary T cells [0124] For immunoblot of SAMHD1 protein in fibroblasts, 300,000 cells were electroplated with VPX (2,4 or 8 pg), VPX ^76A (2 pg), or SAMHD1 mRNA (2,4 or 8 pg) using the NEON Nucleofection System 10 pL kit (Thermo Fisher Scientific), then transferred into 6-well plate. Cells were harvested 3 days later. For immunoblot of SAMHD1 protein in activated primary T cells. 500,000 cells were electroplated with different amount of VPX (2 or 4 pg) or VPX^Q76A (2 pg) mRNA using the NEON Nucleofection System 10 pL kit (Thermo Fisher Scientific), then transferred into 6-well plate. Cells were harvested 3 days later. To generate the desried number of cells for analysis multiple electroporations were performed in parallel. 2 to 3 million cells were harvested in PBS and lysed for 30 min on ice in RIPA buffer (10 mM Tris-Cl [pH 8.0], 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and 140 mM NaCl) supplemented with protease inhibitors. Lysates were clarified by centrifugation (13,000 rpm for 10 min at 4 °C) and the supernatants were collected. Protein samples were quantified with the Bradford assay and resolved by SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF), and probed using the indicated primary antibodies. The membrane was stained with Rabbit anti- Mouse IgG (H+L) HRP-conjugated Secondary Antibody (Thermo), detected with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo) and visualized with X-ray film. The following primary antibodies were used for staining: GAPDH (Santa Cruz, sc-47724) and SAMHD1 (abeam, ab 128107).

[0125] Cell Viability Testing with Trypan Blue Exclusion Method

[0126] Viability testing was carried out for HEK293T cells or fibroblasts supplemented with different concentrations of dNs in their growth media. For each dN concentration, 50K cells were plated in 24-well plate. The cell viability was measured every 24 hours after supplementing with dNs with a different well harvested for each time point. Briefly, prior to trypsinization to release adherent cells, the growth medium containing floating cells from each well was placed into 1.5 ml Eppendorf tube. Adherent cells were released by adding 50ul Trypsin-EDTA (0.25%) solution and incubating at 37 °C for 3 min. The growth medium harvested from the well was used to resuspend the cells by pipetting 2 to 3 times. A single cell suspension was confirmed under the microscope. 20 pL of the cell suspension was transferred to a centrifuge tube and 20 pL of 0.4% trypan blue solution was added. The mixture was incubated at room temperature for 2 minutes. Cell viability within each population was determined using a TC20 cell counter (Bio-Rad).

[0127] Extraction and Measurement of cellular dNTP

[0128] Fibroblasts or primary T cell pellets (~2 million cells) were washed twice with lx Dulbecco’s phosphate-buffered saline and resuspended in 100 pl of ice-cold 60% methanol. Samples were vortexed vigorously to lyse the cells and then heated at 95 °C for 3 min prior to centrifugation at 12,000 g for 30 s. Supernatants were removed by pipet and passed through 3-kDa cutoff centrifugal filters (Amicon Ultra-0.5 ml, Merck) into collection tubes to remove the majority of remaining macromolecules. Next, MeOH and hydrophobic metabolites were removed by washing the extracts twice with 1.4 ml diethyl ether. Residual diethyl ether was evaporated using Speed-Vac centrifugal vacuum evaporator (Savant Instruments, Farmingdale, NY, USA). The dried pellets were subsequently resuspended in dNTP buffer (50 mM Tris-HCl, pH 8.0, and 10 mM MgCh), and the extracts were stored at - 80°C for up to 1 week. Cellular dNTPs were quantified using the Eva-GREEN dNTP quantification method⁴³. The cellular extracts were subjected to a Q5 DNA polymerase and EvaGreen-based assay for dNTPs using dNTP-specific 197-nt templates with conserved primer binding sites as previously described⁴³. The baseline and the end-point fluorescence were read at a temperature above the primer annealing temperature (the temperature for baseline and end-point fluorescence to 75 °C for dATP and dCTP, 78 °C for dTTP, and 73.5 °C for dGTP). For the highest sensitivity, the polymerization reaction time at 66°C was limited to 55 min for dATP, 40 min for dTTP and dCTP, and 20 min for dGTP detection.

[0129] Zebrafish prime editing experiments

[0130] Zebrafish were maintained and bred according to standard protocols. Zebrafish embryos obtained from EK (WT) wild-type in-crosses were used for one cell-stage microinjections of PE RNPs. Prior to injections the tek target sequence was verified by Sanger sequencing. For IxRNP, 12 pM pegRNA (synthesized by IDT) and 6 pM PE protein were combined in nuclease-free water. For 2x and 4X RNP, the amount of pegRNA and protein used were scaled up 2-fold and 4-fold respectively. Complexes were incubated at room temperature for 5 minutes and then 2 nl was injected into single-cell embryos. Injected embryos were incubated at 28.5 °C overnight. Twenty-four hours post injection embryos were assessed for toxicity and genomic DNA was extracted from 20 normally developing embryos using the Qiagen DNeasy Blood and Tissue kit (Qiagen). Injections were performed in three independent replicates.

[0131] In vivo delivery of PEmax mRNA/pegRNA/sgRNA delivery for Pcsk9 editing in mice

[0132] PEmax and PEmax** mRNA were in vitro transcribed and purified as described above. For in vivo mouse experiments, an additional round of purification using cellulose was performed to remove dsRNA contaminants⁶². The purified PEmax/PEmax** mRNA (40pg) was mixed with Pcsk9 synthetic pegRNA (15 g, IDT) and nicking sgRNA (5 g, IDT) in lOmM citrate buffer. This RNA mix constituted the aqueous component for forming RNA-loaded LNPs by microfluidic mixing using the NanoAssemblr Ignite (Precision Nanosystems) with a 3 : 1 aqueous: ethanol flow rate ratio and a total flow rate of 12mL/min. The ethanol component consisted of a lipid mix of D-Lin-MC3-DMA (MedChem Express: HY-112251), DSPC (Sigma-Aldrich: Pl 138), cholesterol (Sigma-Aldrich: C8667), and DMG-PEG at a 50:10:38.5: 1.5 molar ratio⁶³. A lipid to nucleic acid weight ratio of 20 was used to determine mixing volume. After formulation, LNPs were dialyzed (Sigma- Aldrich: PURX35O5O) overnight at 4°C in IxDPBS (Thermo: 14190250), sterile filtered (Thermo: 723-2520), and concentrated (Millipore: UFC8100) to 150j.il per dose. Quality control was performed by dynamic light scattering (Malvern ZetaSizer) to assess particle diameter & RiboGreen (Invitrogen: R11490) quantification of RNA encapsulation efficiency.

[0133] LNPs containing mRNA were delivered by retro-orbital injection into B6 wild type female mice (three doses spaced one week apart). Fresh LNPs were formulated for each dose. Mice were sacrificed one week after the final dose and the liver was harvested. Three liver punches from each mouse liver (one from each lobe) were taken for gDNA extraction. The editing rates were then determined by targeted amplicon deep sequencing.

[0134] Targeted amplicon deep sequencing to assess editing rates

[0135] Genomic DNA was isolated for prime editing analysis from treated cells, zebrafish embryos or mouse liver. Genomic loci spanning each target site were PCR amplified with locus-specific primers carrying tails complementary to the Truseq adapters. 200 ng of genomic DNA was used for the 1^st PCR using Phusion master mix (Thermo) with locus specific primers that contain i5 and i7 adaptor tails. PCR products from the 1^st PCR were used for the 2^nd PCR with i5 primers and i7 primers to complete the adaptors and include the i5 and i7 indices. All primers used for the amplicon sequencing are listed in the Supplementary table. PCR products were purified with Ampure beads (0.9X reaction volume), eluted with 25ul of TE buffer, and quantified by Qubit. Equal molar ratios of each amplicon were pooled and sequenced using Illumina Miniseq. Amplicon sequencing data was analyzed with CRISPResso2. Briefly, demultiplexing and base calling were both performed using bcl2fastq Conversion Software v2.18 (Illumina, Inc.), allowing 1 barcode mismatch with a minimum trimmed read length of 75. Alignment of sequencing reads to each amplicon sequence was performed using CRISPResso2. Since there are multiple base changes or an insertion or deletion in our prime editing samples, we analyze precise editing and indels separately. For single base substitution, samples were analyzed by CRISPResso2 in regular batch mode using the following parameters: “-q 30”, discard indel reads TRUE”, and “-qwc (or — quantification window coordinates)” and “-- expected hdr amplicon seq” providing the desired edited amplicon sequence for each target site. The qwc value specifies the quantification window for indel analysis, including the entire sequence between pegRNA and nicking sgRNA-directed Cas9 nicking sites (when TWIN -PE or PE3 is employed), as well as an additional 10 bp beyond both cut sites. Intended PE editing efficiency was calculated as the percentage of reads with the desired edit without indels (“-discard indel reads TRUE.” mode) out of the total number of reads ((number of desired edit-containing reads) / (number of reference-aligned reads)).

Unintended editing frequency was calculated as the number of discarded reads divided by the total number of reads ((number of indel-containing reads) / (number of reference-aligned reads)). When the desired prime editing outcome comprises multiple base changes or an insertion or deletion, samples were analyzed by CRISPResso2 in regular batch mode using the following parameters: “-q 30”, discard indel reads TRUE”, “-qwc” and “— expected hdr amplicon seq” providing the desired edited amplicon sequence for each target site. For these types of edits, the intended edit was calculated by (the number of HDR- aligned reads) / (number of reference-aligned reads). Unintended edits were calculated as the number of discarded reads divided by the total number of reads ((number of reads “Discarded” reads from the reference-aligned sequences + number of the “Discarded” reads from the HDR-aligned sequences)/(number of reference-aligned reads)). The analysis of scaffold sequences incorporated within the unintended edited sequence population was determined using the scaffold sequence analysis pipeline previously described¹¹, where incorporation of at least 2 bases of the scaffold sequence were required to assign the read as containing a scaffold insertion.

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[0137] The publications (including patent publications), web sites, company names, books, manuals, treatise, and scientific literature referred to herein establish the knowledge that is available to those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter.

[0138] Various embodiments of the present invention may be characterized by the potential claims listed in the paragraphs following this paragraph (and before the actual claims provided at the end of this application). These potential claims form a part of the written description of this application. Accordingly, subject matter of the following potential claims may be presented as actual claims in later proceedings involving this application or any application claiming priority based on this application. Inclusion of such potential claims should not be construed to mean that the actual claims do not cover the subject matter of the potential claims. Thus, a decision to not present these potential claims in later proceedings should not be construed as a donation of the subject matter to the public.

[0139] Without limitation, potential subject matter that may be claimed (prefaced with the letter “P” so as to avoid confusion with the actual claims presented below) includes:

Pl. A prime editor protein, the prime editor protein comprising a Cas9 nickase fused to a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase domain, wherein the M- MLV reverse transcriptase domain comprises a V223M and a L435K mutation.

P2. An isolated nucleic acid encoding the prime editor protein of potential claim Pl.

P3. A programmable prime editing system for modification of a double- stranded target DNA sequence comprising a target strand and a complementary non-target strand, the system comprising: the prime editor protein according to potential claim Pl; and a prime editing guide RNA (pegRNA) comprising a spacer sequence having a region of complementarity to the target strand of the double-stranded target DNA sequence.

P4. The programmable prime editing system according to potential claims 3, wherein the programmable prime editing system further comprises an inhibitor of SAMHDl.

P5. The programmable prime editing system according to potential claim P4, wherein the inhibitor of SAMHDl is selected from the group consisting of Vpx, Vpx (S13E), EBV BGLF4, HHV-6/7 U69, HCMV UL97, and KSHV ORF36.

P6. The prime editing system according to any one of potential claims P3-P5, wherein the programmable prime editing system comprises a nicking sgRNA. P7. A method of modifying a double- stranded target DNA sequence, the method comprising contacting the double-stranded target DNA sequence with the prime editor protein and the pegRNA according to any one of potential claims P3-P6.

P8. A method of modifying a double-stranded target DNA sequence in a cell, the method comprising: sequentially introducing into the cell, in the following order:

(1) a programmable prime editing system, the programmable prime editing system comprising (i) a prime editor protein comprising a Cas9 nickase fused to a M- MLV reverse transcriptase domain and (ii) a pegRNA comprising a spacer sequence having a region of complementarity to a target strand of the double-stranded target DNA sequence; and

(2) a nicking sgRNA.

P9. The method of potential claim P8, wherein the M-MLV reverse transcriptase domain comprises a V223M and a L435K mutation.

PIO. The method according to any one of potential claims P8-P9, wherein the programmable prime editing system further comprises an inhibitor of SAMHD1.

Pl 1. The method of potential claim PIO, wherein the inhibitor of SAMHD1 is selected from the group consisting of Vpx, Vpx (S13E), EBV BGLF4, HHV-6/7 U69, HCMV UL97, and KSHV ORF36.

P12. The method according to any one of potential claims P8-P11, wherein the nicking sgRNA is introduced into the cell 2-36 hours after introducing the programmable prime editing system into the cell.

P13. The method according to any one of potential claims P8-P12, wherein the nicking sgRNA is introduced into the cell 6-24 hours after introducing the programmable prime editing system into the cell. P14. The method according to any one of potential claims P8-P13, wherein the programmable prime editing system is introduced into the cell by a first method selected from the group consisting of electroporation, transfection, viral delivery, virus-like particle delivery, and lipid nanoparticle delivery.

P15. The method according to any one of potential claims P8-P14, wherein the nicking sgRNA is introduced into the cell by a second method selected from the group consisting of electroporation, transfection, viral delivery, virus-like particle delivery, and lipid nanoparticle delivery.

[0140] The embodiments of the invention described above are intended to be merely exemplary; numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended potential claims.

Claims

What is claimed is:

1. A prime editor protein, the prime editor protein comprising a Cas9 nickase fused to a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase domain, wherein the M- MLV reverse transcriptase domain comprises a V223M and a L435K mutation.

2. An isolated nucleic acid encoding the prime editor protein of claim 1.

3. A programmable prime editing system for modification of a double-stranded target DNA sequence comprising a target strand and a complementary non-target strand, the system comprising: the prime editor protein according to claim 1; and a prime editing guide RNA (pegRNA) comprising a spacer sequence having a region of complementarity to the target strand of the double-stranded target DNA sequence.

4. The programmable prime editing system according to claims 3, wherein the programmable prime editing system further comprises an inhibitor of SAMHD1.

5. The programmable prime editing system according to claim 4, wherein the inhibitor of SAMHD1 is selected from the group consisting of Vpx, Vpx (S13E), EBV BGLF4, HHV- 6/7 U69, HCMV UL97, and KSHV ORF36.

6. The prime editing system according to any one of claims 3-5, wherein the programmable prime editing system comprises a nicking sgRNA.

7. A method of modifying a double-stranded target DNA sequence, the method comprising contacting the double-stranded target DNA sequence with the prime editor protein and the pegRNA according to any one of claims 3-6.

8. A method of modifying a double-stranded target DNA sequence in a cell, the method comprising: sequentially introducing into the cell, in the following order:

(1) a programmable prime editing system, the programmable prime editing system comprising (i) a prime editor protein comprising a Cas9 nickase fused to a M- MLV reverse transcriptase domain and (ii) a pegRNA comprising a spacer sequence having a region of complementarity to a target strand of the double-stranded target DNA sequence; and

(2) a nicking sgRNA.

9. The method of claim 8, wherein the M-MLV reverse transcriptase domain comprises a V223M and a L435K mutation.

10. The method according to any one of claims 8-9, wherein the programmable prime editing system further comprises an inhibitor of SAMHD1.

11. The method of claim 10, wherein the inhibitor of SAMHD1 is selected from the group consisting of Vpx, Vpx (S13E), EBV BGLF4, HHV-6/7 U69, HCMV UL97, and KSHV ORF36.

12. The method according to any one of claims 8-11, wherein the nicking sgRNA is introduced into the cell 2-36 hours after introducing the programmable prime editing system into the cell.

13. The method according to any one of claims 8-12, wherein the nicking sgRNA is introduced into the cell 6-24 hours after introducing the programmable prime editing system into the cell.

14. The method according to any one of claims 8-13, wherein the programmable prime editing system is introduced into the cell by a first method selected from the group consisting of electroporation, transfection, viral delivery, virus-like particle delivery, and lipid nanoparticle delivery.

15. The method according to any one of claims 8-14, wherein the nicking sgRNA is introduced into the cell by a second method selected from the group consisting of electroporation, transfection, viral delivery, virus-like particle delivery, and lipid nanoparticle delivery.