Prime editing-mediated readthrough of frameshift mutations (PERF)

Abstract

Aspects of the disclosure relate to systems, compositions, and methods for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA. Additional aspects relate to compositions comprising the prime editing machinery and / or complexes comprising the prime editor and pegRNA that are capable of editing an endogenous tRNA into a suppressor qtRNA. In some aspects, the disclosure further relates to polynucleotides encoding one or more nucleic acid sequences encoding the prime editor and / or pegRNA, cells comprising the polynucleotides and complexes comprising the prime editor and pegRNA, kits comprising any one of the compositions, complexes, polynucleotides, vectors and / or cells disclosed herein, and / or delivery systems for administering any one of the compositions, complexes, polynucleotides, or vectors to a subject in need thereof. Additional aspects relate to methods for inserting a new suppressor qtRNA gene into a target site in a genome using prime editing.

Description

Docket No.: B1195.70187WO00 PRIME EDITING-MEDIATED READTHROUGH OF FRAMESHIFT MUTATIONS (PERF) GOVERNMENT SUPPORT

[0001] This invention was made with government support under R35GM118062 awarded by National Institutes of Health. The government has certain rights in the invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (B119570187WO00-SEQ-JQM.xml; Size: 6,321,277,413 bytes; and Date of Creation: September 19, 2024) filed via optical disc, are herein incorporated by reference in their entirety. BACKGROUND

[0003] Frameshift mutations are caused by a deletion or insertion in a DNA sequence that shifts the way an mRNA sequence is translated. Such mutations generally produce translated proteins that are either too short (e.g., due to creation of a premature termination codon, PTC, in the mRNA) or non-functional. Diminished translation of full-length proteins due to PTCs can induce pathogenic effects in cells and organisms. Indeed, frameshift mutations often result in disease and poor quality of life. Interestingly, many bacteria and viruses utilize suppressor tRNAs with expanded anticodons (e.g., quadruple or quintuple anticodon sequences, qtRNAs) to enable readthrough (e.g., the tRNA binds to a codon with more than 3 nucleotides, and the ribosome continues translating the mRNA into protein). However, such suppressor tRNAs do not naturally occur in the human body. Prime editing allows for precise editing of the genomic DNA encoding tRNAs and may provide a platform for the treatment of diseases associated such mutations. SUMMARY

[0004] Aspects of the disclosure relate to systems, compositions, and methods for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA (e.g., to treat diseases caused by frameshift mutations) with an expanded anticodon sequence (e.g., quadruple or quintuple anticodon sequences, both of which are herein referred to as qtRNAs). Additional aspects relate to 1 12954008Docket No.: B1195.70187WO00 compositions comprising the prime editing machinery (e.g., fusion protein comprising a nucleic acid programmable DNA binding protein and reverse transcriptase, and / or pegRNA, etc.) and / or complexes comprising the prime editor and pegRNA that are capable of editing an anticodon sequence of endogenous tRNA into a suppressor qtRNA with an expanded anticodon sequence. In some aspects, the disclosure further relates to polynucleotides (e.g., for editing said DNA sequences) encoding one or more nucleic acid sequences encoding the prime editor and / or pegRNA, cells comprising the polynucleotides and complexes comprising the prime editor and pegRNA, kits comprising any one of the compositions, complexes, polynucleotides, vectors (e.g., AAV), and / or cells disclosed herein, and / or delivery systems for administering any one of the compositions, complexes, polynucleotides, or vectors to a subject in need thereof (e.g., lipid nanoparticles, AAVs, eVLPs). Additional aspects relate to methods for inserting a new suppressor qtRNA gene into a target site in a genome (e.g., a safe harbor locus site) using prime editing (e.g., SEQ ID NOs: 2199725- 2306988).

[0005] Without wishing to be bound by any particular theory, it is generally known that the genetic code is determined by a combination of tRNAs and aminoacyl tRNA synthetases (AARSs). Messenger RNAs (mRNAs) comprise trinucleotide units, called codons, that code for a particular amino acid. Transfer RNAs (tRNAs) also comprise trinucleotide units, called anticodons, that are complementary to the codon. During translation, tRNAs with the appropriate anticodons (and cognate amino acid) undergo Watson-Crick base pairing to the corresponding codon on the mRNA transcript, enabling accurate codon recognition and protein synthesis. During translation of the genetic code, the mRNA is read in sets of three nucleotides (codons). These three-nucleotide mRNA sequences (e.g., codons) encode for amino acids, and the selection of correct tRNAs helps maintain the precise mRNA reading frame. Genetic mutations, such as the insertion or deletion of one, two, or four nucleotides, leads to a change in the mRNA reading frame. This change, in turn, leads to the formation of a truncated or dysfunctional protein (FIGs.27A, 27B and FIG.28). In some embodiments, these mutations cause certain genetic diseases and cancers. For example, Tay-Sachs disease is caused by a 4- base pair insertion in exon 11 of the HEXA gene. This insertion causes a frameshift mutation and introduces a premature termination codon as found in Ashkenazi patients (Myerowitz 1988). In MUC1, the frameshift mutation is caused by the insertion of a single cytosine within a stretch of cytosines in the variable number of tandem repeats 2 12954008Docket No.: B1195.70187WO00 (VNTR) region of MUC1 (Ogura 2001 and Kirby 2013). Additionally, Crohn’s disease is caused by a cytosine insertion, 3020insC which leads to the formation of a truncated NOD2 protein while a thymine insertion, 3905insT, in exon 20 of the CFTR gene is associated with a severe phenotype in cystic fibrosis (Ogura 2001 and Sanz 2010). Cancers such as colorectal cancer and endometrial carcinomas are reportedly caused by frameshift mutations in DNA mismatch repair gene such as MLH1 or in tumor suppressor gene PTEN (Kadiyska 2006 and Spaanderman 2021). In principle, these mutations can be corrected with suppressor qtRNAs, e.g., suppressor tRNAs with expanded anticodons (e.g., tRNAs that bind to four or five codons).

[0006] As used herein, the term “suppressor qtRNA” refers to any suppressor qtRNA comprising an expanded anticodon sequence (e.g., more than 3 nucleotides). Naturally occurring qtRNAs, such as sufD and sufJ, have been reported in bacteria, and others have shown the possibility of four base pair codon translation in bacteria using natural, evolved, or synthetic quadruplet tRNA (qtRNA).

[0007] Humans (and other species, such as domesticated animals and plants) possess over 500 interspersed tRNA genes, and many of these genes are redundant and dispensable. For example, there are multiple instances of non-synonymous anti-codon mutations being found in healthy study participants, supporting the redundancy and dispensability of certain tRNA genes. Furthermore, one or both copies of the tRNALysCUU gene is deleted in ~50% of humans. For example, using prime editing to convert the CUU anticodon of this tRNALysgene into NCUU, CUUN, NCUUN, NNNN, NNNNN, NNCUU, or CUUNN, respectively, wherein N can be any nucleotide (e.g., A, G, T, or C) would generate a suppressor qtRNALyswith an expanded anticodon capable of reading through frameshift mutation associated with lysine residues.

[0008] As such, various aspects of the present disclosure relate to methods for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA. In some embodiments, the methods comprise contacting the DNA sequence encoding the anticodon sequences of an endogenous tRNA with a prime editor and one or more pegRNA’s. In some embodiments, the prime editor installs an adenine (A), cytosine (C), guanine (G), or a thymine (G) into the anticodon sequence to produce a qtRNA with an expanded anticodon sequence. In some embodiments, the tRNA gene may be any tRNA gene in a host genome, such as those described in Tables 3 or 4 or in U.S. Patent 3 12954008Docket No.: B1195.70187WO00 Provisional Patent Application No:63 / 480495, entitled, “Prime editing-mediated readthrough of premature termination codons (PERT),” filed on January 18, 2023 and U.S. Provisional Patent Application No.: 63 / 579778, entitled, “Prime editing-mediated readthrough of premature termination codons (PERT),” filed on August 30, 2023, both of which are incorporated herein by reference in their entirety.

[0009] Other aspects relate to compositions comprising the prime editing machinery (e.g., fusion protein comprising a nucleic acid programmable DNA binding protein and reverse transcriptase, and / or pegRNA) for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA. The compositions, according to some embodiments, comprises a prime editor (e.g., twinPE). In some embodiments, the prime editor may comprise any suitable prime editor known to the skilled artisan such as those disclosed in Chen and Li, “Prime editing for precise and highly versatile genome manipulation” Nature Review Genetics, 2022, https: / / doi.org / 10.1038 / s41576-022-00541-1, which is incorporated herein by reference in its entirety Exemplary embodiments include, but are not limited to PE1, PE2, PE2*, PEmax, CMP-PE1-V1, hyPE2, IN-PE, ePPE, PE-P3, PE2∆Rnh, sPE, nCas9 and MCP-RT, PE4 / PE5,

[0010] In some embodiments, the prime editor may be a PE2, PE3, PE4, PE5, PE2max, PE3max, PE4max, PE5max, twinPE, or Prime-del, such as those disclosed in U.S. Patent Application U.S.S.N.63 / 022,397, filed May 8, 2020, U.S. Patent Application, U.S.S.N. 63 / 116,785, filed November 20, 2020, and PCT Application, Serial Number PCT / US2021 / 031439, filed May 7, 2021, each of which is incorporated herein by reference in its entirety.

[0011] In some embodiments, the prime editors disclosed herein comprise either an SaCas9 or SpCas9, or a derivative thereof, fused to PEmax or PE6 (e.g, PE6a, PE6b, PE6c, PE6d, PE6e, PE6f, PE6g).

[0012] Other aspects of the present disclosure relate to a pegRNA. The pegRNA, according to some embodiments, may comprise any suitable pegRNA known to the skilled artisan. In some embodiments, the pegRNA further comprises a DNA synthesis template and a primer binding site (PBS). In some embodiments, the pegRNA is configured to bind to a 4 12954008Docket No.: B1195.70187WO00 DNA sequence encoding an anticodon sequence of an endogenous tRNA (e.g., SEQ ID NOs. 1-430 or 2306989-2307418).

[0013] In some embodiments, the prime editing guide RNA (pegRNA) is any suitable pegRNA for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA. In some embodiments the pegRNA comprises a a spacer sequence, a gRNA core, and an extension arm. In some embodiments the extension arm comprises a DNA synthesis template and a primer binding site (PBS).

[0014] In some embodiments, the DNA synthesis template encodes one or more edits (e.g., insertion, deletion, or substitution) to be installed at a target site (e.g., anticodon sequence) in the DNA sequence encoding the endogenous tRNA. In some embodiments, the DNA synthesis template encodes at least one nucleotide insertion to be installed at the target site of the DNA sequence encoding an anticodon sequence (e.g., within the anticodon arm domain). In some embodiments, the DNA synthesis template encodes a sequence that is complementary to any one of the sequences listed SEQ ID NOs: 431- 2199724. The DNA synthesis template may encode other edits according to other embodiments. For example, in some embodiments, the DNA synthesis template encodes one or more nucleotide insertions, deletions, or substitutions to be installed at a target site of the DNA sequence encoding the acceptor stem arm domain, the variable arm domain, the T-arm domain, or the D-arm domain of the endogenous tRNA.

[0015] In some embodiments, the pegRNA is chosen to enable editing of a single specified endogenous tRNA gene without editing other tRNA genes, especially by avoiding the targeting of regions that are highly homologous among tRNA genes. Without wishing to be bound by any particular theory, it is believed that because of prime editing’s exquisite targeting specificity, pegRNAs may be carefully designed to distinguish between on-target and off-target tRNA genes, even ones that differ at most by a few base pairs. For example, as shown in FIG.6, the protospacer of the pegRNA allows the prime editor to specifically bind a particular tRNA within a family of tRNAs having the same anticodon sequence (see boxed region in FIG.6).

[0016] Aspects of the disclosure relate to a complex comprising a prime editor and any pegRNA described herein for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA. In some embodiments, the pegRNA comprises a spacer sequence and an extension arm. In some 5 12954008Docket No.: B1195.70187WO00 embodiments, the pegRNA is any sequence listed in SEQ ID NOs: 431- 2199724. In some embodiments, the pegRNA sequence is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to any sequence listed in SEQ ID NOs: 431- 2199724.

[0017] Other aspects of the disclosure relate to polynucleotides, vectors, cells, pharmaceutical compositions, and kits for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA. For example, in some aspects, the disclosure relates to one or more polynucleotides comprising a first nucleic acid sequence encoding a prime editor and a second nucleic acid sequence encoding a pegRNA. In some embodiments, the pegRNA comprises sequence recited in SEQ ID NOs: 431- 2199724.

[0018] In some aspects, the disclosure relates to pharmaceutical compositions comprising any one of the compositions, pegRNAs, complexes, polynucleotides, vectors, and cells disclosed herein, or any combination thereof, and a pharmaceutically acceptable excipient.

[0019] In some aspects, the disclosure relates to kits comprising any one of the compositions, pegRNAs, complexes, polynucleotides, vectors, and cells disclosed herein, or any combination thereof, and a pharmaceutical excipient, and instructions for editing one or more DNA sequences of an endogenoustRNA by prime editing. In some embodiments, the DNA sequence of the endogenous tRNA is any sequence in SEQ ID NOs.1-430 or 2306989- 2307418.

[0020] Additionally, aspects of the disclosure relate to methods of inserting a new suppressor qtRNA gene into a host genome. Any suitable host genome known to the skilled artisan may be used as contemplated herein (e.g., any prokaryote genome or eukaryote genome). In some embodiments, the host genome is a human genome. In some embodiments, the qtRNA gene is inserted into a safe harbor locus (e.g., ROSA, CCR5, AAVS1, etc.) or general expression site (e.g., ALB) in the host genome, rather than converting an endogenous tRNA gene into a suppressor qtRNA. Without wishing to be bound by any particular theory, this approach requires insertion of a small gene (e.g., <1 kb) rather than a local edit of a subset of endogenous tRNA bases but may offer advantages, such as the lack of dependence on the presence, sequence, and dispensability of an endogenous qtRNA gene in a specific target organism or patient. Further, because tRNAs are short (~200 bp), and typical Pol III promoters for expressing short RNAs are also small (e.g., U6 promoter, 6 12954008Docket No.: B1195.70187WO00 264 bp), it is possible that all of the elements required for suppressor qtRNA expression could be inserted by prime editing methods, such as twin prime editing. Alternatively, in some embodiments, prime editing or twin prime editing is coupled with the use of integrase or recombinase enzymes to perform the insertion as described in U.S. Patent Application, U.S.S.N.63 / 271,700, filed Oct.25, 2021, and in PCT Patent Application Serial Number PCT / US2022 / 078655, filed Oct.25, 2022, both of which are herein incorporated by reference in their entirety. The use of CRISPR-associated transposases (CASTs) and other targeted gene insertion technologies to achieve insertion of a suppressor qtRNA or a suppressor qtRNA expression cassette into the genome is likewise also envisioned.

[0021] In some embodiments, the methods comprise contacting the target site with (i) a prime editor, and (ii) a pegRNAIn some embodiments, the spacer sequence comprises a region of complementarity to a target strand of a double stranded target tRNA gene sequence in the subject, wherein the gRNA core associates with the napDNAbp. In some embodiments, the DNA synthesis template comprises a region of complementarity to the non- target strand of the double-stranded target tRNA gene sequence and encodes a suppressor qtRNA gene sequence to be installed within the target site. In some embodiments, the primer binding site comprises a region of complementarity to a non-target strand of the double- stranded target tRNA gene sequence. In some embodiments, the prime editor and the pegRNA install the suppressor qtRNA gene sequence in the target site in the human genome. In some embodiments, installation of the suppressor qtRNA gene results in the indefinite expression of the suppressor qtRNAs gene.

[0022] Other aspects of the disclosure relate to methods for inserting a DNA sequence encoding a suppressor qtRNA gene into a target site in a human genome using prime editing, the method comprising contacting the target site with (i) a prime editor, (ii) a pegRNA, (iii) a sgRNA, and (iv) a nucleic acid (e.g., a circular plasmid, ssRNA, ssDNA, or dsDNA), wherein the prime editor comprises a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp), a DNA polymerase, and a recombinase. In some embodiments, a nucleic acid sequence of the tRNA is any sequence listed in SEQ ID NOs: 2199725- 2306988.

[0023] Without wishing to bound by any particular theory, recombinases, such as serine integrases (e.g., BxB1), are art recognized enzymes capable of performing site-specific recombination. Site-specific recombination is an art recognized process in which DNA 7 12954008Docket No.: B1195.70187WO00 strand exchange takes place between two DNA segments (e.g., two different double strand DNAs) possessing at least a certain degree of sequence homology. The enzymes recognize and bind to short specific DNA recognition sites (e.g., a first recognition site located on the first double stranded DNA and a second recognition site located on a second double stranded DNA), at which they cleave the DNA backbone, exchange the two DNA helices involved, and rejoin the DNA strands. In some embodiments, the first and second recognition sites comprise identical sequences. In other embodiments, the first and second recognition sites comprise different sequences (e.g. ,attP and attB of phage integrase).

[0024] In some embodiments, the method comprises a nucleic acid (e.g., a circular plasmid, ssRNA, ssDNA, or dsDNA) that encodes the suppressor qtRNA comprising an expanded anticodon sequence that is complementary to a expanded codon sequence in an mRNA sequence (e.g., an mRNA with a frameshift mutation). In some embodiments, the a nucleic acid (e.g., a circular plasmid, ssRNA, ssDNA, or dsDNA) comprises a first recombinase recognition site (e.g., AttP). In some embodiments, the first recombinase recognition site comprises an AttB sequence. In some embodiments, the first recombinase recognition site comprises an AttP. Any suitable AttP or AttB sequence known to the skilled artisan may be used such as those disclosed in Bland et al. “An att site-based recombination reporter system for genome engineering and synthetic DNA assembly” BMC Biotechnology. 17, 62(2017), which is herein incorporated by reference in its entirety.

[0025] In some embodiments, the integrase recombines the a nucleic acid (e.g., a circular plasmid, ssRNA, ssDNA, or dsDNA) comprising the first recombination recognition site with the second recombination site, previously inserted into the host genome at the target site (e.g., safe harbor locus) via prime editing, to permanently insert the desired suppressor qtRNA gene into the human genome at the target site (e.g., ROSA26, CCR5, and AAVS1). In some embodiments, installation of the suppressor qtRNA gene at the target site (e.g., safe harbor locus site or general expression site) results in the indefinite expression of the suppressor qtRNA gene.

[0026] Other aspects of the present disclosure relate to methods of replacing one or more endogenous tRNA genes with a suppressor qtRNA gene. In some embodiments, the methods comprise using prime editing approaches to replace endogenous qtRNA genes with desired suppressor qtRNA genes. In some embodiments, the endogenous tRNA gene is highly expressed, such that replacing it with a suppressor qtRNA gene will enhance expression of 8 12954008Docket No.: B1195.70187WO00 the target suppressor qtRNA protein, and hence, result in increased readthrough efficiency of frameshift mutations.

[0027] Additional aspects relate to methods for treating a disease caused by a frameshift mutation. In some embodiments, the methods comprise converting an endogenous tRNA gene into a suppressor qtRNA gene using prime editing, the method comprising administering to a subject: (i) a prime editor, and (ii) one or more pegRNAs, wherein the suppressor qtRNA gene encodes a suppressor qtRNA capable of binding to an expanded codon (e.g., 4 or 5 codons).

[0028] In some embodiments, the methods comprise installing a suppressor qtRNA gene into a target site in a human genome using prime editing, the method comprising administering to a subject (i) a prime editor and (ii) one or more pegRNAs, wherein the suppressor qtRNA gene encodes a suppressor qtRNA capable of binding to an expanded codon.

[0029] In some embodiments, the disease being treated based on the present disclosure is selected from the group consisting of cancer, Crohn’s disease, cystic fibrosis, HIV, Tay-Sachs disease, Smith-Magenis syndrome, hypertrophic cardiomyopathy, thalassemia, Huntington’s disease, and Alzheimer’s disease.

[0030] The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims. BRIEF DESCRIPTION OF DRAWINGS

[0031] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0032] FIG.1 shows a representative schematic of an exemplary endogenous, dispensable tRNA. Relevant domains include the D-arm domain (e.g., D-loop), acceptor stem domain, T- arm domain (e.g., TΨC loop), variable arm domain (e.g., variable loop), and the anticodon arm domain encoding the anticodon sequence (e.g., anticodon loop).

[0033] FIG.2A shows a schematic illustrating the translation of a normal mRNA transcript into protein. FIG.2B shows a schematic illustrating the translation of an mRNA transcript 9 12954008Docket No.: B1195.70187WO00 comprising a frame shift mutation which results in translation of either a truncated protein or a non-functional protein.

[0034] FIG.3 shows a schematic representation of qtRNA suppression of frameshift mutations within the first seven codons encoding the first seven amino acids of EGFP.

[0035] FIG.4A-4E shows the results of a cellular assay to demonstrate that mammalian cells lack endogenous mechanisms to readthrough expanded codons. FIG.4A shows a schematic of the EGFP reporter plasmid used in the assay. FIG.4B shows another schematic of the EGFP reporter assay illustrating the incorporation of an expanded codon at position Y151 in the EGFR protein. FIG.4C shows a10 schematic of reporter plasmids encoding a GGGG expanded codon, a TAGA expanded codon, a GGG triplet codon, or a TCG triplet codon corresponding to position Y151. FIG.4D shows the various outcomes following translation using triplet and / or quadruplet anticodons to translate the EGFP reporter protein. FIG.4E shows the percentage of translation efficiency of tRNAs with triplet versus quadruple anticodons, relative to wild type pLV-EGFP. FIGs.4F and 4G show quantification of the EGFP fluorescence in HEK293T cells transfected with frameshifted reporter plasmids using flow cytometry.

[0036] FIG.5A-5B shows the results of a cellular reporter assay demonstrating that mammalian cells do not possess endogenous mechanisms to read through expanded codons (FIG.5A) and that exogenously expressed qtRNAs enable readthrough of frameshift mutations in mammalian cells (FIG.5B)

[0037] FIGs.6A-6F show the overexpression of qtRNA glycine and qtRNA serine readthrough cognate quadruplet codon reporter plasmid in HEK 293T cells. FIG.6A shows the sequence of mature Homo sapiens tRNA Gly-CCC-1-1. FIG.6B shows the sequence of mature Homo sapiens tRNA Ser-CGA-1-1. FIG.6C shows readthrough events due to overexpression of three isoforms of qtRNA glycine. FIG.6D shows readthrough event due to overexpression of four isoforms of qtRNA serine. FIGs.6E and 6F show anticodon loop mutations at nucleotides 32, 37, and 38 (black bar) negatively impacted readthrough efficiency.

[0038] FIG.7 shows the sequence alignment of tRNA-Leu-TAA family of tRNAs. The sequence difference in the highlighted region, the protospacer, allows the prime editor to specifically bind to each Leu TAA tRNA uniquely 10 12954008Docket No.: B1195.70187WO00

[0039] FIGs.8A-8B illustrate the anticodon flexibility of qtRNA serine, relative to qtRNA glycine. FIG.8A illustrates an exemplary qtRNA glycine design to decode TAGA and GGGG expanded codons with flow analysis showing the inability of qtRNA glycine to read through a TAGA expanded codon. FIG.8B illustrates an exemplary qtRNA serine design to decode GGGG and TAGA expanded codons with flow analysis showing the ability of qtRNA serine to read through both GGGG and TAGA expanded codons.

[0040] FIGs 9A-9F illustrates the suppression of frameshift mutations in a genome using lentivirally integrated qtRNAs with increasing qtRNA gene number. FIG.9A (top) shows a schematic of lentiviral construct with increasing qtRNA gene number driven by an endogenous leader sequence. FIG.9A (middle) shows a schematic of an exemplary lentiviral construct with increasing qtRNA gene number driven by exogenous promoters. FIG.9A (bottom) shows an exemplary reporter gene construct having an expanded codon at chromosome 20 (20q) corresponding to an expanded codon at amino acid position Y151 of an EGFP protein. FIGs.9B-9D shows readthrough of a GGGG expanded codon with lentivirally integrated qtRNA. FIG.9B shows FACS quantification of frameshift suppression of a GGGG expanded codon with lentivirally integrated qtRNA with increasing qtRNA gene number. FIG.9C shows an exemplary FACS plot of the 1X qtRNA data. FIG.9D shows an exemplary FACS plot of the untransduced reporter cell line. FIG.9E shows FACS plots illustrating that leader sequence driven qtRNA glycine outperforms U6 driven qtRNA in frameshifted reporter cell line. FIG.9F shows FACS plots illustrating the leader sequence driven qtRNA glycine outperforms U6 driven qtRNA in frameshifted reporter cell line even with lower transduction efficiencies.

[0041] FIG.10A and 10B illustrate a more stringent reporter cell line having an additional expanded codon in the linker between the gene encoding mCherry and 2a (FIG.10A) and FACS plots illustrating readthrough events from qtRNA constructs driven by leader sequences or U6 promoters (FIG.10B). As before, qtRNAs sequences operably linked to leader sequences resulted in more readthrough events compared to qtRNAs sequences operably linked to U6 promoters.

[0042] FIG.11A-11C illustrate various lentiviral constructs with different leader sequences and termination sequences for qtRNA glycine-CCCC-2-1. FIG.11A shows a schematic of the various qtRNA glycine constructs tested. FIG.11B shows FACS quantification of frameshift suppression of a CCCC expanded codon with lentivirally integrated qtRNA with 11 12954008Docket No.: B1195.70187WO00 the constructs shown in FIG.11A. FIG.11C shows the raw FACS data for representative constructs shown in FIGs.11A and 11B. DEFINITIONS

[0043] 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. Cas9

[0044] 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 casn1 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 trans-encoded 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 a 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 “gNRA”) can be engineered 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., 12 12954008Docket No.: B1195.70187WO00 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 in 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 M1 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 and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include 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.

[0045] A nuclease-inactivated Cas9 domain may interchangeably be referred to as a “dCas9” protein (for nuclease-“dead” Cas9). Methods for generating a Cas9 domain (or a fragment thereof) having an inactive DNA cleavage domain are known (see, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell.28;152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the 13 12954008Docket No.: B1195.70187WO00 mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science.337:816-821(2012); Qi et al., Cell.28;152(5):1173-83 (2013)). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as “Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.8% identical, or at least about 99.9% identical to wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 2307419). In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 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, or more amino acid changes compared to wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 2307419). In some embodiments, the Cas9 variant comprises a fragment of SEQ ID NO: 2307419 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 2307419). In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 2307419). Cognate Amino Acid

[0046] The term “cognate amino acid” refers to an amino acid that is conjugated to a tRNA molecule comprising an anticodon sequence encoding for said amino acid. CRISPR

[0047] CRISPR is a family of DNA sequences (i.e., CRISPR clusters) in bacteria and archaea that represent snippets of prior infections by a virus that have invaded the prokaryote. The 14 12954008Docket No.: B1195.70187WO00 snippets of DNA are used by the prokaryotic cell to detect and destroy DNA from subsequent attacks by similar viruses and effectively compose, along with an array of CRISPR- associated proteins (including Cas9 and homologs thereof) and CRISPR-associated RNA, a prokaryotic immune defense system. In nature, CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In certain types of CRISPR systems (e.g., type II CRISPR systems), correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cleaves a linear or circular dsDNA target complementary to the RNA. Specifically, 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 “gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species – the guide RNA. 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 is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. CRISPR biology, as well as Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an M1 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 and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the 15 12954008Docket No.: B1195.70187WO00 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.

[0048] In certain types of CRISPR systems (e.g., type II CRISPR systems), correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3- aided processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cleaves a linear or circular nucleic acid target complementary to the RNA. Specifically, 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 to incorporate embodiments of both the crRNA and tracrRNA into a single RNA species—the guide RNA.

[0049] In general, a “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. The tracrRNA of the system is complementary (fully or partially) to the tracr mate sequence present on the guide RNA. DNA synthesis template

[0050] As used herein, the term “DNA synthesis template” refers to the region or portion of the extension arm of a pegRNA that is utilized as a template strand by a polymerase of a prime editor to encode a 3ʹ single-strand DNA flap that contains the desired edit and which then, through the mechanism of prime editing, replaces the corresponding endogenous strand of DNA at the target site. The extension arm, including the DNA synthesis template, may be comprised of DNA or RNA. In the case of RNA, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (e.g., a reverse transcriptase). In the case of DNA, the polymerase of the prime editor can be a DNA-dependent DNA polymerase. In various embodiments the DNA synthesis template may comprise the “edit template” and the 16 12954008Docket No.: B1195.70187WO00 “homology arm”, and all or a portion of the optional 5´ end modifier region, e2. That is, depending on the nature of the e2 region (e.g., whether it includes a hairpin, toe loop, or stem / loop secondary structure), the polymerase may encode none, some, or all of the e2 region as well. Said another way, in the case of a 3ʹ extension arm, the DNA synthesis template can include the portion of the extension arm that spans from the 5ʹ end of the primer binding site (PBS) to 3ʹ end of the gRNA core that may operate as a template for the synthesis of a single-strand of DNA by a polymerase (e.g., a reverse transcriptase). In the case of a 5ʹ extension arm, the DNA synthesis template can include the portion of the extension arm that spans from the 5ʹ end of the pegRNA molecule to the 3ʹ end of the edit template. Preferably, the DNA synthesis template excludes the primer binding site (PBS) of pegRNAs either having a 3ʹ extension arm or a 5ʹ extension arm. Certain embodiments described here refer to an “an RT template,” which is inclusive of the edit template and the homology arm, i.e., the sequence of the pegRNA extension arm which is actually used as a template during DNA synthesis. The term “RT template” is equivalent to the term “DNA synthesis template.” Dual Prime Editing

[0051] As used herein, the terms “dual prime editing”, “twin prime editing (or twinPE)”, and “dual-flap prime editing” are considered equivalent. In the dual-flap prime editing system, two pegRNAs are used to target opposite strands of a genomic site and direct the synthesis of two complementary 3’ flaps containing edited DNA sequence. Unlike classical prime editing, there is no requirement for the pair of edited DNA strands (3’ flaps) to directly compete with 5’ flaps in endogenous genomic DNA, as the complementary edited strand is available for hybridization instead. Since both strands of the duplex are synthesized as edited DNA, the dual-flap prime editing system obviates the need for the replacement of the non-edited complementary DNA strand required by classical prime editing. Instead, cellular DNA repair machinery need only excise the paired 5’ flaps (original genomic DNA) and ligate the paired 3’ flaps (edited DNA) into the locus. Therefore, there is no need to include sequences homologous to genomic DNA in the newly synthesized DNA strands, allowing selective hybridization of the new strands and facilitating edits that contain minimal genomic homology. Nuclease-active versions of prime editors that cut both strands of DNA could also be used to accelerate the removal of the original DNA sequence. 17 12954008Docket No.: B1195.70187WO00 Edit template

[0052] The term “edit template” refers to a portion of the extension arm that encodes the desired edit in the single strand 3ʹ DNA flap that is synthesized by the polymerase, e.g., a DNA-dependent DNA polymerase, RNA-dependent DNA polymerase (e.g., a reverse transcriptase). Certain embodiments described here refer to “an RT template,” which refers to both the edit template and the homology arm together, i.e., the sequence of the pegRNA extension arm which is actually used as a template during DNA synthesis. The term “RT edit template” is also equivalent to the term “DNA synthesis template,” but wherein the RT edit template reflects the use of a prime editor having a polymerase that is a reverse transcriptase, and wherein the DNA synthesis template reflects more broadly the use of a prime editor having any polymerase. Extension arm

[0053] The term “extension arm” refers to a nucleotide sequence component of a pegRNA which provides several functions, including a primer binding site and an edit template for reverse transcriptase. In some embodiments, the extension arm is located at the 3ʹ end of the guide RNA. In other embodiments, the extension arm is located at the 5ʹ end of the guide RNA. In some embodiments, the extension arm also includes a homology arm. In various embodiments, the extension arm comprises the following components in a 5ʹ to 3ʹ direction: the homology arm, the edit template, and the primer binding site. Since polymerization activity of the reverse transcriptase is in the 5ʹ to 3ʹ direction, the preferred arrangement of the homology arm, edit template, and primer binding site is in the 5ʹ to 3ʹ direction such that the reverse transcriptase, once primed by an annealed primer sequence, polymerizes a single strand of DNA using the edit template as a complementary template strand. Further details, such as the length of the extension arm, are described elsewhere herein.

[0054] The extension arm may also be described as comprising generally two regions: a primer binding site (PBS) and a DNA synthesis template, for instance. The primer binding site binds to the 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 3ʹ end on the endogenous nicked strand. As explained herein, 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 3ʹ end (i.e., the 3ʹ of the primer sequence), which then provides a substrate for a polymerase to begin polymerizing a single strand of DNA from the exposed 3ʹ end along the 18 12954008Docket No.: B1195.70187WO00 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 5ʹ 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 3ʹ 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 immediately downstream of the PE-induced nick site. Without being bound by theory, polymerization of the DNA synthesis template continues towards the 5ʹ 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 5ʹ terminus of the pegRNA (e.g., in the case of the 5ʹ 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. Fusion protein

[0055] 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. Another example includes a Cas9 or equivalent thereof to a reverse transcriptase. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference. General expression site 19 12954008Docket No.: B1195.70187WO00

[0056] The term “general expression site” refers to a site in the human genome to which a gene of interest may be inserted, wherein the site is constitutively expressed (e.g., albumin gene, ALB). Guide RNA (“gRNA”)

[0057] 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 the protospacer 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. The Cas9 equivalents may include other napDNAbp from any type of CRISPR system (e.g., type II, V, VI), including Cpf1 (a type-V CRISPR-Cas systems), C2c1 (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system) and C2c3 (a type V CRISPR-Cas system). Further Cas-equivalents are described in Makarova et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299), the contents of which are incorporated herein by reference. Exemplary sequences are and structures of guide RNAs are provided herein. In addition, methods for designing appropriate guide RNA sequences are provided herein. As used herein, the “guide RNA” may also be referred to as a “traditional guide RNA” to contrast it with the modified forms of guide RNA termed “prime editing guide RNAs” (or “pegRNAs”).

[0058] Guide RNAs or pegRNAs may comprise various structural elements that include, but are not limited to:

[0059] Spacer sequence – the sequence in the guide RNA or pegRNA (having about 20 nts in length) which binds to the protospacer in the target DNA.

[0060] gRNA core (or gRNA scaffold or backbone sequence) - refers to the sequence within the gRNA that is responsible for Cas9 binding, it does not include the 20 bp spacer / targeting sequence that is used to guide Cas9 to target DNA.

[0061] Extension arm – a single strand extension at the 3ʹ end or the 5ʹ end of the pegRNA which comprises a primer binding site and a DNA synthesis template sequence that encodes via a polymerase (e.g., a reverse transcriptase) a single stranded DNA flap containing the 20 12954008Docket No.: B1195.70187WO00 genetic change of interest, which then integrates into the endogenous DNA by replacing the corresponding endogenous strand, thereby installing the desired genetic change.

[0062] Transcription terminator – the guide RNA or pegRNA may comprise a transcriptional termination sequence at the 3ʹ of the molecule. Host cell

[0063] The term “host cell,” as used herein, refers to a cell that can host, replicate, and express a vector described herein, e.g., a vector comprising a nucleic acid molecule encoding an MLH1 variant and a fusion protein comprising a Cas9 or Cas9 equivalent and a reverse transcriptase. Linker

[0064] The term “linker,” as used herein, refers to a molecule linking two other molecules or moieties. The linker can be an amino acid sequence in the case of a linker joining two fusion proteins. For example, a Cas9 can be fused to a reverse transcriptase by an amino acid linker sequence. The linker can also be a nucleotide sequence in the case of joining two nucleotide sequences together. For example, in the instant case, the traditional guide RNA is linked via a spacer or linker nucleotide sequence to the RNA extension of a prime editing guide RNA which may comprise a RT template sequence and an RT primer binding site. In other embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 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, 30-35, 35-40, 40- 45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. napDNAbp

[0065] As used herein, the term “nucleic acid programmable DNA binding protein” or “napDNAbp,” of which Cas9 is an example, refer to proteins that use RNA:DNA hybridization to target and bind to specific sequences in a DNA molecule. Each napDNAbp is associated with at least one guide nucleic acid (e.g., guide RNA), which localizes the napDNAbp to a DNA sequence that comprises a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid, or a portion thereof (e.g., the protospacer of a guide RNA). In other words, the guide nucleic-acid “programs” the napDNAbp (e.g., Cas9 or equivalent) to localize and bind to a complementary sequence. 21 12954008Docket No.: B1195.70187WO00

[0066] Without being bound by theory, the binding mechanism of a napDNAbp–guide RNA complex, in general, includes the step of forming an R-loop whereby the napDNAbp induces the unwinding of a double-strand DNA target, thereby separating the strands in the region bound by the napDNAbp. The guide RNA protospacer then hybridizes to the “target strand.” This displaces a “non-target strand” that is complementary to the target strand, which forms the single strand region of the R-loop. In some embodiments, the napDNAbp includes one or more nuclease activities, which then cut the DNA, leaving various types of lesions. For example, the napDNAbp may comprises a nuclease activity that cuts the non-target strand at a first location, and / or cuts the target strand at a second location. Depending on the nuclease activity, the target DNA can be cut to form a “double-stranded break” whereby both strands are cut. In other embodiments, the target DNA can be cut at only a single site, i.e., the DNA is “nicked” on one strand. Exemplary napDNAbp with different nuclease activities include “Cas9 nickase” (“nCas9”) and a deactivated Cas9 having no nuclease activities (“dead Cas9” or “dCas9”). Exemplary sequences for these and other napDNAbp are provided herein. Nickase

[0067] The term “nickase” refers to a Cas9 with one of the two nuclease domains inactivated. This enzyme is capable of cleaving only one strand of a target DNA. Non-cognate amino acid

[0068] The term “non-cognate amino acid” refers to an amino acid that pairs with a tRNA molecule that does not comprise an anticodon sequence encoding said amino acid. Nonsense mutation

[0069] The term “nonsense mutation” refers to a mutation in which a sense codon that corresponds to one of the twenty amino acids specified by the genetic code is changed to a chain-terminating codon (e.g., an opal stop codon, an amber stop codon, or a, ochre stop codon). Nonsense suppressor anticodon sequence

[0070] The term “nonsense suppressor anticodon sequence” refers to an anticodon sequence that is complementary to an opal stop codon (e.g., 5'-UCA-3'), an amber codon (e.g., 5'- CUA-3'), or an ochre stop codon (e.g., 5'-UUA-3'). Nucleic acid molecule

[0071] The term “nucleic acid,” as used herein, refers to a polymer of nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, 22 12954008Docket No.: B1195.70187WO00 uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyl uridine, C5 propynyl cytidine, C5 methylcytidine, 7 deazaadenosine, 7 deazaguanosine, 8 oxoadenosine, 8 oxoguanosine, O(6) methylguanine, 4-acetylcytidine, 5- (carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1-methyl adenosine, 1-methyl guanosine, N6-methyl adenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, 2´-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5ʹ N phosphoramidite linkages). Nuclear localization sequence

[0072] The term “nuclear localization sequence” or “NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport. pegRNA

[0073] As used herein, the terms “prime editing guide RNA” or “pegRNA” or “extended guide RNA” refer 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 compositions described herein. As described herein, the prime editing guide RNA comprise one or more “extended regions” of nucleic acid sequence. The extended regions may comprise, but are not limited to, single-stranded RNA or DNA. Further, the extended regions may occur at the 3´ end of a traditional guide RNA. In other arrangements, the extended regions may occur at the 5´ end of a traditional guide RNA. In still other arrangements, the extended region may occur at an intramolecular region of the traditional guide RNA, for example, in the gRNA core region which associates and / or binds to the napDNAbp. The extended region comprises a “DNA synthesis template” which encodes (by the polymerase 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. The extended region may also comprise other functional sequence elements, such as, but not limited to, a “primer binding site” and a “spacer or linker” sequence, or other structural elements, such as, but not limited 23 12954008Docket No.: B1195.70187WO00 to aptamers, stem loops, hairpins, toe loops (e.g., a 3´ toe loop), or an RNA-protein recruitment domain (e.g., MS2 hairpin).

[0074] 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.

[0075] In certain embodiments, the pegRNAs have a 5ʹ extension arm, a spacer, and a gRNA core. The 5ʹ extension further comprises in the 5ʹ to 3ʹ direction a reverse transcriptase template, a primer binding site, and a linker. The reverse transcriptase template may also be referred to more broadly as the “DNA synthesis template” where the polymerase of a prime editor described herein is not an RT, but another type of polymerase.

[0076] In certain other embodiments, the pegRNAs have a 5ʹ extension arm, a spacer, and a gRNA core. The 5ʹ extension further comprises in the 5ʹ to 3ʹ direction a reverse transcriptase template, a primer binding site, and a linker. The reverse transcriptase template may also be referred to more broadly as the “DNA synthesis template” where the polymerase of a prime editor described herein is not an RT, but another type of polymerase.

[0077] In still other embodiments, the pegRNAs have in the 5ʹ to 3ʹ direction a spacer (1), a gRNA core (2), and an extension arm (3). The extension arm (3) is at the 3ʹ end of the pegRNA. The extension arm (3) further comprises in the 5ʹ to 3ʹ direction a “primer binding site” (A), an “edit template” (B), and a “homology arm” (C). The extension arm (3) may also comprise an optional modifier region at the 3ʹ and 5ʹ ends, which may be the same sequences or different sequences. In addition, the 3ʹ end of the pegRNA may comprise a transcriptional terminator sequence. These sequence elements of the pegRNAs are further described and defined herein.

[0078] In still other embodiments, the pegRNAs have in the 5ʹ to 3ʹ direction an extension arm (3), a spacer (1), and a gRNA core (2). The extension arm (3) is at the 5ʹ end of the pegRNA. The extension arm (3) further comprises in the 3ʹ to 5ʹ direction a “primer binding site” (A), an “edit template” (B), and a “homology arm” (C). The extension arm (3) may also comprise an optional modifier region at the 3ʹ and 5ʹ ends, which may be the same sequences or different sequences. The pegRNAs may also comprise a transcriptional terminator sequence at the 3ʹ end. These sequence elements of the pegRNAs are further described and defined herein. 24 12954008Docket No.: B1195.70187WO00 Polymerase

[0079] As used herein, the term “polymerase” refers to an enzyme that synthesizes a nucleotide strand and that may be used in connection with the prime editor systems described herein. The polymerase can be a “template-dependent” polymerase (i.e., a polymerase that synthesizes a nucleotide strand based on the order of nucleotide bases of a template strand). The polymerase can also be a “template-independent” polymerase (i.e., a polymerase that synthesizes a nucleotide strand without the requirement of a template strand). A polymerase may also be further categorized as a “DNA polymerase” or an “RNA polymerase.” In various embodiments, the prime editor system comprises a DNA polymerase. In various embodiments, the DNA polymerase can be a “DNA-dependent DNA polymerase” (i.e., whereby the template molecule is a strand of DNA). In such cases, the DNA template molecule can be a pegRNA, wherein the extension arm comprises a strand of DNA. In such cases, the pegRNA may be referred to as a chimeric or hybrid pegRNA which comprises an RNA portion (i.e., the guide RNA components, including the spacer and the gRNA core) and a DNA portion (i.e., the extension arm). In various other embodiments, the DNA polymerase can be an “RNA-dependent DNA polymerase” (i.e., whereby the template molecule is a strand of RNA). In such cases, the pegRNA is RNA, i.e., including an RNA extension. The term “polymerase” may also refer to an enzyme that catalyzes the polymerization of nucleotide (i.e., the polymerase activity). Generally, the enzyme will initiate synthesis at the 3'-end of a primer annealed to a polynucleotide template sequence (e.g., such as a primer sequence annealed to the primer binding site of a pegRNA) and will proceed toward the 5´ end of the template strand. A “DNA polymerase” catalyzes the polymerization of deoxynucleotides. As used herein in reference to a DNA polymerase, the term DNA polymerase includes a “functional fragment thereof”. A “functional fragment thereof” refers to any portion of a wild-type or mutant DNA polymerase that encompasses less than the entire amino acid sequence of the polymerase and which retains the ability, under at least one set of conditions, to catalyze the polymerization of a polynucleotide. Such a functional fragment may exist as a separate entity, or it may be a constituent of a larger polypeptide, such as a fusion protein. Premature termination stop codon

[0080] The term “premature termination stop codon” or “PTC” refers to a nonsense mutation in a DNA sequence encoding an mRNA sequence and / or in the mRNA sequence, wherein the 25 12954008Docket No.: B1195.70187WO00 stop codon occurs earlier in the sequence, relative to the non-mutated mRNA sequence, and thus impedes translation of the full-length protein encoded by the mRNA sequence leading to a truncated protein. Premature termination codon may be an ochre stop codon comprising a 5′-UAA-3′ codon sequence, an opal stop codon comprising a 5′-UGA-3′ codon sequence, or an amber stop codon comprising a 5′-UAG-3′ codon sequence. Prime editing

[0081] As used herein, the term “prime editing” refers to an approach for gene editing using napDNAbps, 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. Classical prime editing is described in the inventors publication of Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019), which is incorporated herein by reference in its entirety.

[0082] Prime editing represents a platform for genome editing that is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a nucleic acid programmable DNA binding protein (“napDNAbp”) working in association with a polymerase (i.e., in the form of a fusion protein or otherwise provided in trans with the napDNAbp), wherein the prime editing system is programmed with a prime editing (PE) guide RNA (“pegRNA”) that both specifies the target site and templates the synthesis of the desired edit in the form of a replacement DNA strand by way of an extension (either DNA or RNA) engineered onto a guide RNA (e.g., at the 5ʹ or 3ʹ end, or at an internal portion of a guide RNA). The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same (or is homologous to) sequence as the endogenous strand (immediately downstream of the nick site) of the target site to be edited (with the exception that it includes the desired edit). Through DNA repair and / or replication machinery, the endogenous strand downstream of the nick site is replaced by the newly synthesized replacement strand containing the desired edit. In some cases, prime editing may be thought of as a “search-and-replace” genome editing technology since the prime editors, as described herein, not only search and locate the desired target site to be edited, but at the same time, encode a replacement strand containing a desired edit which is installed in place of the corresponding target site endogenous DNA strand. The prime editors of the present disclosure relate, in part, to the discovery that the mechanism of target-primed reverse 26 12954008Docket No.: B1195.70187WO00 transcription (TPRT) or “prime editing” can be leveraged or adapted for conducting precision CRISPR / Cas-based genome editing with high efficiency and genetic flexibility. TPRT is naturally used by mobile DNA elements, such as mammalian non-LTR retrotransposons and bacterial Group II introns. The inventors have herein used Cas protein-reverse transcriptase fusions or related systems to target a specific DNA sequence with a guide RNA, generate a single strand nick at the target site, and use the nicked DNA as a primer for reverse transcription of an engineered reverse transcriptase template that is integrated with the guide RNA. However, while the concept begins with prime editors that use reverse transcriptase as the DNA polymerase component, the prime editors described herein are not limited to reverse transcriptases but may include the use of virtually any DNA polymerase. Indeed, while the application throughout may refer to prime editors with “reverse transcriptases,” it is set forth here that reverse transcriptases are only one type of DNA polymerase that may work with prime editing. Thus, where ever the specification mentions a “reverse transcriptase,” the person having ordinary skill in the art should appreciate that any suitable DNA polymerase may be used in place of the reverse transcriptase. Thus, in one aspect, the prime editors may comprise Cas9 (or an equivalent napDNAbp) which is programmed to target a DNA sequence by associating it with a specialized guide RNA (i.e., pegRNA) containing a spacer sequence that anneals to a complementary protospacer in the target DNA. The specialized guide RNA also contains new genetic information in the form of an extension that encodes a replacement strand of DNA containing a desired genetic alteration which is used to replace a corresponding endogenous DNA strand at the target site. To transfer information from the pegRNA to the target DNA, the mechanism of prime editing involves nicking the target site in one strand of the DNA to expose a 3′-hydroxyl group. The exposed 3′-hydroxyl group can then be used to prime the DNA polymerization of the edit-encoding extension on pegRNA directly into the target site. In various embodiments, the extension—which provides the template for polymerization of the replacement strand containing the edit—can be formed from RNA or DNA. In the case of an RNA extension, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (such as, a reverse transcriptase). In the case of a DNA extension, the polymerase of the prime editor may be a DNA-dependent DNA polymerase. The newly synthesized strand (i.e., the replacement DNA strand containing the desired edit) that is formed by the herein disclosed prime editors would be homologous to the genomic target sequence (i.e., have the same sequence as) except for the inclusion of a 27 12954008Docket No.: B1195.70187WO00 desired nucleotide change (e.g., a single nucleotide change, a deletion, or an insertion, or a combination thereof). The newly synthesized (or replacement) strand of DNA may also be referred to as a single strand DNA flap, which would compete for hybridization with the complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. In certain embodiments, the system can be combined with the use of an error-prone reverse transcriptase enzyme (e.g., provided as a fusion protein with the Cas9 domain, or provided in trans to the Cas9 domain). The error-prone reverse transcriptase enzyme can introduce alterations during synthesis of the single strand DNA flap. Thus, in certain embodiments, error-prone reverse transcriptase can be utilized to introduce nucleotide changes to the target DNA. Depending on the error-prone reverse transcriptase that is used with the system, the changes can be random or non-random. Resolution of the hybridized intermediate (comprising the single strand DNA flap synthesized by the reverse transcriptase hybridized to the endogenous DNA strand) can include removal of the resulting displaced flap of endogenous DNA (e.g., with a 5ʹ end DNA flap endonuclease, FEN1), ligation of the synthesized single strand DNA flap to the target DNA, and assimilation of the desired nucleotide change as a result of cellular DNA repair and / or replication processes. Because templated DNA synthesis offers single nucleotide precision for the modification of any nucleotide, including insertions and deletions, the scope of this approach is very broad and could foreseeably be used for myriad applications in basic science and therapeutics.

[0083] In various embodiments, prime editing operates by contacting a target DNA molecule (for which a change in the nucleotide sequence is desired to be introduced) with a nucleic acid programmable DNA binding protein (napDNAbp) complexed with a prime editing guide RNA (pegRNA). In various embodiments, the prime editing guide RNA (pegRNA) comprises an extension at the 3´ or 5´ end of the guide RNA, or at an intramolecular location in the guide RNA and encodes the desired nucleotide change (e.g., single nucleotide change, insertion, or deletion). In step (a), the napDNAbp / extended gRNA complex contacts the DNA molecule and the extended gRNA guides the napDNAbp to bind to a target locus. In step (b), a nick in one of the strands of DNA of the target locus is introduced (e.g., by a nuclease or chemical agent), thereby creating an available 3´ end in one of the strands of the target locus. In certain embodiments, the nick is created in the strand of DNA that corresponds to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence, i.e., the “non-target strand.” The nick, however, could be introduced in either of 28 12954008Docket No.: B1195.70187WO00 the strands. That is, the nick could be introduced into the R-loop “target strand” (i.e., the strand hybridized to the protospacer of the extended gRNA) or the “non-target strand” (i.e., the strand forming the single-stranded portion of the R-loop and which is complementary to the target strand). In step (c), the 3´ end of the DNA strand (formed by the nick) interacts with the extended portion of the guide RNA in order to prime reverse transcription (i.e., “target-primed RT”). In certain embodiments, the 3´ end DNA strand hybridizes to a specific RT priming sequence on the extended portion of the guide RNA, i.e., the “reverse transcriptase priming sequence” or “primer binding site” on the pegRNA. In step (d), a reverse transcriptase (or other suitable DNA polymerase) is introduced which synthesizes a single strand of DNA from the 3´ end of the primed site towards the 5´ end of the prime editing guide RNA. The DNA polymerase (e.g., reverse transcriptase) can be fused to the napDNAbp or alternatively can be provided in trans to the napDNAbp. This forms a single- strand DNA flap comprising the desired nucleotide change (e.g., the single base change, insertion, or deletion, or a combination thereof) and which is otherwise homologous to the endogenous DNA at or adjacent to the nick site. In step (e), the napDNAbp and guide RNA are released. Steps (f) and (g) relate to the resolution of the single strand DNA flap such that the desired nucleotide change becomes incorporated into the target locus. This process can be driven towards the desired product formation by removing the corresponding 5´ endogenous DNA flap that forms once the 3´ single strand DNA flap invades and hybridizes to the endogenous DNA sequence. Without being bound by theory, the cells endogenous DNA repair and replication processes resolves the mismatched DNA to incorporate the nucleotide change(s) to form the desired altered product. The process can also be driven towards product formation with “second strand nicking.” This process may introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions.

[0084] The term “prime editor (PE) system” or “prime editor (PE)” or “PE system” or “PE editing system” refers the compositions involved in the method of genome editing using target-primed reverse transcription (TPRT) describe herein, including, but not limited to the napDNAbps, reverse transcriptases, fusion proteins (e.g., comprising napDNAbps and reverse transcriptases), prime editing guide RNAs, and complexes comprising fusion proteins and prime editing guide RNAs, as well as accessory elements, such as second strand nicking components (e.g., second strand sgRNAs) and 5´ endogenous DNA flap removal 29 12954008Docket No.: B1195.70187WO00 endonucleases (e.g., FEN1) for helping to drive the prime editing process towards the edited product formation.

[0085] Although in the embodiments described thus far the pegRNA constitutes a single molecule comprising a guide RNA (which itself comprises a spacer sequence and a gRNA core or scaffold) and a 5ʹ or 3ʹ extension arm comprising the primer binding site and a DNA synthesis template, the pegRNA may also take the form of two individual molecules comprised of a guide RNA and a trans prime editor RNA template (tPERT), which essentially houses the extension arm (including, in particular, the primer binding site and the DNA synthesis domain) and an RNA-protein recruitment domain (e.g., MS2 aptamer or hairpin) in the same molecule which becomes co-localized or recruited to a modified prime editor complex that comprises a tPERT recruiting protein (e.g., MS2cp protein, which binds to the MS2 aptamer). Prime editor

[0086] The term “prime editor” or refers to fusion constructs comprising a napDNAbp (e.g., Cas9 nickase) and a reverse transcriptase that is capable of carrying out prime editing on a target nucleotide sequence in the presence of a pegRNA (or “extended guide RNA”). In some embodiments, simple edits (e.g., modifying the anticodon sequence) may be performed using standard prime editing (e.g., PE2 / PE3) machinery. However, in some embodiments, more complex rewriting of tRNA loci may be desirable (e.g., to change the tRNA composition beyond anticodon changes). In this particular embodiment, dual prime editing (e.g., twinPE) as described in U.S. Patent Application U.S.S.N.63 / 022,397, filed May 8, 2020, U.S. Patent Application U.S.S.N.63 / 116,785, filed November 20, 2020, and PCT Application Serial Number PCT / US2021 / 031439, filed May 7, 2021, all three of which are incorporated by reference in their entirety, may offer greater editing efficiencies. Prime editor complex

[0087] The term “prime editor complex” refers to The term “prime editor” may refer to the fusion protein or to the fusion protein complexed with a pegRNA, and / or further complexed with a second-strand nicking sgRNA. In some embodiments, the prime editor may also refer to the complex comprising a fusion protein (reverse transcriptase fused to a napDNAbp), a pegRNA, and a regular guide RNA capable of directing the second-site nicking step of the non-edited strand as described herein. 30 12954008Docket No.: B1195.70187WO00 Primer binding site

[0088] The term “primer binding site” or “the PBS” refers to the nucleotide sequence located on a pegRNA as a component of the extension arm (typically at the 3ʹ end of the extension arm) and serves to bind to the primer sequence that is formed after Cas9 nicking of the target sequence by the prime editor. As detailed elsewhere, when the Cas9 nickase component of a prime editor nicks one strand of the target DNA sequence, a 3ʹ-ended ssDNA flap is formed, which serves a primer sequence that anneals to the primer binding site on the pegRNA to prime reverse transcription. Protospacer

[0089] As used herein, the term “protospacer” refers to the sequence (~20 bp) in DNA adjacent to the PAM (protospacer adjacent motif) sequence. The protospacer shares the same sequence as the spacer sequence of the guide RNA. The guide RNA anneals to the complement of the protospacer sequence on the target DNA (specifically, one strand thereof, i.e., the “target strand” versus the “non-target strand” of the target DNA sequence). In order for Cas9 to function it also requires a specific protospacer adjacent motif (PAM) that varies depending on the bacterial species of the Cas9 gene. The Cas9 nuclease, derived from S. pyogenes, recognizes a PAM sequence of NGG that is found directly downstream of the target sequence in the genomic DNA, on the non-target strand. The skilled person will appreciate that the literature in the state of the art sometimes refers to the “protospacer” as the ~20-nt target-specific guide sequence on the guide RNA itself, rather than referring to it as a “spacer.” Thus, in some cases, the term “protospacer” as used herein may be used interchangeably with the term “spacer.” The context of the description surrounding the appearance of either “protospacer” or “spacer” will help inform the reader as to whether the term is in reference to the gRNA or the DNA target. Redundant and Dispensable DNA Sequence

[0090] As used herein, the term “redundant and dispensable DNA sequence” refers to a DNA sequence encoding a tRNA gene that has codon degeneracy. Codon degeneracy means that there is more than one codon, and hence anticodon, that specifies a single amino acid. Protospacer adjacent motif (PAM)

[0091] As used herein, the term “protospacer adjacent sequence” or “PAM” refers to an approximately 2-6 base pair DNA sequence that is an important targeting component of a Cas9 nuclease. Typically, the PAM sequence is on either strand, and is downstream in the 5ʹ 31 12954008Docket No.: B1195.70187WO00 to 3ʹ direction of the Cas9 cut site. The canonical PAM sequence (i.e., the PAM sequence that is associated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9) is 5ʹ-NGG-3ʹ wherein “N” is any nucleobase followed by two guanine (“G”) nucleobases. Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms. In addition, any given Cas9 nuclease, e.g., SpCas9, may be modified to alter the PAM specificity of the nuclease such that the nuclease recognizes alternative PAM sequence.

[0092] For example, with reference to the canonical SpCas9 amino acid sequence of SEQ ID NO: 2307419, the PAM sequence can be modified by introducing one or more mutations, including (a) D1135V, R1335Q, and T1337R “the VQR variant”, which alters the PAM specificity to NGAN or NGNG, (b) D1135E, R1335Q, and T1337R “the EQR variant”, which alters the PAM specificity to NGAG, and (c) D1135V, G1218R, R1335E, and T1337R “the VRER variant”, which alters the PAM specificity to NGCG. In addition, the D1135E variant of canonical SpCas9 still recognizes NGG, but it is more selective compared to the wild type SpCas9 protein.

[0093] SpCas9, Streptococcus pyogenes M1, SwissProt Accession No. Q99ZW2, Wild type

[0094] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKAL VRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED YFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFE DREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFL KSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTV KVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN 32 12954008Docket No.: B1195.70187WO00 KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLS ELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR KDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSP TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 2307419).

[0095] It will also be appreciated that Cas9 enzymes from different bacterial species (i.e., Cas9 orthologs) can have varying PAM specificities. For example, Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT or NGRRN. In addition, Cas9 from Neisseria meningitis (NmCas) recognizes NNNNGATT. In another example, Cas9 from Streptococcus thermophilis (StCas9) recognizes NNAGAAW. In still another example, Cas9 from Treponema denticola (TdCas) recognizes NAAAAC. These are examples and are not meant to be limiting. It will be further appreciated that non-SpCas9s bind a variety of PAM sequences, which makes them useful when no suitable SpCas9 PAM sequence is present at the desired target cut site. Furthermore, non-SpCas9s may have other characteristics that make them more useful than SpCas9. For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged into adeno- associated virus (AAV). Further reference may be made to Shah et al., “Protospacer recognition motifs: mixed identities and functional diversity,” RNA Biology, 10(5): 891-899 (which is incorporated herein by reference). Recombinase

[0096] The term “recombinase” refers to any enzyme that catalyzes site-specific recombination events within DNA. In some embodiments, the recombinase is a site-specific recombinase (SSRs). SSRs refer to any enzyme capable of rearranging DNA segments by recognizing and binding to short specific DNA sequences, at which they cleave the DNA backbone, exchange the two DNA helices involved, and rejoin the DNA strands. In some embodiments, the recombinase comprises an integrase (e.g., a serine integrase such as Bxb1). 33 12954008Docket No.: B1195.70187WO00 In some embodiments, the recombinase binds to a recognition site and cleaves the DNA at the recognition site. Reverse transcriptase

[0097] 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. Historically, reverse transcriptase has been used primarily to transcribe mRNA into cDNA which can then be cloned into a vector for further manipulation. 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 5ʹ-3ʹ RNA-directed DNA polymerase activity, 5ʹ-3ʹ DNA-directed DNA polymerase activity, and RNase H activity. RNase H is a processive 5ʹ and 3ʹ ribonuclease specific for the RNA strand for RNA-DNA hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)). Errors in transcription cannot be corrected by reverse transcriptase because known viral reverse transcriptases lack the 3ʹ-5ʹ exonuclease activity necessary for proofreading (Saunders and Saunders, Microbial Genetics Applied to Biotechnology, London: Croom Helm (1987)). A detailed study of the activity of AMV reverse transcriptase and its associated RNase H activity has been presented by Berger et al., Biochemistry 22:2365-2372 (1983). 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. The invention contemplates the use of any such reverse transcriptases, or variants or mutants thereof.

[0098] In addition, the invention contemplates the use of reverse transcriptases that are error- prone, i.e., that may be referred to as error-prone reverse transcriptases or reverse transcriptases that do not support high fidelity incorporation of nucleotides during polymerization. During synthesis of the single-strand DNA flap based on the RT template integrated with the guide RNA, the error-prone reverse transcriptase can introduce one or more nucleotides which are mismatched with the RT template sequence, thereby introducing changes to the nucleotide sequence through erroneous polymerization of the single-strand DNA flap. These errors introduced during synthesis of the single strand DNA flap then become integrated into the double strand molecule through hybridization to the 34 12954008Docket No.: B1195.70187WO00 corresponding endogenous target strand, removal of the endogenous displaced strand, ligation, and then through one more round of endogenous DNA repair and / or sequencing processes. Reverse transcription

[0099] As used herein, the term "reverse transcription" indicates the capability of an enzyme to synthesize a DNA strand (that is, complementary DNA or cDNA) using RNA as a template. In some embodiments, the reverse transcription can be “error-prone reverse transcription,” which refers to the properties of certain reverse transcriptase enzymes which are error-prone in their DNA polymerization activity. Pharmaceutically acceptable carrier

[0100] As used here, the term “pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.). Protein, peptide, and polypeptide

[0101] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. Any of the proteins provided herein may be produced by any method 35 12954008Docket No.: B1195.70187WO00 known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference. Safe harbor locus site

[0102] The term “safe harbor locus site” refers to any site in the genome able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements (i) function predictably and (ii) do not cause alternations of the host genome posing a risk to the host cell or organism. Exemplary embodiments include, but are not limited to, ROSA26, CCR5, and AAVS1. Spacer sequence

[0103] As used herein, the term “spacer sequence” in connection with a guide RNA or a pegRNA refers to the portion of the guide RNA or pegRNA of about 20 nucleotides which contains a nucleotide sequence that shares the same sequence as the protospacer sequence in the target DNA sequence. The spacer sequence anneals to the complement of the protospacer sequence to form a ssRNA / ssDNA hybrid structure at the target site and a corresponding R loop ssDNA structure of the endogenous DNA strand. Suppressor tRNA

[0104] The term “suppressor tRNA” refers to a tRNA (defined elsewhere herein) charged with an amino acid comprising a mutation in the anticodon that allows it to recognize a premature stop codon (defined elsewhere herein as either an amber, ochre, or opal stop codon) on an mRNA and to and insert an amino acid into the amino acid sequence encoded by the mRNA, thus preventing truncation of the amino acid sequence. Suppressor qtRNA

[0105] The term “suppressor qtRNA” refers to a transfer RNA (tRNA) (defined elsewhere herein) charged with an amino acid comprising an expanded anticodon sequence which allows the qtRNA to recognize an expanded codon in an mRNA sequence with a frameshift mutation, thus preserving the normal reading frame. 36 12954008Docket No.: B1195.70187WO00 Target site

[0106] 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 sequence within a nucleic acid molecule to which a complex of the prime editor (PE) and gRNA binds. tRNA

[0107] The terms “tRNA” or “endogenous tRNA” or “unedited tRNA” collectively refer to a transfer RNA as found in nature. tRNA is an art recognized term that refers to a molecule composed of RNA that serves as the physical link between mRNA and the amino acid sequence of proteins. The tRNA structure consists of the following: (i) a 5′-terminal phosphate group, (ii) an acceptor stem made by the base pairing of the 5′-terminal new nucleotide with the 3′-terminal nucleotide (which contains the CCA 3′-terminal group used to attach the amino acid), (iii) a CCA tail at the 3′-end of the tRNA molecule that is covalently bound to an amino acid (herein “aminoacyl-tRNA), (iv) a D arm domain, (v) an anticodon arm comprising an anticodon sequence. The tRNA 5′-to-3′ primary structure contains the anticodon but in reverse order, since 3′-to-5′ directionality is required to read the mRNA from 5′-to-3′, (vi) a T arm domain, and (vii) a variable arm domain Variant

[0108] 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 nucleic acid or amino acid residues as compared to a wild type Cas9 nucleic acid or 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. Vector

[0109] The term “vector,” as used herein, refers to a nucleic acid that can be modified to encode a gene of interest and that is able to enter into a host cell, mutate and replicate within the host cell, and then transfer a replicated form of the vector into another host cell. 37 12954008Docket No.: B1195.70187WO00 Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages and filamentous phage, and conjugative plasmids. Additional suitable vectors will be apparent to those of skill in the art based on the instant disclosure. DETAILED DESCRIPTION

[0110] Aspects of the disclosure relate to compositions, systems, and methods for editing an endogenous tRNA into a suppressor qtRNA using prime editing (e.g., to treat diseases caused by frameshift mutations). Additional aspects relate to compositions comprising the prime editing machinery (e.g., fusion protein comprising a nucleic acid programmable DNA binding protein and reverse transcriptase and / or pegRNA, etc.) and / or complexes comprising the prime editor and pegRNA that are capable of editing an endogenous tRNA into a suppressor qtRNA. In some aspects, the disclosure further relates to polynucleotides encoding one or more nucleic acid sequences encoding the prime editor and / or pegRNA, cells comprising the polynucleotides and complexes comprising the prime editor and pegRNA, kits comprising any one of the compositions, complexes, polynucleotides, vectors (e.g., AAV), and / or cells disclosed herein, and / or delivery systems for administering any one of the compositions, complexes, polynucleotides, vectors, and / or cells to a subject in need thereof (e.g., lipid nanoparticles). Additional aspects relate to methods for inserting a new suppressor qtRNA gene into a target site in a genome (e.g., a safe harbor locus site) using prime editing.

[0111] Various aspects of the present disclosure relate to editing one or more domains of a tRNA to yield a suppressor qtRNA, e.g., a qtRNA. Without wishing to be bound by any particular theory, it is generally believed that a tRNA comprises a structure as shown in FIG. 1. In some embodiments, the endogenous tRNA comprises an acceptor stem domain. In some embodiments, the endogenous tRNA comprises a D loop domain. In some embodiments, the endogenous tRNA comprises a variable loop domain in some embodiments, the endogenous tRNA comprises a TψC loop domain. In some embodiments, the endogenous tRNA comprises an anticodon arm domain. In some embodiments, editing an anticodon sequence within the anticodon arm domain converts an endogenous tRNA to a suppressor qtRNA. Again, without wishing to be bound by any particular theory, it is generally believed that editing the one or more domains may enable charging of the qtRNA 38 12954008Docket No.: B1195.70187WO00 with a non-cognate amino acid and / or improve read-through efficiency of the suppressor qtRNA.

[0112] As used herein, the term “D arm domain” refers to a feature in the tertiary structure of tRNA. Without wishing to be bound by theory, it comprises two D stems and the D loop. The D loop further comprises the base dihydrouridine, for which the arm is named. The D-loops main function is recognition. It is widely believed that it acts as a recognition site for aminoacyl-tRNA synthetase, an enzyme involved in the aminoacylation of the tRNA molecule. Editing of this domain, may according to some embodiments, alter the identity of the amino acid charged to the qtRNA.

[0113] As used herein, the term “T-arm domain” refers to a specialized region of the tRNA which acts as a special recognition site for the ribosome to form a tRNA-ribosome complex during protein biosynthesis (e.g., translation). The T-arm domain is generally believed to have two components: a T-stem and T-loop. There are two T-stems of five base pairs each. The T-loop is often referred to as the TψC arm due to the presence of thymidine, pseudouridine, and cytidine.

[0114] As used herein, the term “anticodon arm domain” refers to a 5-bp stem whose loop contains the anticodon. The anticodon portion of the tRNA binds to the codon sequence in mRNA during translation.

[0115] As used herein, the term “variable arm domain” refers to a loop that is present between the anticodon arm and the TψC arm. The length of the variable arm domain is important in the recognition of the aminoacyl-tRNA synthetase for the tRNA. In some embodiments, the tRNA lacks the variable arm domain. Suppressor qtRNAs via prime editing of endogenous tRNAs for readthrough of frameshift mutations

[0116] Aspects of the present disclosure relate to methods for editing a DNA sequence encoding an endogenous tRNA at one or more target sites for readthrough of frameshift mutations. Without wishing to be bound by any particular theory, it is generally known that the genetic code is determined by a combination of tRNAs and aminoacyl tRNA synthetases (AARSs). Codons are dictated by the three bases in the center of the anticodon loop of each tRNA, which undergo Watson-Crick base pairing to an mRNA transcript during translation, enabling accurate codon recognition. During translation of the genetic code, the mRNA is read in sets of three nucleotides. These three-nucleotide mRNA sequences, or 39 12954008Docket No.: B1195.70187WO00 codons, encode for amino acids, and the selection of correct tRNAs helps maintain the precise mRNA reading frame. Genetic mutations, such as the insertion or deletion of one, two or four nucleotides, leads to a change in the mRNA reading frame. This change, in turn, leads to the formation of a truncated or dysfunctional protein (FIGs. 27A,B and FIG. 28). In some embodiments, these mutations cause certain genetic diseases and cancers, which in principle can be corrected with a tRNA that reads four or five codons (e.g., qtRNAs). As used herein, the term “qtRNA” means a tRNA capable of reading four or more codons. Naturally occurring qtRNAs, such as sufD and sufJ, have been reported in bacteria, and others have shown the possibility of four base pair codon translation in bacteria using natural, evolved, or synthetic quadruplet tRNA (qtRNA). In some embodiments, the qtRNA comprises 4 anticodons and is configured to read four codons. In other embodiments, the qtRNA comprises 5 anticodons and is configured to read five codons.

[0117] Thus, in some embodiments, the methods comprise contacting a DNA sequence encoding an endogenous tRNA at one or more target sites with a prime editor and one or more pegRNA’s. In some embodiments, the prime editor installs one or more modifications in the DNA sequence at the one or more target sites. In some embodiments, the one or more target sites comprise the anticodon loop. Other domains of the tRNA may also be edited according to other embodiments. In some embodiments, the one or more edits (e.g., modifications) are selected from the group consisting of insertions, deletions, and substitution. In some embodiments, the edit is an insertion. In some embodiments, the edits are deletions. In some embodiments, the edits are substitutions.

[0118] In some embodiments, the edited DNA sequence encodes a suppressor qtRNA with an expanded anticodon sequence (e.g., 4, 5, or greater nucleotides). In some embodiments, the qtRNA suppresses one or more frameshift mutations within an mRNA transcript, thus restoring the protein reading frame (FIG. 28). Without wishing to be bound by any particular theory, while prime editing could in principle be used to correct these frameshift mutations, the current disclosure provides a strategy that allows for the correction of nearly all diseases caused by frameshift mutations with a single composition of matter (e.g., a qtRNA). In some embodiments, this involves using prime editing to edit the triplet anticodon loop of an endogenous tRNA to quadruple or quintuple anticodon tRNA (qtRNA).

[0119] In some embodiments, the methods comprise editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a 40 12954008Docket No.: B1195.70187WO00 suppressor qtRNA, comprising contacting the DNA sequence at one or more target sites with a prime editor and one or more pegRNAs, wherein the prime editor installs one or more modifications in the DNA sequence at the one or more target sites, relative to the DNA sequence encoding the endogenous tRNA, thus converting the edited DNA sequence into a DNA sequence encoding a suppressor qtRNA, wherein the one or more pegRNA comprises a spacer sequence, a gRNA core, and an extension arm, and wherein the suppressor qtRNA binds to expanded codons.

[0120] In some embodiments, the methods relate editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA, comprising contacting the DNA sequence at the one or more target sites with a prime editor and one or more pegRNA, wherein the prime editor installs one or more modifications in the DNA sequence at the one or more target sites, relative to the DNA sequence encoding the endogenous tRNA, thus converting the edited DNA sequence into a DNA sequences encoding a suppressor qtRNA, wherein the one or more pegRNA comprises a spacer sequence, a gRNA core, and an extension arm, wherein the spacer sequence and / or extension arms are any sequences listed in SEQ ID NOs: 431-2199724, wherein the DNA sequence is any sequence listed SEQ ID NOs.1-430 or 2306989-2307418.

[0121] In some embodiments, the target site in the DNA sequence encodes one or more domains of the endogenous tRNA. In some embodiments the domain is the D-arm domain of the endogenous tRNA. In some embodiments the domain is a T-arm domain of the endogenous tRNA. Other domains are also possible in other embodiments for example, in some embodiments, the domain is a variable arm domain of the endogenous tRNA. Additionally, or alternatively, in some embodiments the domain is an acceptor stem domain of the tRNA. In some embodiments, the target site in the DNA sequence comprises an anticodon sequence in the anticodon arm domain of the endogenous tRNA.

[0122] As described above, in some embodiments, the method comprises installing one or more modifications at the target site in the DNA sequence. In some embodiments, installing the one or more modifications comprises installing one or more insertions, deletions, or substitutions in one or more of the domains of the endogenous tRNA. For example, in some embodiments, installing the one or more modifications comprises installing one or more nucleotide insertions in the anticodon sequence of the anticodon arm domain. In another set of embodiments, installing the one or more modifications comprises installing a 41 12954008Docket No.: B1195.70187WO00 single nucleotide insertion in the anticodon sequence of the anticodon arm domain. In other embodiments, installing the one or more modifications comprises installing two nucleotide insertions and the anticodon sequence of the anticodon arm domain.

[0123] In some embodiments, installing the one or more modifications comprises installing one or more nucleotide insertions, deletions, or substitutions in the D-arm domain. In some embodiments, installing the one or more modifications comprises installing one or more nucleotide insertions, deletions, or substitutions in an acceptor stem domain. In other embodiments, installing the one or more modifications comprises installing one or more nucleotide insertions, deletions, or substitutions in the T-arm domain. Additionally, or alternatively, in some embodiments, installing the one or more modification comprises installing one or more detail insertions, deletions, or substitutions in the variable arm domain.

[0124] In some embodiments, the method converts an edited DNA sequence into a DNA sequence encoding a suppressor qtRNA. In some embodiments, the edited DNA sequence encodes a suppressor qtRNA comprising an expanded anticodon. In some embodiments, the expanded anticodon comprises four or more nucleotides. For example, in some embodiments the expanded anticodon comprises four nucleotides (e.g., quadruple tRNA). In other embodiments, the expanded anticodon comprises five nucleotides (e.g., quintuple tRNA).

[0125] Those of ordinary skill in the art will appreciate that converting the endogenous tRNA into a qtRNA comprises inserting one or more nucleotides into a DNA sequence encoding the triplet anticodon of the endogenous tRNA (e.g., within the anticodon arm domain). In some embodiments, the one or more nucleotides is inserted at a 5′ end of the DNA sequence. In other embodiments the one or more nucleotides is inserted at a 3′ end of the DNA sequence. Alternatively, or additionally, in some embodiments, the one or more nucleotide is inserted at both a 5′ and 3′ end of the DNA sequence encoding the triplet anticodon. In some embodiments, the expanded anticodon binds to any quadruple or quintuple codon listed in Tables 1 and 2. 42 12954008Docket No.: B1195.70187WO00

[0126] Table 1. Exemplary list of expanded codons that Quadruple tRNAs may bind. Note, that the quadruple tRNA anticodon is complementary to the below listed codons. N may be any nucleotide (e.g., A, T, U, G).43 12954008Docket No.: B1195.70187WO00Table 2. Exemplary list of expanded codons that quintuple tRNAs may bind. Note, that the quintuple tRNA anticodon is complementary to the below listed codons. N may be any nucleotide (e.g., A, T, U, G).44 12954008Docket No.: B1195.70187WO0045 12954008Docket No.: B1195.70187WO0046 12954008Docket No.: B1195.70187WO00

[0127] As described above, various aspects of the present disclosure relate to compositions and methods for editing a DNA sequence encoding an endogenous tRNA by prime editing. In some embodiments, the tRNA gene may be any tRNA gene in a host genome, such as those described in Tables 3 or 4, as shown below. In some embodiments, the tRNA gene comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to any one of SEQ ID NOs 1-430 and 2306989-2307418. In some embodiments, the tRNA gene comprises a nucleic acid sequence that is identical to any one of SEQ ID NOs 1-430 and 2306989-2307418. 47 12954008Docket No.: B1195.70187WO00 Table 3. List of tRNA gene names, genomic coordinates, and nucleic acid sequence.48 12954008Docket No.: B1195.70187WO0049 12954008Docket No.: B1195.70187WO0050 12954008Docket No.: B1195.70187WO0051 12954008Docket No.: B1195.70187WO0052 12954008Docket No.: B1195.70187WO0053 12954008Docket No.: B1195.70187WO0054 12954008Docket No.: B1195.70187WO0055 12954008Docket No.: B1195.70187WO0056 12954008Docket No.: B1195.70187WO0057 12954008Docket No.: B1195.70187WO0058 12954008Docket No.: B1195.70187WO0059 12954008Docket No.: B1195.70187WO0060 12954008Docket No.: B1195.70187WO0061 12954008Docket No.: B1195.70187WO0062 12954008Docket No.: B1195.70187WO0063 12954008Docket No.: B1195.70187WO0064 12954008Docket No.: B1195.70187WO0065 12954008Docket No.: B1195.70187WO0066 12954008Docket No.: B1195.70187WO0067 12954008Docket No.: B1195.70187WO0068 12954008Docket No.: B1195.70187WO0069 12954008Docket No.: B1195.70187WO0070 12954008Docket No.: B1195.70187WO0071 12954008Docket No.: B1195.70187WO0072 12954008Docket No.: B1195.70187WO00Table 4. List of tRNA gene names, genomic coordinates, and nucleic acid sequence. Nucleic acid sequences are complementary to those in Table 373 12954008Docket No.: B1195.70187WO0074 12954008Docket No.: B1195.70187WO0075 12954008Docket No.: B1195.70187WO0076 12954008Docket No.: B1195.70187WO0077 12954008Docket No.: B1195.70187WO0078 12954008Docket No.: B1195.70187WO0079 12954008Docket No.: B1195.70187WO0080 12954008Docket No.: B1195.70187WO0081 12954008Docket No.: B1195.70187WO0082 12954008Docket No.: B1195.70187WO0083 12954008Docket No.: B1195.70187WO0084 12954008Docket No.: B1195.70187WO0085 12954008Docket No.: B1195.70187WO0086 12954008Docket No.: B1195.70187WO0087 12954008Docket No.: B1195.70187WO0088 12954008Docket No.: B1195.70187WO0089 12954008Docket No.: B1195.70187WO0090 12954008Docket No.: B1195.70187WO0091 12954008Docket No.: B1195.70187WO0092 12954008Docket No.: B1195.70187WO0093 12954008Docket No.: B1195.70187WO0094 12954008Docket No.: B1195.70187WO0095 12954008Docket No.: B1195.70187WO0096 12954008Docket No.: B1195.70187WO0097 12954008Docket No.: B1195.70187WO00

[0128] In some embodiments, the methods for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA comprises contacting the DNA sequence at one or more target sites with a prime editor and one or more pegRNAs. In some embodiments, pegRNA comprising a spacer sequence, a gRNA core, and an extension arm. In some embodiments, the pegRNA further comprises a stabilizing 3′-tevopreQi motif. In some embodiments, the pegRNA directs the prime editor to install an edit at the target site located between positions +1 and +40, between positions +5 and +35, between positions +10 and +30, and between positions +15 and +25, relative to a first editable base located 3′ of a pegRNA-directed nick. In some embodiments, the pegRNA directs the prime editor to install an edit at a target site between positions +10 98 12954008Docket No.: B1195.70187WO00 and +20 or between +11 and +17, relative to a first editable base located 3′ of a pegRNA- directed nick. Other installation sites are also possible in other embodiments.

[0129] As mentioned above, the pegRNA comprises an extension arm. In some embodiments, the extension arm of the pegRNA comprises a DNA synthesis template and a primer binding site (PBS). Without wishing to be bound by theory, the DNA synthesis template encodes via a polymerase (e.g., a reverse transcriptase) a single stranded DNA flap containing the genetic change of interest (e.g., single nucleotide insertion), which then integrates into the endogenous DNA sequence by replacing the corresponding endogenous strand, thereby installing the desired genetic change.

[0130] Accordingly, in some embodiments, the DNA synthesis template encodes an expanded anticodon sequence. The expanded anticodon sequence may be any suitable anticodon sequence known to the skilled artisan (e.g., any sequence listed in Tables 1 and 2). In some embodiments, the DNA synthesis template encodes a sequence that is complementary any expanded codon sequence listed in Tables 1 and 2 (e.g., when the extension arm is at a 5′ end of the pegRNA). Likewise, in some embodiments, the DNA synthesis template encodes one or more nucleotide insertions, deletions, or substitutions to be installed at a target site (e.g., anticodon sequence (of the DNA sequence encoding the anticodon arm domain of the endogenous tRNA. In some embodiments, the DNA synthesis template encodes one or more nucleotide insertions to be installed at the target site of the DNA sequence encoding the anticodon arm domain of the tRNA.

[0131] The DNA synthesis template may encode one or more nucleotide insertions, deletions, or substitutions other domains, according to other embodiments. For example, in some embodiments the DNA synthesis template encodes one or more nucleotide insertions deletions or substitutions to be installed at the target site of the DNA sequence encoding the D arm domain, that T-arm domain, the variable arm domain, or the acceptor stem arm domain of the tRNA.

[0132] Additionally, in some embodiments, the DNA synthesis template further encodes one or more PAM-disrupting mutations and / or MMR-evading mutations as described in U.S. Patent Application U.S.S.N.63 / 136,194 filed January 11, 2021 and International Patent Application No. PCT / US2022 / 012054 filed January 11, 2022, both of which are herein incorporated by reference in their entirety. 99 12954008Docket No.: B1195.70187WO00

[0133] In some embodiments, the methods for editing a DNA sequence encoding an endogenous tRNA to produce a DNA sequence encoding a suppressor qtRNA comprises contacting the DNA sequence at one or more target sites with a prime editor. Any suitable prime editor known to the person of ordinary skill in the art may be used in the methods contemplated herein. For example, in some embodiments, the prime editor used to install the edits comprises PE2, PE3, PE4, PE5, PE2max, PE3max, PE4max, PE5max, twinPE, or Prime-del. Other prime editors are also possible. In some embodiments, the prime editors disclosed herein are used in conjugation with a gRNA and / or a second pegRNA to install the desired edits.

[0134] Aspects of the disclosure relate to compositions for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA, wherein said compositions comprise a prime editor and a pegRNA. In some embodiments, the pegRNA comprises a spacer sequence, a core gRNA, and an extension arm comprising a DNA synthesis template and a primer binding site (PBS). In some embodiments, the pegRNA is configured to bind to a DNA sequence encoding an endogenous tRNA.

[0135] In some embodiments, the compositions comprise a prime editor. Any prime editor known in the art may be used to edit the endogenous tRNA into a suppressor qtRNA. For example, in some embodiments, the prime editor used to install the edits comprises PE2, PE3, PE4, PE5, PE2max, PE3max, PE4max, PE5max, twinPE, or Prime-del. Other prime editors are also possible. In some embodiments, the prime editors disclosed herein are used in conjugation with a gRNA and / or a second pegRNA to install the desired edits.

[0136] In some embodiments, the compositions comprise one or more pegRNAs comprises a spacer sequence, a core gRNA, and an extension arm comprising a DNA synthesis template and a primer binding site. In some embodiments, the pegRNA is tailored to maximize editing efficiency. In some embodiments, the DNA synthesis template encodes one or more edits to be installed at a target site in the DNA sequence encoding the endogenous tRNA. In some embodiments, the one or more edits are selected from the group consisting of insertions, deletions, and substitutions. In some embodiments, the DNA synthesis template encodes at least one nucleotide insertion to be installed at the target site of the DNA sequence encoding an anticodon sequence of the endogenous tRNA. In some embodiments, the DNA synthesis template encodes one or more nucleotide insertions, 100 12954008Docket No.: B1195.70187WO00 deletions, or substitutions to be installed at the target site of the DNA sequence encoding the D-arm domain, the T-arm domain, the variable arm domain, or the acceptor stem arm domain of the tRNA.

[0137] The DNA synthesis template may encode other edits in other embodiments. For example, the DNA synthesis template may further encode a PAM-disrupting mutation and / or an MMR-evading mutation, relative to an endogenous tRNA, in some embodiments.

[0138] In some embodiments, the compositions further comprise a sgRNA and / or a second pegRNA, for example, as may be needed for PE3 and twinPE prime editors, respectively.

[0139] Some aspects of the present disclosure relate to methods using twinPE prime editors to edit one or more domains of the endogenous qtRNA . In some embodiments, the methods comprise editing both strands of a DNA sequence encoding the endogenous tRNA at a target site to be edited. Target sites include, but are not limited to, the D-arm domain, T- arm domain, acceptor stem domain, variable arm domain, and the anticodon arm domain comprising the anticodon sequence of the endogenous tRNA . In some embodiments, the methods comprise contacting the DNA sequence with a first prime editor complex and a second prime editor complex. Each of the first and second prime editor complexes comprise (1) a prime editor (e.g., PE2) comprising (i) a napDNAbp and a polymerase (e.g., a polypeptide having an RNA-dependent DNA polymerase activity) and (2) a pegRNA comprising a spacer sequence, gRNA core, an extension arm comprising a DNA synthesis template and a primer binding site.

[0140] In some embodiments, the DNA synthesis template of the pegRNA of the first prime editor complex encodes a first single stranded DNA sequence. In some embodiments, the DNA synthesis template of the pegRNA of the second prime editor complex encodes a second single-stranded DNA sequence. In some embodiments, the first single strand DNA sequence and the second single stranded DNA sequence encode one or more nucleotide insertion to be installed at the target site of the DNA sequence encoding the anticodon sequence of the anticodon arm domain of the endogenous tRNA . In some embodiments, the first single strand DNA sequence and the second single stranded DNA sequence further encode PAM-disrupting mutations and / or MMR-evading mutations.

[0141] In some embodiments, the first single-stranded DNA sequence and the second single-stranded DNA sequence each comprises a region of complementarity to each other, 101 12954008Docket No.: B1195.70187WO00 such that they form a duplex comprising the edited portion, relative to the DNA sequence at the target site to be edited. In some embodiments, the duplex is integrated into the target site to be edited. In some embodiments, integrating the duplex into the target site installs the expanded anticodon at the target site of the DNA sequence encoding the triple anticodon sequence of the endogenous tRNA.

[0142] In some embodiments, integrating the duplex into the target site further installs a PAM-disrupting mutation and / or an MMR-evading mutation in the DNA sequence.

[0143] Other aspects of the disclosure relate to compositions comprising twinPEs for editing endogenous tRNAs into suppressor qtRNAs . In some embodiments, the compositions comprise a first and second prime editor complex. In some embodiments, the first and second prime editor complexes comprises (1) a prime editor comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp), and (ii) a polypeptide having an RNA-dependent DNA polymerase activity; and (2) a pegRNA comprising a spacer sequence, gRNA core, an extension arm comprising a DNA synthesis template and a primer binding site (PBS). In some embodiments, the DNA synthesis template of the pegRNA of the first prime editor complex encodes a first single-stranded DNA sequence and the DNA synthesis template of the pegRNA of the second prime editor complex encodes a second single- stranded DNA sequence. In some embodiments, the first single-stranded DNA sequence and the second single-stranded DNA sequence each comprises a region of complementarity to the other. In some embodiments, the first single-stranded DNA sequence and the second single- stranded DNA sequence form a duplex comprising an edited portion as compared to the DNA sequence at the target site to be edited, which integrates into the target site to be edited.

[0144] Aspects of the disclosure relate to pegRNAs for editing a DNA sequence encoding an endogenous tRNA by prime editing into a suppressor qtRNA. In some embodiments, the pegRNA comprises a spacer sequence, a gRNA core, and an extension arm comprising a DNA synthesis template and a primer binding site. In some embodiments, the pegRNA further comprises a stabilizing 3′-tevopreQi motif. In some embodiments, the pegRNA is configured to bind to a DNA sequence encoding an endogenous tRNA.

[0145] Any suitable pegRNA for editing a DNA sequence encoding an endogenous tRNA by prime editing into a suppressor qtRNA that is known to the skilled artisan may be used to edit the DNA sequence. In some embodiments, the pegRNA comprises a 3′- tevopreQi motif. Without wishing to be bound by any particular theory, such motifs are 102 12954008Docket No.: B1195.70187WO00 known in the art to enhance prime editing efficiencies. In some embodiments, the pegRNA comprises a PAM sequence. In some embodiments the PAM sequence is and NGG. In some embodiments, the spacer sequence and / or extension arms of the pegRNA’s disclosed herein may be any subsequence listed within the sequences listed in SEQ ID NOs: 431- 2199724.

[0146] In some embodiments, the DNA synthesis template encodes an expanded anticodon sequence. The expanded anticodon sequence may be any suitable anticodon sequence known to the skilled artisan (e.g., any sequence listed in Tables 1 and 2). In some embodiments, the DNA synthesis template encodes a sequence that is complementary to any expanded codon sequence listed in Tables 1 and 2 (e.g., when the extension arm is at a 5′ end of the pegRNA). Likewise, in some embodiments, the DNA synthesis template encodes one or more nucleotide insertions, deletions, or substitutions to be installed at a target site (e.g., anticodon sequence( of the DNA sequence encoding the anticodon arm domain of the endogenous tRNA. In some embodiments, the DNA synthesis template encodes one or more nucleotide insertions to be installed at the target site of the DNA sequence encoding the anticodon arm domain of the tRNA. In some embodiments, the DNA synthesis template encodes at least one nucleotide insertion to be installed at the target site.

[0147] The DNA synthesis template may encode one or more nucleotide insertions, deletions, or substitutions other domains, according to other embodiments. For example, in some embodiments the DNA synthesis template encodes one or more nucleotide insertions deletions or substitutions to be installed at the target site of the DNA sequence encoding the D arm domain, that T-arm domain, the variable arm domain, or the acceptor stem arm domain of the tRNA, relative to the endogenous tRNA. Additionally, in some embodiments, the DNA synthesis template further encodes one or more PAM-disrupting mutation and / or MMR- evading mutations.

[0148] Other aspects of the disclosure relate to complex comprising a prime editor and a pegRNA for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA, wherein the pegRNA comprises a spacer sequence and an extension arm, wherein the spacer sequence is configured to bind to the DNA sequence encoding the endogenous tRNA. The complex may comprise any prime editor and pegRNA disclosed herein.

[0149] Additionally, some aspects of the disclosure relate to a polynucleotide for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA 103 12954008Docket No.: B1195.70187WO00 sequence encoding a suppressor qtRNA, the polynucleotide comprising a first nucleic acid sequence encoding a prime editor and a second nucleic acid sequence encoding a pegRNA, wherein the pegRNA is configured to bind to a DNA sequence encoding an endogenous tRNA.

[0150] In some embodiments, second nucleic acid sequence encodes a pegRNA comprising a sequence with at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% sequence identity to any sequence listed in SEQ ID NOs: 431- 2199724. Suppressor qtRNA via replacing endogenous tRNAs using prime editing for readthrough of frameshift mutations.

[0151] As described elsewhere herein, aspects of the disclosure relate to methods using prime editing to replace endogenous tRNAs with suppressor qtRNAs. Without wishing to be bound by any particular theory, it is possible that repurposing certain tRNAs into suppressor qtRNAs may negatively perturb cell function. As such, certain embodiments relate to overwriting an existing RNA with a suppressor qtRNA . The skilled artisan will appreciate that an ideal target tRNA candidate for conversion to a suppressor qtRNA may not be expressed in a target tissue, and thus would likely elicit poor levels of readthrough. To address this limitation, highly expressed tRNA genes, e.g., tRNA genes in a target organ or tissue, are overwritten with the sequence of an optimized suppressor qtRNA candidate sequence.

[0152] In some embodiments, the endogenous tRNA to be edited has a plurality of isodecoders that may be edited. In some embodiments, the endogenous tRNA has greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, greater than or equal to 10, greater than or equal to 15, greater than or equal to 20, greater than or equal to 25, greater than or equal to 30, greater than or equal to 35, greater than or equal 40, greater than or equal to 45, or greater than or equal to 50 isodecoders. In some embodiments, the endogenous tRNA has less than or equal to 50, less than or equal to 45, less than or equal to 40, less than or equal to 35, less than or equal to 30, less than or equal to 25, less than or equal to 20, less than or equal to 15, less than or equal to 10, less than or equal to 9, less than or equal to 8, less than or equal to 7, less than or equal to 6, less than or equal to 5, less than or equal to 4, less than or equal to 3, or less than or equal 104 12954008Docket No.: B1195.70187WO00 to 2 isodecoders. Any method known in the art by the skilled artisan may be used to replace an endogenous tRNA gene with a suppressor qtRNA gene. In some embodiments, standard prime editing techniques are used to install the desired edits. In some embodiments, twin prime editing (twinPE), also known as “dual flap” prime editing, is used to install the edits. Without wishing to be bound by any particular theory, it is generally believed that twinPE may provide higher editing efficiencies due to the large size (e.g., length) of the desired edit. In some embodiments, the prime editor used to install the edits comprises PE2, PE3, PE4, PE5, PE2max, PE3max, PE4max, PE5max, twinPE, or Prime-del. Suppressor qtRNAs via gene insertion for readthrough of frameshift mutations

[0153] Aspects of the disclosure relate to methods for inserting a new suppressor qtRNA gene into a target site of an organism’s genome. Without wishing to be bound by theory, this approach requires insertion of a small gene rather than a local edit of a subset of endogenous tRNA bases, but may offer complementary advantages such as the lack of dependence on the presence, sequence, and dispensability of an endogenous tRNA gene in a specific target organism or patient. Any suitable method known in the art may be used to insert the new suppressor qtRNA gene into the target site. Exemplary methods, include but are not limited to, prime editing methods (e.g., twinPE), prime editing methods coupled with integrase or recombinase enzymes, CRISPR-associated transposases (CASTs) and other targeted gene insertion technologies to achieve insertion of a suppressor qtRNA or a suppressor qtRNA expression cassette into the human genome is likewise also envisioned.

[0154] Accordingly, in some embodiments, the methods relate to inserting a DNA sequence encoding a suppressor qtRNA gene into a target site in a host genome using prime editing, the method comprising contacting the target site with (i) a prime editor, (ii) a pegRNA, (iii) a sgRNA, and (iv) a nucleic acid (e.g., a circular plasmid, ssRNA, ssDNA, or dsDNA), wherein the prime editor comprises a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp), a DNA polymerase, and a recombinase.

[0155] In some embodiments, the nucleic acid is any nucleic acid that can be subsequently converted into double stranded DNA (e.g., for use as a substrate for a recombinase). In some embodiments, the nucleic acid is selected from the group consisting of a plasmid (e.g., circular or linear), ssRNA, ssDNA, and dsDNA. In some embodiments, the nucleic acid is a circular plasmid. In some embodiments, the nucleic acid is a ssRNA. In 105 12954008Docket No.: B1195.70187WO00 some embodiments, the nucleic acid is a ssDNA. In other embodiments, the nucleic acid is dsDNA.

[0156] In other embodiments, the methods relate to inserting a DNA sequence encoding a suppressor qtRNA sequence into a target site in a host genome using twinPE. In some embodiments, the method comprises contacting the target site with (i) a prime editor and (ii) a pegRNA. In some embodiments, the prime editor comprises a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a DNA polymerase. In some embodiments, the pegRNA comprises a spacer sequence, a gRNA core, and an extension arm comprising a DNA synthesis template and a primer binding site (PBS). In some embodiments, the spacer sequence comprises a region of complementarity to a target strand of a double stranded target tRNA gene sequence in the subject. In some embodiments, the gRNA core associates with the napDNAbp. In some embodiments, the DNA synthesis template comprises a region of complementarity to the non-target strand of the double- stranded target tRNA gene sequence and encodes a suppressor qtRNA gene sequence to be installed within the target site. In some embodiments, the primer binding site comprises a region of complementarity to a non-target strand of the double-stranded target tRNA gene sequence. In some embodiments, the prime editor and the pegRNA install the suppressor qtRNA gene sequence in the target site in the human genome.

[0157] In some embodiments, the pegRNA comprises a spacer sequence, a gRNA core, and an extension arm comprising a DNA synthesis template and a primer binding site (PBS). In some embodiments, the spacer sequence comprises a region of complementarity to a target strand (e.g., protospacer sequence) of a double stranded target tRNA gene sequence in the subject. In some embodiments, the gRNA core associates with the napDNAbp. In some embodiments, the DNA synthesis template comprises a region of complementarity to the non-target strand of the double-stranded target tRNA gene sequence and encodes the suppressor qtRNA gene sequence to be installed within the target site. In some embodiments, the primer binding site comprises a region of complementarity to a non-target strand of the double-stranded target tRNA gene sequence.

[0158] In some embodiments, the prime editor and the pegRNA install the suppressor qtRNA gene sequence in the target site in the genome (e.g., human genome). In some embodiments, installation of the suppressor qtRNA gene results in the indefinite expression of the suppressor qtRNAs gene. 106 12954008Docket No.: B1195.70187WO00

[0159] In some embodiments, the suppressor qtRNA gene encodes for a suppressor qtRNA with an expanded anticodon. Those of ordinary skill in the art will appreciate that converting the endogenous tRNA into a qtRNA comprises inserting one or more nucleotides into a DNA sequence encoding the triplet anticodon of the endogenous tRNA (e.g., within the anticodon arm domain). In some embodiments, the one or more nucleotides is inserted at a 5′ end of the DNA sequence. In other embodiments the one or more nucleotides is inserted at a 3′ end of the DNA sequence. Alternatively, or additionally, in some embodiments, the one or more nucleotide is inserted at both a 5′ and 3′ end of the DNA sequence encoding the triplet anticodon. In some embodiments, the expanded anticodon binds to any quadruple or quintuple codon listed in Tables 1 and 2.

[0160] In some embodiments, the suppressor qtRNA encodes for one or more mutations, relative to an endogenous tRNA . For example, in some embodiments, the suppressor qtRNA encodes for a tRNA with an expanded anticodon, but includes one or more mutations in one or more domains that results in the suppressor qtRNA being charged with a non-cognate amino acid. Any mutation known in the art that results in amino acid misincorporation is envisioned herein. For example, in some embodiments, the suppressor qtRNA gene encodes a Lys-tRNA-CUU comprising a C70U mutation in the acceptor stem domain. In this embodiment, expression of the suppressor qtRNA gene would result in production of Ala-tRNA-CCU, instead of the endogenous Lys-tRNA-CUU. Other mutations are also possible in other embodiments. For example, in some embodiments, the suppressor qtRNA gene encodes a single base nucleotide insertion in a variable arm domain, relative to an endogenous tRNA .

[0161] In some embodiments, the suppressor qtRNA gene may be inserted into any suitable target site within the genome. In some embodiments, the target site is a safe harbor locus site. Without wishing to be bound by theory, genomic safe harbor locus sites are sites in the genome that are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function properly and do not cause alternations of the host genome posing a risk to the host cell or organism.

[0162] In some embodiments, any suitable safe harbor locus site (e.g., any currently known site or yet to be determined sites) may be used as the target site for gene insertion. In some embodiments, the safer harbor locus site comprises the ROSA26 gene, the AAVS1 gene, or the CCR5 gene. 107 12954008Docket No.: B1195.70187WO00

[0163] In some embodiments, the target site is a general expression site. Any suitable general expression site (e.g., any currently known site or yet to be determined sites) may be used as the target site for gene insertion. For example, in some embodiments, the general expression site comprises the albumin gene (ALB gene).

[0164] Other aspects of the disclosure relate to inserting a suppressor qtRNA gene into a genome using a prime editor fusion protein comprising a napDNAbp, a polymerase, and a recombinase and a pegRNA. Without wishing to bound by theory, recombinases, such as serine integrases (e.g., Bxb1) are art recognized enzymes capable of performing site- specific recombination. Site-specific recombination is an art recognized process in which DNA strand exchange takes place between 2 DNA segments (e.g., 2 different double strand DNAs) possessing at least a certain degree of sequence homology. The enzymes recognize and bind to short specific DNA recognition sites (e.g., a first recognition site located on the first double stranded DNA and a second recognition site located on a second double stranded DNA), at which they cleave the DNA backbone, exchange the two DNA helices involved, and rejoin the DNA strands. In some embodiments, the first and second recognition sites comprise identical sequences. In other embodiments, the first and second recognition sites comprise different sequences (e.g. ,attP and attB of phage integrase).

[0165] In some embodiments, the method comprises a nucleic acid (e.g., a circular plasmid, ssRNA, ssDNA, or dsDNA)that encodes the suppressor qtRNA gene to be inserted into the target site. In some embodiments, the suppressor qtRNA gene comprises an expanded anticodon sequence that is complementary to any one of the expanded codon sequences listed in Tables 1 and 2.

[0166] In some embodiments, the nucleic acid (e.g., a circular plasmid, ssRNA, ssDNA, or dsDNA) comprises a first recombinase recognition site (e.g., AttP). In some embodiments, the first recombinase recognition site comprises an AttB sequence. In some embodiments, the first recombinase recognition site comprises an AttP. Any suitable AttP or AttB sequence known to the skilled artisan may be used such as those disclosed in Bland et al. “An att site-based recombination reporter system for genome engineering and synthetic DNA assembly” BMC Biotechnology.17, 62(2017), which is herein incorporated by reference in its entirety.

[0167] In some embodiments, a pegRNA comprises a spacer sequence, a gRNA core, and an extension arm comprising a DNA synthesis template and a primer binding site (PBS). 108 12954008Docket No.: B1195.70187WO00 Those of skill in the art will understand that the pegRNA guides the prime editor to the target site and encodes the edit to be installed into the human genome. In some embodiments, the DNA synthesis template encodes a single stranded DNA sequence encoding a second recombinase recognition site (e.g. a AttP or AttB). In some embodiments, the second recombinase recognition site comprises an AttB sequence. In some embodiments, the second recombinase recognition site comprises an AttP sequence. Any suitable AttP or AttB sequence known to the skilled artisan may be used such as those disclosed in Bland et al. “An att site-based recombination reporter system for genome engineering and synthetic DNA assembly” BMC Biotechnology.17, 62(2017).

[0168] In some embodiments, the method comprises placing the integrase in contact with the first recombination recognition site and second recombination recognition site. Upon being placed in contact, the integrase recombines the nucleic acid (e.g., a circular plasmid, ssRNA, ssDNA, or dsDNA) comprising the first recombination recognition site with the second recombination site that was previously inserted into the human genome at the target site (e.g., safe harbor locus) via prime editing. In certain embodiments, this permanently inserts the desired suppressor qtRNA gene into the human genome at the target site (e.g., ROSA26, CCR5, and AAVS1). In some embodiments, installation of the suppressor qtRNA gene at the target site (e.g., safe harbor locus site or general expression site) results in the indefinite expression of the suppressor qtRNAs gene.

[0169] Other aspects relate to methods for treating a disease caused by frameshift mutations, the method comprising mutating an endogenous tRNA gene into a suppressor qtRNA gene using prime editing, the method comprising administering to a subject (i) a prime editor and (ii) a pegRNA, wherein the suppressor qtRNA gene encodes a suppressor qtRNA molecule comprising an anticodon sequence comprising ochre stop codon, an opal stop codon, or an amber stop codon.

[0170] Additional aspects of the disclosure relate to methods for treating a disease caused by frameshift mutations. In some embodiments, the method comprises converting an endogenous tRNA gene into a suppressor qtRNA gene using prime editing (e.g., twinPE). In some embodiments, the method comprises administering to a subject (i) a prime editor and (ii) one or more pegRNAs, wherein the suppressor qtRNA gene encodes a suppressor qtRNA with an expanded anticodon for readthrough of frameshift mutations. 109 12954008Docket No.: B1195.70187WO00

[0171] In other embodiments, the methods comprise installing a suppressor qtRNA gene into a target site in a human genome using prime editing. In some embodiments, the methods comprise administering to a subject (i) a prime editor and (ii) one or more pegRNAs, wherein the suppressor qtRNA gene encodes a suppressor qtRNA capable of binding to an expanded codon.

[0172] In some embodiments, the disease is any disease caused by a frameshift mutation. Exemplary embodiments, include but are not limited to, cancer, Crohn’s disease, cystic fibrosis, HIV, Tay-Sachs disease, Smith-Magenis syndrome, hypertrophic cardiomyopathy, thalassemia, Huntington’s disease, MUC1 kidney disease, and Alzheimer's disease.

[0173] Other aspects of the disclosure relate to a prime editing guide RNA (pegRNA), for example, to guide a Cas9n to a target DNA sequence. In some embodiments, the pegRNA comprises a spacer sequence, a core sequence, and an extension arm sequence. The extension arm sequence, according to some embodiments, encodes a primer binding site sequence (PBS), an expanded anticodon sequence, and a flap sequence (e.g., 3′ or 5′ flap sequence).

[0174] In some embodiments, the spacer sequence comprises a region of complementarity to a target strand of a double stranded target tRNA gene sequence encoding a triple anticodon sequence. In other embodiments, the spacer sequence comprises a region of complementarity to a non-target strand of a double stranded target tRNA gene sequence encoding a triplet anticodon sequence.

[0175] The PBS, in some embodiments, comprises a region of complementarity to a non-target strand of the double-stranded target tRNA gene sequence, the non-target strand comprising a 3′ end generated from a nick site in the R-loop of the non-target strand. In other embodiments, the PBS comprises a region of complementarity to a target strand of the double-stranded target tRNA gene sequence, the target strand comprising a 5´end generated from a nick site in R-loop of the target strand.

[0176] Additionally, in some embodiments, the PBS is between 8 to 16 nucleotides downstream of, and adjacent to, an expanded anticodon sequence. In some embodiments, the PBS is located at greater than or equal to 8 nucleotides, greater than or equal to 9 nucleotides, greater than or equal to 10 nucleotides, greater than or equal to 11 nucleotides, greater than or equal to 12 nucleotides, greater than or equal to 13 nucleotides, greater than or equal to 14 nucleotides, greater than or equal to 15 nucleotides, or greater than or equal to 16 nucleotides 110 12954008Docket No.: B1195.70187WO00 downstream of, and adjacent to, an expanded anticodon sequence. In some embodiments, the PBS is located at less than or equal to 16 nucleotides, less than or equal to 15 nucleotides, less than or equal to 14 nucleotides, less than or equal to 13 nucleotides, less than or equal to 12 nucleotides, less than or equal to 11 nucleotides, less than or equal to 10 nucleotides, less than or equal to 9 nucleotides, or less than or equal to 8 nucleotides downstream of, and adjacent to, an expanded anticodon sequence.

[0177] The expanded anticodon sequence, according to some embodiments, comprises 4 nucleotides having a first nucleotide position, a second nucleotide position, a third nucleotide position, and a fourth nucleotide position. In some embodiments, the expanded anticodon sequence comprises an A, G, T, or C in the first nucleotide position, the second nucleotide positions, the third nucleotide position, and the fourth nucleotide position. This means that the expanded anticodon sequence may encode 44= 256 possible anticodon sequences.

[0178] In other embodiments, the expanded anticodon sequence comprises 5 nucleotides having a first nucleotide position, a second nucleotide position, a third nucleotide position, a fourth nucleotide position, and a fifth nucleotide position. In some embodiments, the expanded anticodon sequence comprises an A, G, T, or C in the first nucleotide position, the second nucleotide positions, the third nucleotide position, the fourth nucleotide position, and the fifth nucleotide position. This means that the expanded anticodon sequence may encode 45= 1024 possible anticodon sequences.

[0179] In some embodiments, the pegRNA further comprises a 3′-flap or 5′ flap sequence. The 3′-flap sequence is complementary to at least a portion of the target strand, according to some embodiments. The 5′-flap sequence is complementary to at least a portion of the non-target strand. Additionally, in some embodiments, the 3′-flap or 5′-flap sequence is between 3 and 30 nucleotides upstream of, and adjacent to, the expanded anticodon sequence. In some embodiments, the 3′-flap or 5′-flap sequence is greater than or equal to 3 nucleotides, greater than or equal to 4 nucleotides, greater than or equal to 5 nucleotides, greater than or equal to 6 nucleotides, greater than or equal to 7 nucleotides, greater than or equal to 8 nucleotides, greater than or equal to 9 nucleotides, greater than or equal to 10 nucleotides, greater than or equal to 11 nucleotides, greater than or equal to 12 nucleotides, greater than or equal to 13 nucleotides, greater than or equal to 14 nucleotides, greater than or equal to 15 nucleotides, greater than or equal to 16 nucleotides, greater than or equal to 17 111 12954008Docket No.: B1195.70187WO00 nucleotides, greater than or equal to 18 nucleotides, greater than or equal to 19 nucleotides, greater than or equal to 20 nucleotides, greater than or equal to 21 nucleotides, greater than or equal to 22 nucleotides, greater than or equal to 23 nucleotides, greater than or equal to 24 nucleotides, greater than or equal to 25 nucleotides, greater than or equal to 26 nucleotides, greater than or equal to 27 nucleotides, greater than or equal to 28 nucleotides, greater than or equal to 29 nucleotides, or greater than or equal to 30 nucleotides upstream of, and adjacent to, the expanded anticodon sequence. In some embodiments, the 3′-flap or 5′-flap sequence is less than or equal to 30 nucleotides, less than or equal to 29 nucleotides, less than or equal to 28 nucleotides, less than or equal to 27 nucleotides, less than or equal to 26 nucleotides, less than or equal to 25 nucleotides, less than or equal to 24 nucleotides, less than or equal to 23 nucleotides, less than or equal to 22 nucleotides, less than or equal to 21 nucleotides, less than or equal to 20 nucleotides, less than or equal to 19 nucleotides, less than or equal to 18 nucleotides, less than or equal to 17 nucleotides, less than or equal to 16 nucleotides, less than or equal to 15 nucleotides, less than or equal to 14 nucleotides, less than or equal to 13 nucleotides, less than or equal to 12 nucleotides, less than or equal to 11 nucleotides, less than or equal to 10 nucleotides, less than or equal to 9 nucleotides, less than or equal to 8 nucleotides, less than or equal to 7 nucleotides, less than or equal to 6 nucleotides, less than or equal to 5 nucleotides, less than or equal to 4 nucleotides, or less than or equal to 3 nucleotides upstream of, and adjacent to, the expanded anticodon sequence. Prime editors

[0180] The present disclosure contemplates using prime editors comprising fusion proteins, wherein the fusion proteins comprise a nucleic acid programmable DNA binding protein (napDNAbp) domain and a polymerase (e.g., reverse transcriptase) domain. Any suitable napDNAbp and polymerase known in the art may be combined into a single fusion protein with any suitable structural configuration, in accordance with some embodiments. For example, the fusion protein may comprise, from the N-terminus to the C-terminus direction, a napDNAbp fused to a polymerase. In other embodiments, the fusion protein may comprise from the N-terminus to the C-terminus direction, a polymerase fused to a napDNAbp. The fused domain may optionally be joined by a linker, e.g., an amino acid sequence. In other embodiments, the fusion proteins may comprise the structure NH2- [napDNAbp]-[ polymerase]-COOH; or NH2-[polymerase]-[napDNAbp]-COOH, wherein each instance of “]-[“ indicates the presence of an optional linker sequence. In embodiments 112 12954008Docket No.: B1195.70187WO00 wherein the polymerase is a reverse transcriptase, the fusion proteins may comprise the structure NH2-[napDNAbp]-[RT]-COOH; or NH2-[RT]-[napDNAbp]-COOH, wherein each instance of indicates the presence of an optional linker sequence.

[0181] Since prime editors, and hence napDNAbps and polymerases, are well-known in the art, and the amino acid sequences are readily available, this disclosure is not meant in any way to be limited to those specific napDNAbps and / or polymerases identified herein. Non-limiting examples of prime editors contemplated herein may be found in U.S. Provisional Application No.62 / 820,813, U.S. Provisional Application No.62 / 858,958, U.S. Provisional Application No.62 / 889,996, U.S. Provisional Application No.62 / 922,654, U.S. Provisional Application No.62 / 913,553, U.S. Provisional Application No.62 / 973,558, U.S. Provisional Application No.62 / 931,195, U.S. Provisional Application No.62 / 944,231, U.S. Provisional Application No.62 / 974,537, U.S. Provisional Application No.62 / 991,069, U.S. Provisional Application No.63 / 100,548, U.S. Provisional Application No.63 / 022,397, U.S. Provisional Application No.63 / 116,785, International PCT Application No. PCT / US2020 / 023721, International PCT Application No. PCT / US2020 / 023553, International PCT Application No. PCT / US2020 / 023583, International PCT Application No. PCT / US2020 / 023730, International PCT Application No. PCT / US2020 / 023713, International PCT Application No. PCT / US2020 / 023712, International PCT Application No. PCT / US2020 / 023727, International PCT Application No. PCT / US2020 / 023724, International PCT Application No. PCT / US2020 / 023725, International PCT Application No. PCT / US2020 / 023728, International PCT Application No. PCT / US2020 / 023732, International PCT Application No. PCT / US2020 / 023723, and International PCT Application No. PCT / US2021 / 031439, all of which are herein incorporated by reference in their entirety.

[0182] In some embodiments, the napDNAbp domain and the polymerase domain are fused together without a linker. In other embodiments, the napDNAbp domain is fused to the polymerase domain via a linker. Any suitable linker known in the art may be used to fuse the napDNAbp domain and the polymerase domain. For example, in some embodiments, the linker is a peptide, a polypeptide, a protein, a nucleic acid, a polymer, a polysaccharide, or any combination thereof.

[0183] In some embodiments, the fusion proteins may comprise any suitable structural configuration. For example, the fusion protein may comprise from the N-terminus to the C-terminus direction, a napDNAbp fused to a polymerase (e.g., DNA-dependent DNA 113 12954008Docket No.: B1195.70187WO00 polymerase or RNA-dependent DNA polymerase, such as, reverse transcriptase). In other embodiments, the fusion protein may comprise from the N-terminus to the C-terminus direction, a polymerase (e.g., a reverse transcriptase) fused to a napDNAbp. The fused domain may optionally be joined by a linker, e.g., an amino acid sequence. In other embodiments, the fusion proteins may comprise the structure NH2-[napDNAbp]-[ polymerase]-COOH; or NH2-[polymerase]-[napDNAbp]-COOH, wherein each instance of “]-[“ indicates the presence of an optional linker sequence. In embodiments wherein the polymerase is a reverse transcriptase, the fusion proteins may comprise the structure NH2- [napDNAbp]-[RT]-COOH; or NH2-[RT]-[napDNAbp]-COOH, wherein each instance of “]-[“ indicates the presence of an optional linker sequence.

[0184] In various embodiments, the prime editor fusion protein may have the following structure (referred to herein as “PE1”), which includes a Cas9 variant comprising an H840A mutation (i.e., a Cas9 nickase) and an M-MLV RT wild type, as well as an N- terminal NLS sequence (19 amino acids) and an amino acid linker (32 amino acids) that joins the C-terminus of the Cas9 nickase domain to the N-terminus of the RT domain. The PE1 fusion protein has the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(wt)].

[0185] In some embodiments, the prime editor fusion protein (referred to herein as “PE2”) comprises a Cas9(H840A) and a variant MMLV RT having the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] and a desired pegRNA.

[0186] In one set of embodiments, a prime editor of the present disclosure may be a PE1, PE2, PE3 or TwinPE editor as described in Anzalone et al., “Search-and-replace genome editing without double-strand breaks or donor DNA,” Nature.2019 Dec; 576(7785): 149- 157; Anzalone et al., “Programmable deletion, replacement, integration, and inversion of large DNA sequences with twin prime editing,” Nat. Biotechnol.2022 May; 40(5):731-740; and by Choi et al., “Precise genomic deletions using paired prime editing,” Nat. Biotechnol. 2022 Feb; 40(2):218-226, all of which are herein incorporated by reference in their entirety. In some embodiments, TwinPE comprises a pair of PE2 editors and two pegRNAs that target opposite strands of a double stranded nucleic acid (e.g., DNA).

[0187] In some embodiments, a PE3 prime editor comprises PE2 machinery and an additional sgRNA. 114 12954008Docket No.: B1195.70187WO00

[0188] In some embodiments, a TwinPE editor comprises a first prime editor complex and a second prime editor complex. In some embodiments, the first prime editor complex comprises a first prime editor comprising a first nucleic acid programmable DNA binding protein (first napDNAbp) and a first polypeptide comprising an RNA-dependent DNA polymerase activity. The first prime editor complex further comprises a first prime editing guide RNA (first pegRNA) that binds to a first binding site on a first strand of the double- stranded DNA sequence upstream of the target site to be edited. In some embodiments, the first prime editor complex is a first PE2 editor. In some embodiments, the second prime editor complex comprises a second prime editor comprising a second nucleic acid programmable DNA binding protein (second napDNAbp) and a second polypeptide comprising an RNA-dependent DNA polymerase activity. The second prime editor complex further comprises a second prime editing guide RNA (second pegRNA) that binds to a second binding site on a second strand of the double-stranded DNA sequence upstream of the target site to be edited. In some embodiments, the second prime editor complex is a second PE2 editor. In some embodiments, the first pegRNA comprises a first DNA synthesis template encoding a first single-stranded DNA sequence and the second pegRNA comprises a second DNA synthesis template encoding a second single-stranded DNA sequence. In some embodiments, the first and the second single-stranded DNA sequence each comprise a region of complementarity to the other. In some embodiments, the first single-stranded DNA sequence and the second single-stranded DNA sequence form a duplex comprising an edited portion as compared to the DNA sequence at the target site to be edited. napDNAbp domain

[0189] In some embodiments, a prime editor comprises a (napDNAbp) domain. Any suitable napDNAbp domain known in the art may be used in the prime editors described herein, such as those described in detail in United State Patent Application 63 / 136,194, titled “Prime editor variants, constructs, and methods of using the same” by David Liu, et al., filed on January 11, 2021 and by Nidhi et al in “Novel CRISPR-Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives”, Int. J. Mol. Sci.2021 Apr; 22(7): 3327, both of which are incorporated herein by reference in their entirety. For example, in various embodiments, the napDNAbp may be any suitable Cas protein known in the art, including by not limited to Cas 1, Cas 2, Cas 3, Cas 4, Cas 5, Cas 6, Cas 7, Cas 8, Cas 9, Cas 10, Cas 11, Cas 12, Cas 12a (e.g., Cpf1), Cas 12b, Cas 12b2, Cas 115 12954008Docket No.: B1195.70187WO00 13a, Cas 13b, Cas 13d, Cas 14a, Cas X (e.g, Cas 12e), Cas Y (e.g., Cas12d), C2c1, C2c3. C2c4. C2c8, C2c5, C2c10, C2c9, C2c2, C2c7, and C2c6. In some embodiments, the Cas may be any class 1, type I; class 1 type III; class 1, type IV; class 2, type II; class 2, type V; or class 2, type VI. In certain embodiments, the Cas is a Class 2 CRISPR-Cas system, including any type II, type V, or type VI CRISPR-Cas enzyme.

[0190] Given the rapid development of CRISPR-Cas as a tool for genome editing, there have been constant developments in the nomenclature used to describe and / or identify CRISPR-Cas enzymes, such as Cas9 and Cas9 orthologs. This application references CRISPR-Cas enzymes with nomenclature that may be old and / or new as described in United State Patent Application 63 / 136,194 (described elsewhere herein) or Makarova et al., The CRISPR Journal, Vol.1, No.5, 2018, which is incorporated herein by reference in its entirety.

[0191] Other napDNAbps are also possible in other embodiments. For example, in some embodiments, the napDNAbp comprises the canonical SpCas9, or any ortholog Cas9 protein, or any variant Cas9 protein —including any naturally occurring variant, mutant, or otherwise engineered version of Cas9 — that is known or that may be made or evolved through a directed evolutionary or otherwise mutagenic process. In various embodiments, the Cas9 or Cas9 variants have a nickase activity, i.e., only cleave one strand of the target DNA sequence. In other embodiments, the Cas9 or Cas9 variants have inactive nucleases, i.e., are “dead” Cas9 proteins. Other variant Cas9 proteins that may be used are those having a smaller molecular weight than the canonical SpCas9 (e.g., for easier delivery) or having modified or rearranged primary amino acid structure (e.g., the circular permutant formats). In other embodiments, still, the Cas9 or Cas9 variants are high-fidelity variants that have weakened DNA phosphate backbone interaction with the nuclease, resulting in genome-wide specificity and undetectable off-target effects.

[0192] In some embodiments, the Cas9 protein can be a wild type Cas9 ortholog from another bacterial species different from the canonical Cas9 from S. pyogenes. For example, the following Cas9 orthologs can be used in connection with the prime editor constructs described in this specification. In addition, any variant Cas9 orthologs having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any of the below orthologs may also be used with the present prime editors. 116 12954008Docket No.: B1195.70187WO00

[0193] Exemplary embodiments include, but are not limited to, LfCas9 (Lactobacillus fermentum wild type, GenBank: SNX31424.11), SaCas9 (Staphylococcus aureus wild type, GenBank: AYD60528.1), SaCas9 (Staphylococcus aureus), StCas9 (Streptococcus thermophilus, UniProtKB / Swiss-Prot: G3ECR1.2 Wild type), LcCas9 (Lactobacillus crispatus, NCBI Reference Sequence: WP_133478044.1, Wild type), PdCas9 (Pedicoccus damnosus, NCBI Reference Sequence: WP_062913273.1, Wild type), FnCas9 (Fusobaterium nucleatum, NCBI Reference Sequence: WP_060798984.1), EcCas9 (Enterococcus cecorum, NCBI Reference Sequence: WP_047338501.1, Wild type), AhCas9 (Anaerostipes hadrus, NCBI Reference Sequence: WP_044924278.1, Wild type), KvCas9 (Kandleria vitulina, NCBI Reference Sequence: WP_031589969.1, Wild type), EfCas9 (Enterococcus faecalis, NCBI Reference Sequence: WP_016631044.1, Wild type), Staphylococcus aureus Cas9, Geobacillus thermodenitrificans Cas9, and ScCas9 (S. canis, 1375 AA, 159.2 kDa), Streptococcus pyogenes Cas9 (SpCas9), SaCas-HF (e.g., a high-fidelity variant of SaCas9 with genome wide activity in human cells), KKHSaCas9 (e.g., which recognizes a 5’- NNGRRT-3’ PAM), microABE1744 (e.g., an engineered SaCas9 variant with significantly improved on-target editing), Streptococcus thermophilus Cas9 (StCas9), Neisseria meningitidis Cas9 (NmCas9), Francisella novicida Cas9 (FnCas9), Campylobacter jejuni (CjCas9), Streptococcus canis Cas9 (ScCas9), Staphylococcus auricularis Cas9 (SauriCas9),

[0194] In certain embodiments, the prime editors described herein may include a dead Cas9, e.g., dead SpCas9, which has no or reduced nuclease activity due to one or more mutations (e.g., D10A and H840A) that inactive both nuclease domains of Cas9, namely the RuvC domain (which cleaves the non-protospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand).

[0195] In some embodiments, the prime editors described herein comprise a Cas9 nickase. The term “Cas9 nickase” of “nCas9” refers to a variant of Cas9 which is capable of introducing a single-strand break in a double strand DNA molecule target. In some embodiments, the Cas9 nickase comprises only a single functioning nuclease domain.

[0196] In various embodiments described herein, the prime editors comprising a napDNAbp, such as a Cas9 protein, are “programmable” by way of their becoming complexed with a guide RNA (or a pegRNA, as the case may be), which guides the Cas9 protein to a target site on the DNA which possess a sequence that is complementary to the spacer portion of the gRNA (or pegRNA) and also which possesses the required PAM 117 12954008Docket No.: B1195.70187WO00 sequence. However, in certain embodiment envisioned here, the napDNAbp may be substituted with a different type of programmable protein, such as a zinc finger nuclease or a transcription activator-like effector nuclease (TALEN). See U.S. Ser. No.12 / 965,590; U.S. Ser. No.13 / 426,991 (U.S. Pat. No.8,450,471); U.S. Ser. No.13 / 427,040 (U.S. Pat. No. 8,440,431); U.S. Ser. No.13 / 427,137 (U.S. Pat. No.8,440,432); and U.S. Ser. No. 13 / 738,381, all of which are incorporated by reference herein in their entirety. In addition, TALENS are described in WO 2015 / 027134, US 9,181,535, Boch et al., "Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors", Science, vol.326, pp.1509-1512 (2009), Bogdanove et al., TAL Effectors: Customizable Proteins for DNA Targeting, Science, vol.333, pp.1843-1846 (2011), Cade et al., "Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs", Nucleic Acids Research, vol.40, pp.8001-8010 (2012), and Cermak et al., "Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting", Nucleic Acids Research, vol.39, No.17, e82 (2011), each of which are incorporated herein by reference. See also, for example, in Carroll et al., “Genome Engineering with Zinc-Finger Nucleases,” Genetics, Aug 2011, Vol.188: 773-782; Durai et al., “Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells,” Nucleic Acids Res, 2005, Vol.33: 5978-90; and Gaj et al., “ZFN, TALEN, and CRISPR / Cas-based methods for genome engineering,” Trends Biotechnol.2013, Vol.31: 397-405, each of which are incorporated herein by reference in their entireties. Polymerase domain

[0197] In some embodiments, the prime editors disclosed herein comprise a polymerase domain or a variant thereof (e.g., DNA-dependent DNA polymerase or RNA- dependent DNA polymerase, such as, reverse transcriptase). In some cases, the polymerase, or variant thereof, may be provided as a fusion protein with a napDNAbp or other programmable nuclease, or provided in trans.

[0198] Any polymerase known in the art may be used in the prime editors with the methods and compositions disclosed herein. The polymerases may be wild type polymerases, functional fragments, mutants, variants, or truncated variants, and the like. The polymerases may include wild type polymerases from eukaryotic, prokaryotic, archael, or viral organisms, and / or the polymerases may be modified by genetic engineering, mutagenesis, or directed evolution-based processes. The polymerases may include T7 DNA polymerase, T5 DNA 118 12954008Docket No.: B1195.70187WO00 polymerase, T4 DNA polymerase, Klenow fragment DNA polymerase, DNA polymerase III and the like. The polymerases may also be thermostable, and may include Taq, Tne, Tma, Pfu, Tfl, Tth, Stoffel fragment, VENT® and DEEPVENT® DNA polymerases, KOD, Tgo, JDF3, and mutants, variants and derivatives thereof (see U.S. Pat. No.5,436,149; U.S. Pat. No.4,889,818; U.S. Pat. No.4,965,185; U.S. Pat. No.5,079,352; U.S. Pat. No.5,614,365; U.S. Pat. No.5,374,553; U.S. Pat. No.5,270,179; U.S. Pat. No.5,047,342; U.S. Pat. No. 5,512,462; WO 92 / 06188; WO 92 / 06200; WO 96 / 10640; Barnes, W. M., Gene 112:29-35 (1992); Lawyer, F. C., et al., PCR Meth. Appl.2:275-287 (1993); Flaman, J.-M, et al., Nuc. Acids Res.22(15):3259-3260 (1994), each of which are incorporated by reference).

[0199] In some embodiments, the polymerases used in the methods and compositions disclosed herein are “template-dependent” polymerase (since the polymerases are intended to rely on the DNA synthesis template to specify the sequence of the DNA strand under synthesis during prime editing. As used herein, the term “template DNA molecule” refers to that strand of a nucleic acid from which a complementary nucleic acid strand is synthesized by a DNA polymerase, for example, in a primer extension reaction of the DNA synthesis template of a PegRNA.

[0200] The disclosure contemplates any wild type polymerase obtained from any naturally-occurring organism or virus, or obtained from a commercial or non-commercial source. In addition, the polymerases usable in the prime editors can include any naturally- occurring mutant polymerase, engineered mutant polymerase, or other variant polymerase, including truncated variants that retain function. The polymerases usable herein may also be engineered to contain specific amino acid substitutions, such as those specifically disclosed herein. In certain preferred embodiments, the polymerases usable in the prime editors utilized in the methods and compositions of the present disclosure are template-based polymerases, i.e., they synthesize nucleotide sequences in a template-dependent manner.

[0201] In some embodiments, the polymerase is a DNA polymerase (e.g., a “DNA- dependent DNA polymerase” whereby the template molecule is a strand of DNA). In some embodiments, the polymerase is an RNA polymerase. In various other embodiments, the DNA polymerase can be an “RNA-dependent DNA polymerase” (i.e., whereby the template molecule is a strand of RNA). The term “polymerase” may also refer to an enzyme that catalyzes the polymerization of nucleotide (i.e., the polymerase activity). Generally, the enzyme will initiate synthesis at the 3'-end of a primer annealed to a polynucleotide template 119 12954008Docket No.: B1195.70187WO00 sequence (e.g., such as a primer sequence annealed to the primer binding site of a PegRNA), and will proceed toward the 5´ end of the template strand.

[0202] In some embodiments, the DNA polymerase is a “functional fragment thereof”. A “functional fragment thereof” refers to any portion of a wild-type or mutant DNA polymerase that encompasses less than the entire amino acid sequence of the polymerase and which retains the ability, under at least one set of conditions, to catalyze the polymerization of a polynucleotide. Such a functional fragment may exist as a separate entity, or it may be a constituent of a larger polypeptide, such as a fusion protein.

[0203] In some embodiments, the polymerase is a reverse transcriptase (RT). RTs are art recognized enzymes with RNA- and DNA-dependent DNA polymerization activity, and an RNaseH activity that catalyzes the cleavage of RNA in RNA-DNA hybrids. In some embodiments, the RT is mutated to disable the RNaseH domain (e.g., to prevent unintended damage to the mRNA). In other embodiments, still, the RNaseH domain is truncated.

[0204] Any of the wild type, variant, and / or mutant forms of reverse transcriptases known in the art or which can be made using methods known in the art may be used in the compositions and methods as disclosed herein. For example, in some embodiments, the RT is a wild type RT. Non-limiting examples of RTs include Moloney Murine Leukemia Virus (M-MLV); Human Immunodeficiency Virus (HIV) reverse transcriptase and avian Sarcoma- Leukosis Virus (ASLV) reverse transcriptase, which includes but is not limited to Rous Sarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV reverse transcriptase, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV reverse transcriptase, Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A reverse transcriptase, Avian Sarcoma Virus UR2 Helper Virus UR2AV reverse transcriptase, Avian Sarcoma Virus Y73 Helper Virus YAV reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase, and Myeloblastosis Associated Virus (MAV). pegRNAs

[0205] In some embodiments, the compositions and methods for prime editing contemplated herein comprise at least one pegRNA. Any suitable pegRNA architecture known in the art may be used in any one of the compositions and methods for prime editing disclosed herein, such as those described in U.S. Provisional Application U.S.S.N. 63 / 255,897, U.S. Provisional Application U.S.S.N.63 / 231,230, U.S. Provisional Application 120 12954008Docket No.: B1195.70187WO00 U.S.S.N.63 / 194,913, U.S. Provisional Application U.S.S.N.63 / 194,865, U.S. Provisional Application U.S.S.N.63 / 176,180, U.S. Provisional Application U.S.S.N.63 / 176,202, and U.S. Provisional Application U.S.S.N.63 / 136,194, U.S. Provisional Application No. 63 / 022,397, U.S. Provisional Application No.63 / 116,785, International Patent Application No. PCT / US2021 / 031439, and International Patent Application No. PCT / US2022 / 012054, the entire contents each of which is incorporated herein by reference in their entireties.

[0206] In some embodiments, the pegRNA comprises a spacer sequence, gRNA core, a DNA synthesis template, and a primer binding site. As used herein, the term “spacer sequence” in connection with a guide RNA or a pegRNA refers to the portion of the guide RNA or pegRNA of about 20 nucleotides which contains a nucleotide sequence that shares the same sequence as the protospacer sequence in the target DNA sequence. The spacer sequence anneals to the complement of the protospacer sequence to form a ssRNA / ssDNA hybrid structure at the target site and a corresponding R loop ssDNA structure of the endogenous DNA strand.

[0207] In some embodiments, the pegRNA comprises a gRNA core.

[0208] In some embodiments, an extended guide RNA usable in the prime editing system utilized in the methods and compositions disclosed herein whereby a traditional guide RNA includes a ~20 nt protospacer sequence and a gRNA core region, which binds with the napDNAbp. In this embodiment, the guide RNA includes an extended RNA segment at the 5´ end, i.e., a 5´ extension. In this embodiment, the 5´extension includes a reverse transcription template sequence, a reverse transcription primer binding site, and an optional 5-20 nucleotide linker sequence. The RT primer binding site hybrizes to the free 3ʹ end that is formed after a nick is formed in the non-target strand of the R-loop, thereby priming reverse transcriptase for DNA polymerization in the 5´-3´ direction.

[0209] In another embodiment, an extended guide RNA usable in the prime editing system utilized in the methods and compositions disclosed herein whereby a traditional guide RNA includes a ~20 nt protospacer sequence and a gRNA core, which binds with the napDNAbp. In this embodiment, the guide RNA includes an extended RNA segment at the 3´ end, i.e., a 3´ extension. In this embodiment, the 3´extension includes a reverse transcription template sequence, and a reverse transcription primer binding site. The RT primer binding site hybrizes to the free 3´ end that is formed after a nick is formed in the non- 121 12954008Docket No.: B1195.70187WO00 target strand of the R-loop, thereby priming reverse transcriptase for DNA polymerization in the 5´-3´ direction.

[0210] In another embodiment, an extend guide RNA usable in the prime editing system utilized in the methods and compositions disclosed herein whereby a traditional guide RNA includes a ~20 nt protospacer sequence and a gRNA core, which binds with the napDNAbp. In this embodiment, the guide RNA includes an extended RNA segment at an intermolecular position within the gRNA core, i.e., an intramolecular extension. In this embodiment, the intramolecular extension includes a reverse transcription template sequence, and a reverse transcription primer binding site. The RT primer binding site hybrizes to the free 3´ end that is formed after a nick is formed in the non-target strand of the R-loop, thereby priming reverse transcriptase for DNA polymerization in the 5´-3´ direction.

[0211] In one embodiment, the position of the intermolecular RNA extension is not in the protospacer sequence of the guide RNA. In another embodiment, the position of the intermolecular RNA extension in the gRNA core. In still another embodiment, the position of the intermolecular RNA extension is any with the guide RNA molecule except within the protospacer sequence, or at a position which disrupts the protospacer sequence.

[0212] In one embodiment, the intermolecular RNA extension is inserted downstream from the 3´ end of the protospacer sequence. In another embodiment, the intermolecular RNA extension is inserted at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides downstream of the 3´ end of the protospacer sequence.

[0213] In other embodiments, the intermolecular RNA extension is inserted into the gRNA, which refers to the portion of the guide RNA corresponding or comprising the tracrRNA, which binds and / or interacts with the Cas9 protein or equivalent thereof (i.e, a different napDNAbp). Preferably the insertion of the intermolecular RNA extension does not disrupt or minimally disrupts the interaction betweeen the tracrRNA portion and the napDNAbp. 122 12954008Docket No.: B1195.70187WO00

[0214] The length of the RNA extension (which includes at least the RT template and primer binding site, e.g., see FIG.3) can be any useful length. In various embodiments, the RNA extension is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

[0215] The RT template sequence can also be any suitable length. For example, the RT template sequence can be at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

[0216] In still other embodiments, wherein the reverse transcription primer binding site sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

[0217] In other embodiments, the optional linker or spacer sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 123 12954008Docket No.: B1195.70187WO00 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

[0218] The RT template sequence, in certain embodiments, encodes a single-stranded DNA molecule which is homologous to the non-target strand (and thus, complementary to the corresponding site of the target strand) but includes one or more nucleotide changes. The least one nucleotide change may include one or more single-base nucleotide changes, one or more deletions, and one or more insertions.

[0219] The synthesized single-stranded DNA product of the RT template sequence is homologous to the non-target strand and contains one or more nucleotide changes. The single-stranded DNA product of the RT template sequence hybridizes in equilibrium with the complementary target strand sequence, thereby displacing the homologous endogenous target strand sequence. The displaced endogenous strand may be referred to in some embodiments as a 5´ endogenous DNA flap species. This 5´ endogenous DNA flap species can be removed by a 5´ flap endonuclease (e.g., FEN1) and the single-stranded DNA product, now hybridized to the endogenous target strand, may be ligated, thereby creating a mismatch between the endogenous sequence and the newly synthesized strand. The mismatch may be resolved by the cell’s innate DNA repair and / or replication processes.

[0220] In various embodiments, the nucleotide sequence of the RT template sequence corresponds to the nucleotide sequence of the non-target strand which becomes displaced as the 5´ flap species and which overlaps with the site to be edited.

[0221] In various embodiments of the extended guide RNAs, the reverse transcription template sequence may encode a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to a nick site, wherein the single-strand DNA flap comprises a desired nucleotide change. The single-stranded DNA flap may displace an endogenous single-strand DNA at the nick site. The displaced endogenous single-strand DNA at the nick site can have a 5´ end and form an endogenous flap, which can be excised by the cell. In various embodiments, excision of the 5´ end endogenous flap can help drive 124 12954008Docket No.: B1195.70187WO00 product formation since removing the 5´ end endogenous flap encourages hybridization of the single-strand 3´ DNA flap to the corresponding complementary DNA strand, and the incorporation or assimilation of the desired nucleotide change carried by the single-strand 3´ DNA flap into the target DNA.

[0222] In various embodiments of the extended guide RNAs, the cellular repair of the single-strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.

[0223] In still other embodiments, the desired nucleotide change is installed in an editing window that is between about -5 to +5 of the nick site, or between about -10 to +10 of the nick site, or between about -20 to +20 of the nick site, or between about -30 to +30 of the nick site, or between about -40 to + 40 of the nick site, or between about -50 to +50 of the nick site, or between about -60 to +60 of the nick site, or between about -70 to +70 of the nick site, or between about -80 to +80 of the nick site, or between about -90 to +90 of the nick site, or between about -100 to +100 of the nick site, or between about -200 to +200 of the nick site.

[0224] In other embodiments, the desired nucleotide change is installed in an editing window that is between about +1 to +2 from the nick site, or about +1 to +3, +1 to +4, +1 to +5, +1 to +6, +1 to +7, +1 to +8, +1 to +9, +1 to +10, +1 to +11, +1 to +12, +1 to +13, +1 to +14, +1 to +15, +1 to +16, +1 to +17, +1 to +18, +1 to +19, +1 to +20, +1 to +21, +1 to +22, +1 to +23, +1 to +24, +1 to +25, +1 to +26, +1 to +27, +1 to +28, +1 to +29, +1 to +30, +1 to +31, +1 to +32, +1 to +33, +1 to +34, +1 to +35, +1 to +36, +1 to +37, +1 to +38, +1 to +39, +1 to +40, +1 to +41, +1 to +42, +1 to +43, +1 to +44, +1 to +45, +1 to +46, +1 to +47, +1 to +48, +1 to +49, +1 to +50, +1 to +51, +1 to +52, +1 to +53, +1 to +54, +1 to +55, +1 to +56, +1 to +57, +1 to +58, +1 to +59, +1 to +60, +1 to +61, +1 to +62, +1 to +63, +1 to +64, +1 to +65, +1 to +66, +1 to +67, +1 to +68, +1 to +69, +1 to +70, +1 to +71, +1 to +72, +1 to +73, +1 to +74, +1 to +75, +1 to +76, +1 to +77, +1 to +78, +1 to +79, +1 to +80, +1 to +81, +1 to +82, +1 to +83, +1 to +84, +1 to +85, +1 to +86, +1 to +87, +1 to +88, +1 to +89, +1 to +90, +1 to +90, +1 to +91, +1 to +92, +1 to +93, +1 to +94, +1 to +95, +1 to +96, +1 to +97, +1 to +98, +1 to +99, +1 to +100, +1 to +101, +1 to +102, +1 to +103, +1 to +104, +1 to +105, +1 to +106, +1 to +107, +1 to +108, +1 to +109, +1 to +110, +1 to +111, +1 to +112, +1 to +113, +1 to +114, +1 to +115, +1 to +116, +1 to +117, +1 to +118, +1 to +119, +1 to +120, +1 to +121, +1 to +122, +1 to +123, +1 to +124, or +1 to +125 from the nick site. 125 12954008Docket No.: B1195.70187WO00

[0225] In still other embodiments, the desired nucleotide change is installed in an editing window that is between about +1 to +2 from the nick site, or about +1 to +5, +1 to +10, +1 to +15, +1 to +20, +1 to +25, +1 to +30, +1 to +35, +1 to +40, +1 to +45, +1 to +50, +1 to +55, +1 to +100, +1 to +105, +1 to +110, +1 to +115, +1 to +120, +1 to +125, +1 to +130, +1 to +135, +1 to +140, +1 to +145, +1 to +150, +1 to +155, +1 to +160, +1 to +165, +1 to +170, +1 to +175, +1 to +180, +1 to +185, +1 to +190, +1 to +195, or +1 to +200, from the nick site.

[0226] In various aspects, the extended guide RNAs are modified versions of a guide RNA. Guide RNAs maybe naturally occurring, expressed from an encoding nucleic acid, or synthesized chemically. Methods are well known in the art for obtaining or otherwise synthesizing guide RNAs and for determining the appropriate sequence of the guide RNA, including the protospacer sequence which interacts and hybridizes with the target strand of a genomic target site of interest.

[0227] In various embodiments, the particular design aspects of a guide RNA sequence will depend upon the nucleotide sequence of a genomic target site of interest (i.e., the desired site to be edited) and the type of napDNAbp (e.g., Cas9 protein) present in the prime editing systems utilized in the methods and compositions described herein, among other factors, such as PAM sequence locations, percent G / C content in the target sequence, the degree of microhomology regions, secondary structures, etc.

[0228] In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a napDNAbp (e.g., a Cas9, Cas9 homolog, or Cas9 variant) to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 126 12954008Docket No.: B1195.70187WO00 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.

[0229] In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a prime editor to a target sequence may be assessed by any suitable assay. For example, the components of a prime editor, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of a prime editor disclosed herein, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a prime editor, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art.

[0230] A guide sequence may be selected to target any target sequence. In some embodiments, the target sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome. For example, for the S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG where NNNNNNNNNNNNXGG (N is A, G, T, or C; and X can be anything). A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG where NNNNNNNNNNNXGG (N is A, G, T, or C; and X can be anything). For the S. thermophilus CRISPR1Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW where NNNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T). A unique target sequence in a genome may include an S. thermophilus CRISPR 1 Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXXAGAAW where NNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T). For the S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGGXG where NNNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything). A unique target 127 12954008Docket No.: B1195.70187WO00 sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGGXG where NNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything). In each of these sequences “M” may be A, G, T, or C, and need not be considered in identifying a sequence as unique.

[0231] In some embodiments, a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res.9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62). Further algorithms may be found in U.S. Provisional Application, U.S. Ser. No.61 / 836,080, entitled, “Methods, systems, and apparatus for identifying target sequences for Cas enzymes or CRISPR-Cas systems for target sequences and conveying results thereof,” filed June 17, 2013 ); incorporated herein by reference.

[0232] In general, a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a complex at a target sequence, wherein the complex comprises the tracr mate sequence hybridized to the tracr sequence. In general, degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the tracr sequence or tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary 128 12954008Docket No.: B1195.70187WO00 structure, such as a hairpin. Preferred loop forming sequences for use in hairpin structures are four nucleotides in length, and most preferably have the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternative sequences. The sequences preferably include a nucleotide triplet (for example, AAA), and an additional nucleotide (for example C or G). Examples of loop forming sequences include CAAA and AAAG. In an embodiment of the invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In preferred embodiments, the transcript has two, three, four or five hairpins. In a further embodiment of the invention, the transcript has at most five hairpins. In some embodiments, the single transcript further includes a transcription termination sequence; preferably this is a polyT sequence, for example six T nucleotides. Further non-limiting examples of single polynucleotides comprising a guide sequence, a tracr mate sequence, and a tracr sequence are as follows (listed 5′ to 3′), where “N” represents a base of a guide sequence, the first block of lower case letters represent the tracr mate sequence, and the second block of lower case letters represent the tracr sequence, and the final poly-T sequence represents the transcription terminator: (1)NNNNNNNNGTTTTTGTACTCTCAAGATTTAGAAATAAATCTTGCAGAAGCTACA AAGATAAGGCTTCATGCCGAAATCAACACCCTGTCATTTTATGGCAGGGTGTTTTC GTTATTTAATTTTTT (SEQ ID NO: 2307420);

[0233] (2)NNNNNNNNNNNNNNNNNNGTTTTTGTACTCTCAGAAATGCAGAA GCTACAAAGATAAGGCTTCATGCCGAAATCAACACCCTGTCATTTTATGGCAGGGT GTTTTCGTTATTTAATTTTTT (SEQ ID NO: 2307421);

[0234] (3)NNNNNNNNNNNNNNNNNNNNGTTTTTGTACTCTCAGAAATGCAG AAGCTACAAAGATAAGGCTTCATGCCGAAATCAACACCCTGTCATTTTATGGCAGG GTGTTTTTT (SEQ ID NO: 2307422);

[0235] (4)NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAG TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTT TTT (SEQ ID NO: 2307423);

[0236] (5)NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAG TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGTTTTTTT (SEQ ID NO: 2307424); AND 129 12954008Docket No.: B1195.70187WO00

[0237] (6) NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCATTTTTTTT (SEQ ID NO: 2307425).

[0238] In some embodiments, sequences (1) to (3) are used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4) to (6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a separate transcript from a transcript comprising the tracr mate sequence.

[0239] It will be apparent to those of skill in the art that in order to target any of the fusion proteins comprising a Cas9 domain and a single-stranded DNA binding protein, as disclosed herein, to a target site, e.g., a site comprising a point mutation to be edited, it is typically necessary to co-express the fusion protein together with a guide RNA, e.g., an sgRNA. As explained in more detail elsewhere herein, a guide RNA typically comprises a tracrRNA framework allowing for Cas9 binding, and a guide sequence, which confers sequence specificity to the Cas9:nucleic acid editing enzyme / domain fusion protein.

[0240] In some embodiments, the guide RNA comprises a structure 5ʹ-[guide sequence]- GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAAGGCUAGUCCGUUAUCAACU UGAAAAAGUGGCACCGAGUCGGUGCUUUUU-3ʹ (SEQ ID NO: 2307426), wherein the guide sequence comprises a sequence that is complementary to the target sequence. The guide sequence is typically 20 nucleotides long. The sequences of suitable guide RNAs for targeting Cas9:nucleic acid editing enzyme / domain fusion proteins to specific genomic target sites will be apparent to those of skill in the art based on the instant disclosure. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting any of the provided fusion proteins to specific target sequences are provided herein. Additional guide sequences are well known in the art and can be used with the prime editors utilized in the methods and compositions described herein.

[0241] In some embodiments, a pegRNA comprises three main component elements ordered in the 5ʹ to 3ʹ direction, namely: a spacer, a gRNA core, and an extension arm at the 3ʹ end. The extension arm may further be divided into the following structural elements in the 5ʹ to 3ʹ direction, namely: a primer binding site (A), an edit template (B), and a homology 130 12954008Docket No.: B1195.70187WO00 arm (C). In addition, the pegRNA may comprise an optional 3ʹ end modifier region (e1) and an optional 5ʹ end modifier region (e2). Still further, the pegRNA may comprise a transcriptional termination signal at the 3ʹ end of the pegRNA (not depicted). These structural elements are further defined herein. The depiction of the structure of the pegRNA is not meant to be limiting and embraces variations in the arrangement of the elements. For example, the optional sequence modifiers (e1) and (e2) could be positioned within or between any of the other regions shown, and not limited to being located at the 3ʹ and 5ʹ ends.

[0242] In some embodiments, a pegRNA contemplated herein and may be designed in accordance with the methodology defined in Example 2. The pegRNA comprises three main component elements ordered in the 5ʹ to 3ʹ direction, namely: a spacer, a gRNA core, and an extension arm at the 3ʹ end. The extension arm may further be divided into the following structural elements in the 5ʹ to 3ʹ direction, namely: a primer binding site (A), an edit template (B), and a homology arm (C). In addition, the pegRNA may comprise an optional 3ʹ end modifier region (e1) and an optional 5ʹ end modifier region (e2). Still further, the pegRNA may comprise a transcriptional termination signal on the 3ʹ end of the pegRNA (not depicted). These structural elements are further defined herein. The depiction of the structure of the pegRNA is not meant to be limiting and embraces variations in the arrangement of the elements. For example, the optional sequence modifiers (e1) and (e2) could be positioned within or between any of the other regions shown, and not limited to being located at the 3ʹ and 5ʹ ends.

[0243] The pegRNAs may also include additional design improvements that may modify the properties and / or characteristics of pegRNAs thereby improving the efficacy of prime editing. In various embodiments, these improvements may belong to one or more of a number of different categories, including but not limited to: (1) designs to enable efficient expression of functional pegRNAs from non-polymerase III (pol III) promoters, which would enable the expression of longer pegRNAs without burdensome sequence requirements; (2) improvements to the core, Cas9-binding pegRNA scaffold, which could improve efficacy; (3) modifications to the pegRNA to improve RT processivity, enabling the insertion of longer sequences at targeted genomic loci; and (4) addition of RNA motifs to the 5ʹ or 3ʹ termini of the pegRNA that improve pegRNA stability, enhance RT processivity, prevent misfolding of the pegRNA, or recruit additional factors important for genome editing. 131 12954008Docket No.: B1195.70187WO00

[0244] In one embodiment, pegRNA could be designed with polIII promoters to improve the expression of longer-length pegRNA with larger extension arms. sgRNAs are typically expressed from the U6 snRNA promoter. This promoter recruits pol III to express the associated RNA and is useful for expression of short RNAs that are retained within the nucleus. However, pol III is not highly processive and is unable to express RNAs longer than a few hundred nucleotides in length at the levels required for efficient genome editing. Additionally, pol III can stall or terminate at stretches of U’s, potentially limiting the sequence diversity that could be inserted using a pegRNA. Other promoters that recruit polymerase II (such as pCMV) or polymerase I (such as the U1 snRNA promoter) have been examined for their ability to express longer sgRNAs. However, these promoters are typically partially transcribed, which would result in extra sequence 5ʹ of the spacer in the expressed pegRNA, which has been shown to result in markedly reduced Cas9:sgRNA activity in a site- dependent manner. Additionally, while pol III-transcribed pegRNAs can simply terminate in a run of 6-7 U’s, pegRNAs transcribed from pol II or pol I would require a different termination signal. Often such signals also result in polyadenylation, which would result in undesired transport of the pegRNA from the nucleus. Similarly, RNAs expressed from pol II promoters such as pCMV are typically 5ʹ-capped, also resulting in their nuclear export.

[0245] Previously, Rinn and coworkers screened a variety of expression platforms for the production of long-noncoding RNA- (lncRNA) tagged sgRNAs183. These platforms include RNAs expressed from pCMV and that terminate in the ENE element from the MALAT1 ncRNA from humans184, the PAN ENE element from KSHV185, or the 3ʹ box from U1 snRNA186. Notably, the MALAT1 ncRNA and PAN ENEs form triple helices protecting the polyA-tail184, 187. These constructs could also enhance RNA stability. It is contemplated that these expression systems will also enable the expression of longer pegRNAs.

[0246] In addition, a series of methods have been designed for the cleavage of the portion of the pol II promoter that would be transcribed as part of the pegRNA, adding either a self-cleaving ribozyme such as the hammerhead188, pistol189, hatchet189, hairpin190, VS191, twister192, or twister sister192ribozymes, or other self-cleaving elements to process the transcribed guide, or a hairpin that is recognized by Csy4193and also leads to processing of the guide. Also, it is hypothesized that incorporation of multiple ENE motifs could lead to improved pegRNA expression and stability, as previously demonstrated for the KSHV PAN RNA and element185. It is also anticipated that circularizing the pegRNA in the form of a 132 12954008Docket No.: B1195.70187WO00 circular intronic RNA (ciRNA) could also lead to enhanced RNA expression and stability, as well as nuclear localization194.

[0247] In various other embodiments, the pegRNA may be improved by introducing improvements to the scaffold or core sequences. This can be done by introducing known

[0248] The core, Cas9-binding pegRNA scaffold can likely be improved to enhance PE activity. Several such approaches have already been demonstrated. For instance, the first pairing element of the scaffold (P1) contains a GTTTT-AAAAC pairing element. Such runs of Ts have been shown to result in pol III pausing and premature termination of the RNA transcript. Rational mutation of one of the T-A pairs to a G-C pair in this portion of P1 has been shown to enhance sgRNA activity, suggesting this approach would also be feasible for pegRNAs195. Additionally, increasing the length of P1 has also been shown to enhance sgRNA folding and lead to improved activity195, suggesting it as another avenue for the improvement of pegRNA activity.

[0249] In various other embodiments, the pegRNA may be improved by introducing modifications to the edit template region. As the size of the insertion templated by the pegRNA increases, it is more likely to be degraded by endonucleases, undergo spontaneous hydrolysis, or fold into secondary structures unable to be reverse-transcribed by the RT or that disrupt folding of the pegRNA scaffold and subsequent Cas9-RT binding. Accordingly, it is likely that modification to the template of the pegRNA might be necessary to affect large insertions, such as the insertion of whole genes. Some strategies to do so include the incorporation of modified nucleotides within a synthetic or semi-synthetic pegRNA that render the RNA more resistant to degradation or hydrolysis or less likely to adopt inhibitory secondary structures196. Such modifications could include 8-aza-7-deazaguanosine, which would reduce RNA secondary structure in G-rich sequences; locked-nucleic acids (LNA) that reduce degradation and enhance certain kinds of RNA secondary structure; 2’-O-methyl, 2’- fluoro, or 2’-O-methoxyethoxy modifications that enhance RNA stability. Such modifications could also be included elsewhere in the pegRNA to enhance stability and activity. Alternatively or additionally, the template of the pegRNA could be designed such that it both encodes for a desired protein product and is also more likely to adopt simple secondary structures that are able to be unfolded by the RT. Such simple structures would act as a thermodynamic sink, making it less likely that more complicated structures that would prevent reverse transcription would occur. Finally, one could also split the template into two, 133 12954008Docket No.: B1195.70187WO00 separate pegRNAs. In such a design, a PE would be used to initiate transcription and also recruit a separate template RNA to the targeted site via an RNA-binding protein fused to Cas9 or an RNA recognition element on the pegRNA itself such as the MS2 aptamer. The RT could either directly bind to this separate template RNA, or initiate reverse transcription on the original pegRNA before swapping to the second template. Such an approach could enable long insertions by both preventing misfolding of the pegRNA upon addition of the long template and also by not requiring dissociation of Cas9 from the genome for long insertions to occur, which could possibly be inhibiting PE-based long insertions.

[0250] In still other embodiments, the pegRNA may be improved by introducing additional RNA motifs at the 5ʹ and 3ʹ termini of the pegRNAs, or even at positions therein between (e.g., in the gRNA core region, or the the spacer). Several such motifs - such as the PAN ENE from KSHV and the ENE from MALAT1 were discussed above as possible means to terminate expression of longer pegRNAs from non-pol III promoters. These elements form RNA triple helices that engulf the polyA tail, resulting in their being retained within the nucleus184, 187. However, by forming complex structures at the 3ʹ terminus of the pegRNA that occlude the terminal nucleotide, these structures would also likely help prevent exonuclease-mediated degradation of pegRNAs.

[0251] Other structural elements inserted at the 3ʹ terminus could also enhance RNA stability, albeit without enabling termination from non-pol III promoters. Such motifs could include hairpins or RNA quadruplexes that would occlude the 3ʹ terminus197, or self-cleaving ribozymes such as HDV that would result in the formation of a 2’-3ʹ-cyclic phosphate at the 3ʹ terminus and also potentially render the pegRNA less likely to be degraded by exonucleases198. Inducing the pegRNA to cyclize via incomplete splicing - to form a ciRNA - could also increase pegRNA stability and result in the pegRNA being retained within the nucleus194.

[0252] Additional RNA motifs could also improve RT processivity or enhance pegRNA activity by enhancing RT binding to the DNA-RNA duplex. Addition of the native sequence bound by the RT in its cognate retroviral genome could enhance RT activity199. This could include the native primer binding site (PBS), polypurine tract (PPT), or kissing loops involved in retroviral genome dimerization and initiation of transcription199.

[0253] Addition of dimerization motifs - such as kissing loops or a GNRA tetraloop / tetraloop receptor pair200- at the 5ʹ and 3ʹ termini of the pegRNA could also result 134 12954008Docket No.: B1195.70187WO00 in effective circularization of the pegRNA, improving stability. Additionally, it is envisioned that addition of these motifs could enable the physical separation of the pegRNA spacer and primer, prevention occlusion of the spacer which would hinder PE activity. Short 5ʹ extensions or 3´ extensions to the pegRNA that form a small toehold hairpin in the spacer region or along the primer binding site could also compete favorably against the annealing of intracomplementary regions along the length of the pegRNA, e.g., the interaction between the spacer and the primer binding site that can occur.Finally, kissing loops could also be used to recruit other template RNAs to the genomic site and enable swapping of RT activity from one RNA to the other.

[0254] pegRNA scaffolds could be further improved via directed evolution, in an analogous fashion to how SpCas9 and prime editors (PE) have been improved. Directed evolution could enhance pegRNA recognition by Cas9 or evolved Cas9 variants. Additionally, it is likely that different pegRNA scaffold sequences would be optimal at different genomic loci, either enhancing PE activity at the site in question, reducing off-target activities, or both. Finally, evolution of pegRNA scaffolds to which other RNA motifs have been added would almost certainly improve the activity of the fused pegRNA relative to the unevolved, fusion RNA. For instance, evolution of allosteric ribozymes composed of c-di- GMP-I aptamers and hammerhead ribozymes led to dramatically improved activity202, suggesting that evolution would improve the activity of hammerhead-pegRNA fusions as well. In addition, while Cas9 currently does not generally tolerate 5ʹ extension of the sgRNA, directed evolution will likely generate enabling mutations that mitigate this intolerance, allowing additional RNA motifs to be utilized.

[0255] The present disclosure contemplates any such ways to further improve the efficacy of the prime editing systems utilized in the methods and compositions disclosed here.

[0256] In various embodiments, it may be advantageous to limit the appearance of consecutive sequence of Ts from the extension arm as consecutive series of T’s may limit the capacity of the pegRNA to be transcribed. For example, strings of at least consecutive three T’s, at least consecutive four T’s, at least consecutive five T’s, at least consecutive six T’s, at least consecutive seven T’s, at least consecutive eight T’s, at least consecutive nine T’s, at least consecutive ten T’s, at least consecutiv eleven T’s, at least consecutive twelve T’s, at least consecutive thirteen T’s , at least consecutive fourteen T’s, or at least consecutive 135 12954008Docket No.: B1195.70187WO00 fifteen T’s should be avoided when designing the pegRNA, or should be at least removed from the final designed sequence. In one embodiment, one can avoid the includes of unwanted strings of consecutive T’s in pegRNA extension arms but avoiding target sites that are rich in consecutive A:T nucleobase pairs. Linkers, NLS, and other PE elements Linkers

[0257] In some embodiments, a linker joins a gRNA binding domain of an RNA- programmable nuclease and the catalytic domain of a polymerase (e.g., a reverse transcriptase). In some embodiments, a linker joins a dCas9 and reverse transcriptase. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 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, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

[0258] The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polpeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5- pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises 136 12954008Docket No.: B1195.70187WO00 amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may included funtionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0259] In some other embodiments, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 2307427), (G)n (SEQ ID NO: 2307428), (EAAAK)n (SEQ ID NO: 2307429), (GGS)n(SEQ ID NO: 2307430), (SGGS)n(SEQ ID NO: 2307431), (XP)n(SEQ ID NO: 2307432), or any combination thereof, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, the linker comprises the amino acid sequence (GGS)n (SEQ ID NO: 2307433), wherein n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 2307434). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 2307435). In some embodiments, the linker comprises the amino acid sequence SGGSGGSGGS (SEQ ID NO: 2307436). In some embodiments, the linker comprises the amino acid sequence SGGS (SEQ ID NO: 2307437). In other embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSSG GS (SEQ ID NO: 2307438). Other sequences are also possible.

[0260] In particular, the following linkers can be used in various embodiments to join prime editor domains with one another:

[0261] GGS;

[0262] GGSGGS (SEQ ID NO: 2307440);

[0263] GGSGGSGGS (SEQ ID NO: 2307441);

[0264] SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 2307442);

[0265] SGSETPGTSESATPES (SEQ ID NO: 2307434);

[0266] SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKL DGSGSGGSSGGS (SEQ ID NO: 2307438).

[0267] The PE fusion proteins may also comprise various other domains besides the napDNAbp (e.g., Cas9 domain) and the polymerase domain (e.g., RT domain). For example, 137 12954008Docket No.: B1195.70187WO00 in the case where the napDNAbp is a Cas9 and the polymerase is a RT, the PE fusion proteins may comprise one or more linkers that join the Cas9 domain with the RT domain. The linkers may also join other functional domains, such as nuclear localization sequences (NLS) or a FEN1 (or other flap endonuclease) to the PE fusion proteins or a domain thereof, or a recombinase (e.g., an integrase). Nuclear localization sequences (NLS)

[0268] In various embodiments, the PE fusion proteins may comprise one or more nuclear localization sequences (NLS), which help promote translocation of a protein into the cell nucleus. Such sequences are well-known in the art and thus, are not provided herein.

[0269] The PE fusion proteins may comprise any known NLS sequence, including any of those described in Cokol et al., “Finding nuclear localization signals,” EMBO Rep., 2000, 1(5): 411-415 and Freitas et al., “Mechanisms and Signals for the Nuclear Import of Proteins,” Current Genomics, 2009, 10(8): 550-7, Plank et al., International PCT application PCT / EP2000 / 011690, filed November 23, 2000, published as WO / 2001 / 038547 on May 31, 2001, the contents of which are incorporated herein by reference.

[0270] In various embodiments, the prime editors and constructs encoding the prime editors utilized in the methods and compositions disclosed herein further comprise one or more, preferably, at least two nuclear localization signals. In certain embodiments, the prime editors comprise at least two NLSs. In embodiments with at least two NLSs, the NLSs can be the same NLSs or they can be different NLSs. In addition, the NLSs may be expressed as part of a fusion protein with the remaining portions of the prime editors. In some embodiments, one or more of the NLSs are bipartite NLSs (“bpNLS”). In certain embodiments, the disclosed fusion proteins comprise two bipartite NLSs. In some embodiments, the disclosed fusion proteins comprise more than two bipartite NLSs.

[0271] The location of the NLS fusion can be at the N-terminus, the C-terminus, or within a sequence of a prime editor (e.g., inserted between the encoded napDNAbp component (e.g., Cas9) and a polymerase domain (e.g., a reverse transcriptase domain).

[0272] The NLSs may be any known NLS sequence in the art. The NLSs may also be any future-discovered NLSs for nuclear localization. The NLSs also may be any naturally-occurring NLS, or any non-naturally occurring NLS (e.g., an NLS with one or more desired mutations). 138 12954008Docket No.: B1195.70187WO00

[0273] Most NLSs can be classified in three general groups: (i) a monopartite NLS exemplified by the SV40 large T antigen NLS (PKKKRKV (SEQ ID NO: 2307443)); (ii) a bipartite motif consisting of two basic domains separated by a variable number of spacer amino acids and exemplified by the Xenopus nucleoplasmin NLS (KRXXXXXXXXXXKKKL (SEQ ID NO: 2307444)); and (iii) noncanonical sequences such as M9 of the hnRNP Al protein, the influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS (Dingwall and Laskey 1991).

[0274] The present disclosure contemplates any suitable means by which to modify a prime editor to include one or more NLSs. In one aspect, the prime editors may be engineered to express a prime editor protein that is translationally fused at its N-terminus or its C-terminus (or both) to one or more NLSs, i.e., to form a prime editor-NLS fusion construct. In other embodiments, the prime editor-encoding nucleotide sequence may be genetically modified to incorporate a reading frame that encodes one or more NLSs in an internal region of the encoded prime editor. In addition, the NLSs may include various amino acid linkers or spacer regions encoded between the prime editor and the N-terminally, C- terminally, or internally-attached NLS amino acid sequence, e.g, and in the central region of proteins. Thus, the present disclosure also provides for nucleotide constructs, vectors, and host cells for expressing fusion proteins that comprise a prime editor and one or more NLSs.

[0275] The prime editors utilized in the methods and compositions described herein may also comprise nuclear localization signals which are linked to a prime editor through one or more linkers, e.g., and polymeric, amino acid, nucleic acid, polysaccharide, chemical, or nucleic acid linker element. The linkers within the contemplated scope of the disclosure are not intented to have any limitations and can be any suitable type of molecule (e.g., polymer, amino acid, polysaccharide, nucleic acid, lipid, or any synthetic chemical linker domain) and be joined to the prime editor by any suitable strategy that effectuates forming a bond (e.g., covalent linkage, hydrogen bonding) between the prime editor and the one or more NLSs. Flap endonucleases

[0276] In various embodiments, the PE fusion proteins may comprise one or more flap endonucleases (e.g., FEN1), which refers to an enzyme that catalyzes the removal of 5ʹ single strand DNA flaps. These are naturally occurring enzymes that process the removal of 5ʹ flaps formed during cellular processes, including DNA replication. The prime editing utilized in the methods and compositions described herein may utilize endogenously supplied 139 12954008Docket No.: B1195.70187WO00 flap endonucleases or those provided in trans to remove the 5ʹ flap of endogenous DNA formed at the target site during prime editing. Flap endonucleases are known in the art and can be found described in Patel et al., “Flap endonucleases pass 5ʹ-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5ʹ-ends,” Nucleic Acids Research, 2012, 40(10): 4507-4519 and Tsutakawa et al., “Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily,” Cell, 2011, 145(2): 198-211 (each of which are incorporated herein by reference).

[0277] Other endonucleases that may be utilized by the instant methods to facilitate removal of the 5´ end single strand DNA flap include, but are not limited to (1) trex 2, (2) exo1 endonuclease (e.g., Keijzers et al., Biosci Rep.2015, 35(3): e00206) Additional PE elements

[0278] In certain embodiments, the prime editors utilized in the methods and compositions described herein may comprise an inhibitor of base repair. The term “inhibitor of base repair” or “IBR” refers to a protein that is capable in inhibiting the activity of a nucleic acid repair enzyme, for example a base excision repair enzyme. In some embodiments, the IBR is an inhibitor of OGG base excision repair. In some embodiments, the IBR is an inhibitor of base excision repair (“iBER”). Exemplary inhibitors of base excision repair include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGG1, hNEIL1, T7 EndoI, T4PDG, UDG, hSMUG1, and hAAG. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is an iBER that may be a catalytically inactive glycosylase or catalytically inactive dioxygenase or a small molecule or peptide inhibitor of an oxidase, or variants threreof. In some embodiments, the IBR is an iBER that may be a TDG inhibitor, MBD4 inhibitor or an inhibitor of an AlkBH enzyme. In some embodiments, the IBR is an iBER that comprises a catalytically inactive TDG or catalytically inactive MBD4. An exemplary catalytically inactive TDG is an N140A mutant of SEQ ID NO: 2307445 (human TDG).

[0279] TDG (human) (wild-type)

[0280] MEAENAGSYSLQQAQAFYTFPFQQLMAEAPNMAVVNEQQMPEEVPAPA PAQEPVQEAPKGRKRKPRTTEPKQPVEPKKPVESKKSGKSAKSKEKQEKITDTFKVK RKVDRFNGVSEAELLTKTLPDILTFNLDIVIIGINPGLMAAYKGHHYPGPGNHFWKCL FMSGLSEVQLNHMDDHTLPGKYGIGFTNMVERTTPGSKDLSSKEFREGGRILVQKLQ KYQPRIAVFNGKCIYEIFSKEVFGVKVKNLEFGLQPHKIPDTETLCYVMPSSSARCAQ 140 12954008Docket No.: B1195.70187WO00 FPRAQDKVHYYIKLKDLRDQLKGIERNMDVQEVQYTFDLQLAQEDAKKMAVKEEK YDPGYEAAYGGAYGENPCSSEPCGFSSNGLIESVELRGESAFSGIPNGQWMTQSFTD QIPSFSNHCGTQEQEEESHA (SEQ ID NO: 2307445)

[0281] In some embodiments, the fusion proteins described herein may comprise one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the prime editor components). A fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Other exemplary features that may be present are localization sequences, such as cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins.

[0282] Examples of protein domains that may be fused to a prime editor or component thereof (e.g., the napDNAbp domain, the polymerase domain, or the NLS domain) include, without limitation, epitope tags, and reporter gene sequences. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta- glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A prime editor may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a prime editor are described in US Patent Publication No.2011 / 0059502, published March 10, 2011 and incorporated herein by reference in its entirety.

[0283] In an aspect of the disclosure, a reporter gene which includes, but is not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP), may be 141 12954008Docket No.: B1195.70187WO00 introduced into a cell to encode a gene product which serves as a marker by which to measure the alteration or modification of expression of the gene product. In certain embodiments of the disclosure the gene product is luciferase. In a further embodiment of the disclosure the expression of the gene product is decreased.

[0284] Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags , biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein comprises one or more His tags.

[0285] In some embodiments of the present disclosure, the activity of the prime editing system may be temporally regulated by adjusting the residence time, the amount, and / or the activity of the expressed components of the PE system. For example, as described herein, the PE may be fused with a protein domain that is capable of modifying the intracellular half-life of the PE. In certain embodiments involving two or more vectors (e.g., a vector system in which the components described herein are encoded on two or more separate vectors), the activity of the PE system may be temporally regulated by controlling the timing in which the vectors are delivered. For example, in some embodiments a vector encoding the nuclease system may deliver the PE prior to the vector encoding the template. In other embodiments, the vector encoding the pegRNA may deliver the guide prior to the vector encoding the PE system. In some embodiments, the vectors encoding the PE system and pegRNA are delivered simultaneously. In certain embodiments, the simultaneously delivered vectors temporally deliver, e.g., the PE, pegRNA, and / or second strand guide RNA components. In further embodiments, the RNA (such as, e.g., the nuclease transcript) transcribed from the coding sequence on the vectors may further comprise at least one element that is capable of modifying the intracellular half-life of the RNA and / or modulating translational control. In some embodiments, the half-life of the RNA may be increased. In some embodiments, the half-life of the RNA may be decreased. In some embodiments, the element may be capable of increasing the stability of the RNA. In some embodiments, the element may be capable of decreasing the stability of the RNA. In some embodiments, the 142 12954008Docket No.: B1195.70187WO00 element may be within the 3' UTR of the RNA. In some embodiments, the element may include a polyadenylation signal (PA). In some embodiments, the element may include a cap, e.g., an upstream mRNA or pegRNA end. In some embodiments, the RNA may comprise no PA such that it is subject to quicker degradation in the cell after transcription. In some embodiments, the element may include at least one AU-rich element (ARE). The AREs may be bound by ARE binding proteins (ARE-BPs) in a manner that is dependent upon tissue type, cell type, timing, cellular localization, and environment. In some embodiments the destabilizing element may promote RNA decay, affect RNA stability, or activate translation. In some embodiments, the ARE may comprise 50 to 150 nucleotides in length. In some embodiments, the ARE may comprise at least one copy of the sequence AUUUA. In some embodiments, at least one ARE may be added to the 3' UTR of the RNA. In some embodiments, the element may be a Woodchuck Hepatitis Virus (WHP).

[0286] Posttranscriptional Regulatory Element (WPRE), which creates a tertiary structure to enhance expression from the transcript. In further embodiments, the element is a modified and / or truncated WPRE sequence that is capable of enhancing expression from the transcript, as described, for example in Zufferey et al., J Virol, 73(4): 2886-92 (1999) and Flajolet et al., J Virol, 72(7): 6175-80 (1998). In some embodiments, the WPRE or equivalent may be added to the 3' UTR of the RNA. In some embodiments, the element may be selected from other RNA sequence motifs that are enriched in either fast- or slow-decaying transcripts.

[0287] In some embodiments, the vector encoding the PE or the pegRNA may be self- destroyed via cleavage of a target sequence present on the vector by the PE system. The cleavage may prevent continued transcription of a PE or a pegRNA from the vector. Although transcription may occur on the linearized vector for some amount of time, the expressed transcripts or proteins subject to intracellular degradation will have less time to produce off- target effects without continued supply from expression of the encoding vectors. Kits, cells, and vectors Kits

[0288] The compositions of the present disclosure may be assembled into kits. In some embodiments, the kit comprises nucleic acid vectors for the expression of a prime editor. In other embodiments, the kit further comprises appropriate guide nucleotide sequences (e.g., pegRNAs and second-site gRNAs) or nucleic acid vectors for the expression 143 12954008Docket No.: B1195.70187WO00 of such guide nucleotide sequences, to target the Cas9 protein or prime editor to the desired target sequence.

[0289] The kit described herein may include one or more containers housing components for performing the methods described herein and optionally instructions for use. Any of the kits described herein may further comprise components needed for performing the assay methods. Each component of the kits, where applicable, may be provided in liquid form (e.g., in solution) or in solid form, (e.g., a dry powder). In certain cases, some of the components may be reconstitutable or otherwise processible (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water), which may or may not be provided with the kit.

[0290] In some embodiments, the kits may optionally include instructions and / or promotion for use of the components provided. As used herein, “instructions” can define a component of instruction and / or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and / or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which can also reflect approval by the agency of manufacture, use or sale for animal administration. As used herein, “promoted” includes all methods of doing business including methods of education, hospital and other clinical instruction, scientific inquiry, drug discovery or development, academic research, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with the disclosure. Additionally, the kits may include other components depending on the specific application, as described herein.

[0291] The kits may contain any one or more of the components described herein in one or more containers. The components may be prepared sterilely, packaged in a syringe and shipped refrigerated. Alternatively it may be housed in a vial or other container for storage. A second container may have other components prepared sterilely. Alternatively the kits may include the active agents premixed and shipped in a vial, tube, or other container. 144 12954008Docket No.: B1195.70187WO00

[0292] The kits may have a variety of forms, such as a blister pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag. The kits may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kits may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration, etc. Some aspects of this disclosure provide kits comprising a nucleic acid construct comprising a nucleotide sequence encoding the various components of the prime editing system utilized in the methods and compositions described herein (e.g., including, but not limited to, the napDNAbps, reverse transcriptases, polymerases, fusion proteins (e.g., comprising napDNAbps and reverse transcriptases (or more broadly, polymerases), extended guide RNAs, and complexes comprising fusion proteins and extended guide RNAs, as well as accessory elements, such as second strand nicking components (e.g., second strand nicking gRNA) and 5´ endogenous DNA flap removal endonucleases for helping to drive the prime editing process towards the edited product formation). In some embodiments, the nucleotide sequence(s) comprises a heterologous promoter (or more than a single promoter) that drives expression of the prime editing system components.

[0293] Other aspects of this disclosure provide kits comprising one or more nucleic acid constructs encoding the various components of the prime editing systems utilized in the methods and compositions described herein, e.g., the comprising a nucleotide sequence encoding the components of the prime editing system capable of modifying a target DNA sequence. In some embodiments, the nucleotide sequence comprises a heterologous promoter that drives expression of the prime editing system components.

[0294] Some aspects of this disclosure provides kits comprising a nucleic acid construct, comprising (a) a nucleotide sequence encoding a napDNAbp (e.g., a Cas9 domain) fused to a reverse transcriptase and (b) a heterologous promoter that drives expression of the sequence of (a). Cells 145 12954008Docket No.: B1195.70187WO00

[0295] Cells that may contain any of the compositions described herein include prokaryotic cells and eukaryotic cells. The methods described herein are used to deliver a prime editor and a pegRNA into a eukaryotic cell (e.g., a mammalian cell, such as a human cell). In some embodiments, the cell is in vitro (e.g., cultured cell). In some embodiments, the cell is in vivo (e.g., in a subject such as a human subject). In some embodiments, the cell is ex vivo (e.g., isolated from a subject and may be administered back to the same or a different subject).

[0296] Mammalian cells of the present disclosure include human cells, primate cells (e.g., vero cells), rat cells (e.g., GH3 cells, OC23 cells) or mouse cells (e.g., MC3T3 cells). There are a variety of human cell lines, including, without limitation, human embryonic kidney (HEK) cells, HeLa cells, cancer cells from the National Cancer Institute's 60 cancer cell lines (NCI60), DU145 (prostate cancer) cells, Lncap (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-438 (breast cancer) cells, PC3 (prostate cancer) cells, T47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, SHSY5Y human neuroblastoma cells (cloned from a myeloma) and Saos-2 (bone cancer) cells. In some embodiments, rAAV vectors are delivered into human embryonic kidney (HEK) cells (e.g., HEK 293 or HEK 293T cells). In some embodiments, rAAV vectors are delivered into stem cells (e.g., human stem cells) such as, for example, pluripotent stem cells (e.g., human pluripotent stem cells including human induced pluripotent stem cells (hiPSCs)). A stem cell refers to a cell with the ability to divide for indefinite periods in culture and to give rise to specialized cells. A pluripotent stem cell refers to a type of stem cell that is capable of differentiating into all tissues of an organism, but not alone capable of sustaining full organismal development. A human induced pluripotent stem cell refers to a somatic (e.g., mature or adult) cell that has been reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells (see, e.g., Takahashi and Yamanaka, Cell 126 (4): 663–76, 2006, incorporated by reference herein). Human induced pluripotent stem cell cells express stem cell markers and are capable of generating cells characteristic of all three germ layers (ectoderm, endoderm, mesoderm).

[0297] Additional non-limiting examples of cell lines that may be used in accordance with the present disclosure include 293-T, 293-T, 3T3, 4T1, 721, 9L, A-549, A172, A20, A253, A2780, A2780ADR, A2780cis, A431, ALC, B16, B35, BCP-1, BEAS-2B, bEnd.3, 146 12954008Docket No.: B1195.70187WO00 BHK-21, BR 293, BxPC3, C2C12, C3H-10T1 / 2, C6, C6 / 36, Cal-27, CGR8, CHO, CML T1, CMT, COR-L23, COR-L23 / 5010, COR-L23 / CPR, COR-L23 / R23, COS-7, COV-434, CT26, D17, DH82, DU145, DuCaP, E14Tg2a, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, Hepa1c1c7, High Five cells, HL-60, HMEC, HT-29, HUVEC, J558L cells, Jurkat, JY cells, K562 cells, KCL22, KG1, Ku812, KYO1, LNCap, Ma-Mel 1, 2, 3....48, MC-38, MCF-10A, MCF-7, MDA-MB-231, MDA-MB-435, MDA- MB-468, MDCK II, MG63, MONO-MAC 6, MOR / 0.2R, MRC5, MTD-1A, MyEnd, NALM- 1, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NW-145, OPCN / OPCT Peer, PNT-1A / PNT 2, PTK2, Raji, RBL cells, RenCa, RIN-5F, RMA / RMAS, S2, Saos-2 cells, Sf21, Sf9, SiHa, SKBR3, SKOV-3, T-47D, T2, T84, THP1, U373, U87, U937, VCaP, WM39, WT-49, X63, YAC-1 and YAR cells.

[0298] Some aspects of this disclosure provide cells comprising any of the constructs disclosed herein. In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD- 3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB / 3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A 172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293. BxPC3. C3H-10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr − / −, COR-L23, COR-L23 / CPR, COR-L23 / 5010, COR-L23 / R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, 147 12954008Docket No.: B1195.70187WO00 MDA-MB-435, MDCK II, MDCK 11, MOR / 0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI- H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT cell lines, Peer, PNT-1A / PNT 2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof.

[0299] Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassus, Va.)). In some embodiments, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with the components of a CRISPR system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a CRISPR complex, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds. Vectors

[0300] Some aspects of the present disclosure relate to using recombinant virus vectors (e.g., adeno-associated virus vectors, adenovirus vectors, or herpes simplex virus vectors) for the delivery of the prime editors and pegRNAs as described herein into a cell. In the case of a split-PE approach, the N-terminal portion of a PE fusion protein and the C- terminal portion of a PE fusion are delivered by separate recombinant virus vectors (e.g., adeno-associated virus vectors, adenovirus vectors, or herpes simplex virus vectors) into the same cell, since the full-length Cas9 protein or prime editors exceeds the packaging limit of various virus vectors, e.g., rAAV (~4.9 kb).

[0301] In some embodiments, the vectors used herein may encode the PE fusion proteins, or any of the components thereof (e.g., napDNAbp, linkers, or polymerases). In addition, the vectors used herein may encode the pegRNAs, and / or the accessory gRNA for second strand nicking. The vectors may be capable of driving expression of one or more coding sequences in a cell. In some embodiments, the cell may be a prokaryotic cell, such as, e.g., a bacterial cell. In some embodiments, the cell may be a eukaryotic cell, such as, e.g., a yeast, plant, insect, or mammalian cell. In some embodiments, the eukaryotic cell may be a 148 12954008Docket No.: B1195.70187WO00 mammalian cell. In some embodiments, the eukaryotic cell may be a rodent cell. In some embodiments, the eukaryotic cell may be a human cell. Suitable promoters to drive expression in different types of cells are known in the art. In some embodiments, the promoter may be wild-type. In other embodiments, the promoter may be modified for more efficient or efficacious expression. In yet other embodiments, the promoter may be truncated yet retain its function. For example, the promoter may have a normal size or a reduced size that is suitable for proper packaging of the vector into a virus.

[0302] In some embodiments, the promoters that may be used in the prime editor vectors may be constitutive, inducible, or tissue-specific. In some embodiments, the promoters may be a constitutive promoters. Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-alpha (EFla) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, a functional fragment thereof, or a combination of any of the foregoing. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EFla promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On® promoter (Clontech). In some embodiments, the promoter may be a tissue- specific promoter. In some embodiments, the tissue-specific promoter is exclusively or predominantly expressed in liver tissue. Non-limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase- 1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM- 2 promoter, INF-β promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.

[0303] In some embodiments, the prime editor pegRNA vectors (e.g., including any vectors encoding the prime editor fusion protein and / or the pegRNAs, and / or the accessory second strand nicking gRNAs) may comprise inducible promoters to start expression only 149 12954008Docket No.: B1195.70187WO00 after it is delivered to a target cell. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On® promoter (Clontech).

[0304] In additional embodiments, the prime editor vectors (e.g., including any vectors encoding the prime editor fusion protein and / or the pegRNAs, and / or the accessory second strand nicking gRNAs) may comprise tissue- specific promoters to start expression only after it is delivered into a specific tissue. Non-limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase- 1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM- 2 promoter, INF-β promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.

[0305] In some embodiments, the nucleotide sequence encoding the pegRNA (or any guide RNAs used in connection with prime editing) may be operably linked to at least one transcriptional or translational control sequence. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to at least one promoter. In some embodiments, the promoter may be recognized by RNA polymerase III (Pol III). Non- limiting examples of Pol III promoters include U6, HI and tRNA promoters. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human U6 promoter. In other embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human HI promoter. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human tRNA promoter. In embodiments with more than one guide RNA, the promoters used to drive expression may be the same or different. In some embodiments, the nucleotide encoding the crRNA of the guide RNA and the nucleotide encoding the tracr RNA of the guide RNA may be provided on the same vector. In some embodiments, the nucleotide encoding the crRNA and the nucleotide encoding the tracr RNA may be driven by the same promoter. In some embodiments, the crRNA and tracr RNA may be transcribed into a single transcript. For example, the crRNA and tracr RNA may be processed from the single transcript to form a double-molecule guide RNA. Alternatively, the crRNA and tracr RNA may be transcribed into a single-molecule guide RNA. 150 12954008Docket No.: B1195.70187WO00

[0306] In some embodiments, the nucleotide sequence encoding the guide RNA may be located on the same vector comprising the nucleotide sequence encoding the PE fusion protein. In some embodiments, expression of the guide RNA and of the PE fusion protein may be driven by their corresponding promoters. In some embodiments, expression of the guide RNA may be driven by the same promoter that drives expression of the PE fusion protein. In some embodiments, the guide RNA and the PE fusion protein transcript may be contained within a single transcript. For example, the guide RNA may be within an untranslated region (UTR) of the Cas9 protein transcript. In some embodiments, the guide RNA may be within the 5´ UTR of the PE fusion protein transcript. In other embodiments, the guide RNA may be within the 3' UTR of the PE fusion protein transcript. In some embodiments, the intracellular half-life of the PE fusion protein transcript may be reduced by containing the guide RNA within its 3' UTR and thereby shortening the length of its 3' UTR. In additional embodiments, the guide RNA may be within an intron of the PE fusion protein transcript. In some embodiments, suitable splice sites may be added at the intron within which the guide RNA is located such that the guide RNA is properly spliced out of the transcript. In some embodiments, expression of the Cas9 protein and the guide RNA in close proximity on the same vector may facilitate more efficient formation of the CRISPR complex.

[0307] The vector system may comprise one vector, or two vectors, or three vectors, or four vectors, or five vector, or more. In some embodiments, the vector system may comprise one single vector, which encodes both the PE fusion protein and the pegRNA. In other embodiments, the vector system may comprise two vectors, wherein one vector encodes the PE fusion protein, and the other encodes the pegRNA. Virus-lipid particle (VLPs)

[0308] Aspects of the disclosure relate to delivering a prime editing system using VLPs, such as those described in U.S. Patent Application U.S.S.N.63 / 298,626 filed on January 11, 2022, which is herein incorporated by reference in its entirety. In various embodiments, the eVLPs (e.g., PE-VLPs) consist of a supra-molecular assembly comprising (a) an envelope comprising (i) a lipid membrane (e.g., single-layer or bi-layer membrane) and a (ii) viral envelope glycoprotein (e.g., VSV-G) and (b) a multi-protein core region enclosed by the envelope and comprising (i) a Gag protein, (ii) a Gag-Pro-Pol protein (with the “Pro” component referring to a protease), and (iii) a Gag-cargo fusion protein comprising a Gag 151 12954008Docket No.: B1195.70187WO00 protein fused to a cargo protein (e.g., a napDNAbp or PE or a split PE) via a cleavable linker (e.g., a protease-cleavable linker). In various embodiments, the cargo protein is a napDNAbp (e.g., Cas9). In other embodiments, the cargo protein is a prime editor. In various embodiments (e.g., FIG.2A) the PE may be split into a Cas9 domain and a reverse transcriptase domain as separate fusion proteins each with Gag. In various embodiments, the split domains of PE may comprise split-intein sequences which allows the split domains to re-form a PE once delivered to a cell. In various other embodiments, the multi-protein core region of the VLPs further comprises one or more pegRNA molecules and / or second-site nicking guide RNA which are complexed with the napDNAbp or the prime editor to form a ribonucleoprotein (RNP).

[0309] In various embodiments, the VLPs are prepared in a producer cell that is transiently transformed with plasmid DNA that encodes the various protein and nucleic acid (pegRNAs and guide RNAs) components of the VLPs. Without being bound by theory, the components self-assemble at the cell membrane and bud out in accordance with the naturally occurring mechanism of budding (e.g., retroviral budding or the budding mechanism of other envelope viruses) in order to release from the cell fully-matured VLPs. Once formed, the Gag-Pol-Pro cleaves the protease-sensitive linker of the Gag-cargo (i.e., [Gag]-[cleavable linker]-[cargo], wherein the cargo can be PE-RNP or a napDNAbp RNP) thereby releasing the PE RNP and / or napDNAbp RNA, as the case may be, within the VLP. Once the VLP is administered to a recipient cell and taken up by said recipient cell, the contents of the VLP are released, e.g., released PE RNP and / or napDNAbp RNP. Once in the cell, the RNPs may translocate to the nuclease of the cell (in particular, where NLSs are included on the RNPs), where DNA editing may occur at target sites specified by the guide RNA. Various embodiments comprise one or more improvements.

[0310] In one improvement, the protease-cleavable linker is optimized to improve cleavage efficiency after VLP maturation, as demonstrated herein for v.2 VLPs (or “second generation” VLPs).

[0311] In another improvement, the Gag-cargo fusion (e.g., Gag-BE) further comprises one or more nuclear export signals at one or more locations along the length of the fusion polypeptide protein which may be joined by a cleavable linker such that during VLP assembly in the producer cell, the Gag-cargo fusions (due to presence of competing NLS signals) do not accumulate in the nucleus of the producer cells but instead are available in the 152 12954008Docket No.: B1195.70187WO00 cytoplasm to undergo the VLP assembly process at the cell membrane. Once inside the matured VLPs following release from the producer cell, the NES may be cleaved by Gag- Pro-Pol thereby separating the cargo (e.g., napDNAbp or a PE) from the NES. Upon delivery to a recipient cell, therefore, the cargo (e.g., napDNAbp or PE, typically flanked with one or more NLS elements) will not comprise an NES element, which may otherwise prohibit the transport of the cargo into the nuclease and hinder gene editing activity. This is exemplified as v.3 VLPs described herein (or “third generation” VLPs).

[0312] In other embodiments, the eVLPs disclosed herein may comprise split PE domains contained in a single all-in-one VLP system or in a two-particle system whereby each PE half domain is formed in separate VLPs. See FIG.3A.

[0313] In one aspect, the present disclosure provides a eVLP comprising an (a) envelope and (b) a multi-protein core, wherein the envelope comprises a lipid membrane (e.g., a lipid mono or bi-layer membrane) and a viral envelope glycoprotein and wherein the multi-protein core comprises a Gag (e.g., a retroviral Gag), a group-specific antigen (gag) protease (pro) polyprotein (i.e., “Gag-Pro-Pol”) and a fusion protein comprising a Gag-cargo (e.g., Gag-napDNAbp or Gag-PE). In various embodiments, the Gag-cargo may comprise a ribonucleoprotein cargo, e.g., a napDNAbp or a PE complexed with a guide RNA. In still further embodiments, the Gag-cargo (e.g., Gag fused to a napDNAbp or a PE) may comprise one or more NLS sequences and / or one or more NES sequences to regulate the cellular location of the cargo in a cell. An NLS sequence will facilitate the transport of the cargo into the cell’s nuclease to facilitate editing. A NES will do the opposite, i.e., transport the cargo out from the nucleus, and / or prevent the transport of the cargo into the nucleus. In certain embodiments, the NES may be coupled to the fusion protein by a cleavable linker (e.g., a protease linker) such that during assembly in a producer cell, the NES signals operates to keep the cargo in the cytoplasm and available for the packaging process. However, once matured VLPs are budded out or released from a producer cell in a mature form, the cleavable linker joining the NES may be cleaved, thereby removing the association of NES with the cargo. Thus, without an NES, the cargo will translocate to the nuclease with its NLS sequences, thereby facilitating editing. Various napDNAbps may be used in the systems of the present disclosure. In some embodiments, the napDNAbp is a Cas9 protein (e.g., a Cas9 nickase, dead Cas9 (dCas9), or another Cas9 variant as described herein). In some embodiments, the Cas9 protein is bound to a guide RNA (gRNA). The fusion protein may 153 12954008Docket No.: B1195.70187WO00 further comprise other protein domains, such as effector domains. In some embodiments, the fusion protein further comprises a deaminase domain (e.g., an adenosine deaminase domain or a cytosine deaminase domain). In certain embodiments, the fusion protein comprises a prime editor, such as PE2, PE3, or PEmax prime editor, or any of the other prime editors described herein or known in the art.

[0314] In some embodiments, the fusion protein comprises more than one NES (e.g., two NES, three NES, four NES, five NES, six NES, seven NES, eight NES, nine NES, or ten or more NES). In certain embodiments, the fusion protein further comprises a nuclear localization sequence (NLS), or more than one NLS (e.g., two NLS, three NLS, four NLS, five NLS, six NLS, seven NLS, eight NLS, nine NLS, or ten or more NLS). In certain embodiments, the fusion protein may comprise at least one NES and one NLS.

[0315] The Gag-cargo fusion proteins described herein comprise one or more cleavable linkers. In one embodiment, the Gag-cargo fusion proteins comprise a cleavable linker joining the Gag to the cargo, such that once the Gag-cargo fusion has been packaged in mature VLPs (which will also contain the Gag-Pro-Pol, the protease activity can cleave the Gag-cargo cleavable linker, thereby releasing the cargo. In some embodiments, a cleavable linker may also be provided in such a location such that when the cleavable linker is cleaved (e.g., by the Gag-Pro-Pol protein), the NES is separated away from the cargo protein. Such an arrangement of the fusion protein allows the fusion protein to be exported from the nucleus of a producing cell during BE-VLP production, and the NES can later be cleaved from the fusion protein after delivery to a target cell, releasing the PE (or release of split PE half domains from the same or a two-particle system) and allowing it to enter the nucleus of the target cell. In some embodiments, the cleavable linker comprises a protease cleavage site (e.g., a Moloney murine leukemia virus (MMLV) protease cleavage site or a Friend murine leukemia virus (FMLV) protease cleavage site). Various protease cleavage sites can be used in the fusion proteins of the present disclosure. In certain embodiments, the protease cleavage site comprises the amino acid sequence TSTLLMENSS (SEQ ID NO: 2307446), PRSSLYPALTP (SEQ ID NO: 2307447), VQALVLTQ (SEQ ID NO: 2307448), PLQVLTLNIERR (SEQ ID NO: 2307449), or an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 2307446-2307449. In some embodiments, the cleavable linker of the fusion protein is cleaved by the protease of the gag-pro polyprotein. In certain embodiments, the cleavable linker of the fusion protein is not cleaved by the protease of the 154 12954008Docket No.: B1195.70187WO00 gag-pro polyprotein until the PE-VLP has been assembled and delivered into a target cell. In some embodiments, the gag-pro polyprotein of the PE-VLPs described herein comprises an MMLV gag-pro polyprotein or an FMLV gag-pro polyprotein. In some embodiments, the gag nucleocapsid protein of the fusion protein in the PE-VLPs described herein comprises an MMLV gag nucleocapsid protein or an FMLV gag nucleocapsid protein.

[0316] In certain embodiments, the fusion protein comprises the following non- limiting structures:

[0317] [gag nucleocapsid protein]-[1X-3X NES]-[cleavable linker]-[NLS]-[RT domain]-[napDNAbp]-[NLS], wherein ]-[comprises an optional linker (e.g., an amino acid linker, or any of the linkers provided herein);

[0318] [1X-3X NES]-[gag nucleocapsid protein]-[cleavable linker]-[NLS]-[RT domain]-[napDNAbp]-[NLS], wherein ]-[comprises an optional linker (e.g., an amino acid linker, or any of the linkers provided herein); or

[0319] [gag nucleocapsid protein]-[1X-3X NES]-[cleavable linker]-[NLS]-[RT domain]-[napDNAbp]-[NLS]-[cleavable linker]-[1X-3X NES], wherein ]-[ comprises an optional linker (e.g., an amino acid linker, or any of the linkers provided herein).

[0320] The eVLPs (e.g., the PE-VLPs) provided by the present disclosure comprise an outer encapsulation layer (or envelope layer) comprising a viral envelope glycoprotein. Any viral envelope glycoprotein described herein, or known in the art, may be used in the PE-VLPs of the present disclosure. In some embodiments, the viral envelope glycoprotein is an adenoviral envelope glycoprotein, an adeno-associated viral envelope glycoprotein, a retroviral envelope glycoprotein, or a lentiviral envelope glycoprotein. In certain embodiments, the viral envelope glycoprotein is a retroviral envelope glycoprotein. In some embodiments, the viral envelope glycoprotein is a vesicular stomatitis virus G protein (VSV- G), a baboon retroviral envelope glycoprotein (BaEVRless), a FuG-B2 envelope glycoprotein, an HIV-1 envelope glycoprotein, or an ecotropic murine leukemia virus (MLV) envelope glycoprotein. In some embodiments, the viral envelope glycoprotein targets the system to a particular cell type (e.g., immune cells, neural cells, retinal pigment epithelium cells, etc.). For example, using different envelope glycoproteins in the eVLPs described herein may alter their cellular tropism, allowing the PE-VLPs to be targeted to specific cell types. In some embodiments, the viral envelope glycoprotein is a VSV-G protein, and the VSV-G protein targets the system to retinal pigment epithelium (RPE) cells. In some 155 12954008Docket No.: B1195.70187WO00 embodiments, the viral envelope glycoprotein is an HIV-1 envelope glycoprotein, and the HIV-1 envelope glycoprotein targets the system to CD4+ cells. In some embodiments, the viral envelope glycoprotein is a FuG-B2 envelope glycoprotein, and the FuG-B2 envelope glycoprotein targets the system to neurons.

[0321] It will be appreciated that general methods are known in the art for producing viral vector particles, which generally contain coding nucleic acids of interest, may also be used for producing the virus-derived particles according to the present invention, which do not contain coding nucleic acids of interest but instead are designed to deliver a protein cargo (e.g., a PE RNP).

[0322] Conventional viral vector particles encompass retroviral, lentiviral, adenoviral and adeno-associated viral vector particles that are well known in the art. For a review of various viral vector particles that may be used, the one skilled in the art may notably refer to Kushnir et al. (2012, Vaccine, Vol.31: 58-83), Zeltons (2013, Mol Biotechnol, Vol.53: 92- 107), Ludwig et al. (2007, Curr Opin Biotechnol, Vol.18(no 6): 537-55) and Naskalaska et al. (2015, Vol.64 (no 1): 3-13). Further, references to various methods using virus-derived particles for delivering proteins to cells are found by the one skilled in the art in the article of Maetzig et al. (2012, Current Gene therapy, Vol.12: 389-409) as well as the article of Kaczmarczyk et al. (2011, Proc Natl Acad Sci USA, Vol.108 (no 41): 16998-17003).

[0323] Generally, a virus-like particle that is used according to the present disclosure, which virus-like particle may also be termed “virus-derived particle, ” is formed by one or more virus-derived structural protein(s) and / or one more virus-derived envelope protein.

[0324] A virus-like particle that is used according to the present invention is replication incompetent in a host cell wherein it has entered.

[0325] In preferred embodiments, a virus-like particle is formed by one or more retrovirus-derived structural protein(s) and optionally one or more virus-derived envelope protein(s).

[0326] In preferred embodiments, the virus-derived structural protein is a retroviral Gag protein or a peptide fragment thereof. As it is known in the art, Gag and Gag / pol precursors are expressed from full length genomic RNA as polyproteins, which require proteolytic cleavage, mediated by the retroviral protease (PR), to acquire a functional conformation. Further, Gag, which is structurally conserved among the retroviruses, is composed of at least three protein units: matrix protein (MA), capsid protein (CA) and 156 12954008Docket No.: B1195.70187WO00 nucleocapsid protein (NC), whereas Pol consists of the retroviral protease, (PR), the retrotranscriptase (RT) and the integrase (IN).

[0327] In some embodiments, a virus-derived particle comprises a retroviral Gag protein but does not comprise a Pol protein.

[0328] As it is known in the art, the host range of retroviral vector, including lentiviral vectors, may be expanded or altered by a process known as pseudotyping. Pseudotyped lentiviral vectors consist of viral vector particles bearing glycoproteins derived from other enveloped viruses. Such pseudotyped viral vector particles possess the tropism of the virus from which the glycoprotein is derived.

[0329] In some embodiments, a virus-like particle is a pseudotyped virus-like particle comprising one or more viral structural protein(s) or viral envelope protein(s) imparting a tropism to the said virus-like particle for certain eukaryotic cells. A pseudotyped virus-like particle as described herein may comprise, as the viral protein used for pseudotyping, a viral envelope protein selected in a group comprising VSV-G protein, Measles virus HA protein, Measles virus F protein, Influenza virus HA protein, Moloney virus MLV-A protein, Moloney virus MLV-E protein, Baboon Endogenous retrovirus (BAEV) envelope protein, Ebola virus glycoprotein and foamy virus envelope protein, or a combination of two or more of these viral envelope proteins.

[0330] A well-known illustration of pseudotyping viral vector particles consists of the pseudotyping of viral vector particles with the vesicular stomatitis virus glycoprotein (VSV- G). For the pseudotyping of viral vector particles, the one skilled in the art may notably refer to Yee et al. (1994, Proc Natl Acad Sci, USA, Vol.91: 9564-9568) Cronin et al. (2005, Curr Gene Ther, Vol.5(no 4): 387-398), which are incorporated herein by reference.

[0331] For producing virus-like particles, and more precisely VSV-G pseudotypes virus-like particles, for delivering protein(s) of interest into target cells, the one skilled in the art may refer to Mangeot et al. (2011, Molecular Therapy, Vol.19 (no 9): 1656-1666).

[0332] In some embodiments, a virus-like particle further comprises a viral envelope protein, wherein either (i) the said viral envelope protein originates from the same virus as the viral structural protein, e.g., originates from the same virus as the viral Gag protein, or (ii) the said viral envelope protein originates from a virus distinct from the virus from which originates the viral structural protein, e.g. originates from a virus distinct from the virus from which originates the viral Gag protein. 157 12954008Docket No.: B1195.70187WO00

[0333] As it is readily understood by the one skilled in the art, a virus-like particle that is used according to the disclosure may be selected in a group comprising Moloney murine leukemia virus-derived vector particles, Bovine immunodeficiency virus-derived particles, Simian immunodeficiency virus-derived vector particles, Feline immunodeficiency virus-derived vector particles, Human immunodeficiency virus-derived vector particles, Equine infection anemia virus-derived vector particles, Caprine arthritis encephalitis virus- derived vector particle, Baboon endogenous virus-derived vector particles, Rabies virus- derived vector particles, Influenza virus-derived vector particles, Norovirus-derived vector particles, Respiratory syncytial virus-derived vector particles, Hepatitis A virus-derived vector particles, Hepatitis B virus-derived vector particles, Hepatitis E virus-derived vector particles, Newcastle disease virus-derived vector particles, Norwalk virus-derived vector particles, Parvovirus-derived vector particles, Papillomavirus-derived vector particles, Yeast retrotransposon-derived vector particles, Measles virus-derived vector particles, and bacteriophage-derived vector particles.

[0334] In particular, a virus-like particle that is used according to the invention is a retrovirus-derived particle. Such retrovirus may be selected among Moloney murine leukemia virus, Bovine immunodeficiency virus, Simian immunodeficiency virus, Feline immunodeficiency virus, Human immunodeficiency virus, Equine infection anemia virus, and Caprine arthritis encephalitis virus.

[0335] In another embodiment, a virus-like particle that is used according to the disclosure is a lentivirus-derived particle. Lentiviruses belong to the retroviruses family, and have the unique ability of being able to infect non-dividing cells.

[0336] Such lentivirus may be selected among Bovine immunodeficiency virus, Simian immunodeficiency virus, Feline immunodeficiency virus, Human immunodeficiency virus, Equine infection anemia virus, and Caprine arthritis encephalitis virus.

[0337] For preparing Moloney murine leukemia virus-derived vector particles, one skilled in the art may refer to the methods disclosed by Sharma et al. (1997, Proc Natl Acad Sci USA, Vol.94: 10803+-10808), Guibingua et al. (2002, Molecular Therapy, Vol.5(no 5): 538-546), which are incorporated herein by reference. Moloney murine leukemia virus- derived (MLV-derived) vector particles may be selected in a group comprising MLV-A- derived vector particles and MLV-E-derived vector particles. 158 12954008Docket No.: B1195.70187WO00

[0338] For preparing Bovine Immunodeficiency virus-derived vector particles, the one skilled in the art may refer to the methods disclosed by Rasmussen et al. (1990, Virology, Vol.178(no 2): 435-451), which is incorporated herein by reference.

[0339] For preparing Simian immunodeficiency virus-derived vector particles, including VSV-G pseudotyped SIV virus-derived particles, the one skilled in the art may notably refer to the methods disclosed by Mangeot et al. (2000, Journal of Virology, Vol. 71(no 18): 8307-8315), Negre et al. (2000, Gene Therapy, Vol.7: 1613-1623) Mangeot et al. (2004, Nucleic Acids Research, Vol.32 (no 12), e102), which are incorporated herein by reference.

[0340] For preparing Feline Immunodeficiency virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Saenz et al. (2012, Cold Spring Harb Protoc, (1): 71-76; 2012, Cold Spring Harb Protoc, (1): 124-125; 2012, Cold Spring Harb Protoc, (1): 118-123), which are incorporated herein by reference.

[0341] For preparing Human immunodeficiency virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Jalaguier et al. (2011, PlosOne, Vol.6(no 11), e28314), Cervera et al. (J Biotechnol, Vol.166(no 4): 152-165), Tang et al. (2012, Journal of Virology, Vol.86(no 14): 7662-7676), which are incorporated herein by reference.

[0342] For preparing Equine infection anemia virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Olsen (1998, Gene Ther, Vol. 5(no 11): 1481-1487), which are incorporated herein by reference.

[0343] For preparing Caprine arthritis encephalitis virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Mselli-Lakhal et al. (2006, J Virol Methods, Vol.136(no 1-2): 177-184), which are incorporated herein by reference.

[0344] For preparing Baboon endogenous virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Girard-Gagnepain et al. (2014, Blood, Vol.124(no 8): 1221-1231), which is incorporated herein by reference.

[0345] For preparing Rabies virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Kang et al. (2015, Viruses, Vol.7: 1134-1152, doi:10.3390 / v7031134), Fontana et al. (2014, Vaccine, Vol.32(no 24): 2799-27804) or to the 159 12954008Docket No.: B1195.70187WO00 PCT application published under no WO 2012 / 0618, which is incorporated herein by reference.

[0346] For preparing Influenza virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Quan et al. (2012, Virology, Vol.430: 127- 135) and to Latham et al. (2001, Journal of Virology, Vol.75(no 13): 6154-6155), which is incorporated herein by reference.

[0347] For preparing Norovirus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Tomé-Amat et al., (2014, Microbial Cell Factories, Vol.13: 134-142), which is incorporated herein by reference.

[0348] For preparing Respiratory syncytial virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Walpita et al. (2015, PlosOne, DOI: 10.1371 / journal.pone.0130755), which is incorporated herein by reference.

[0349] For preparing Hepatitis B virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Hong et al. (2013, Vol.87(no 12): 6615- 6624), which is incorporated herein by reference.

[0350] For preparing Hepatitis E virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Li et al. (1997, Journal of Virology, Vol. 71(no 10): 7207-7213), which is incorporated herein by reference.

[0351] For preparing Newcastle disease virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Murawski et al. (2010, Journal of Virology, Vol.84(no 2): 1110-1123), which is incorporated herein by reference.

[0352] For preparing Norwalk virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Herbst-Kralovetz et al. (2010, Expert Rev Vaccines, Vol.9(no 3): 299-307), which is incorporated herein by reference.

[0353] For preparing Parvovirus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Ogasawara et al. (2006, In Vivo, Vol.20: 319- 324), which is incorporated herein by reference.

[0354] For preparing Papillomavirus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Wang et al. (2013, Expert Rev Vaccines, Vol.12(no 2): doi:10.1586 / erv.12.151), which is incorporated herein by reference.

[0355] A virus-like particle that is used herein comprises a Gag protein, and most preferably a Gag protein originating from a virus selected in a group comprising Rous 160 12954008Docket No.: B1195.70187WO00 Sarcoma Virus (RSV) Feline Immunodeficiency Virus (FIV), Simian Immunodeficiency Virus (SIV), Moloney Leukemia Virus (MLV) and Human Immunodeficiency Viruses (HIV-1 and HIV-2) especially Human Immunodeficiency Virus of type 1 (HIV-1).

[0356] In some embodiments, a virus-like particle may also comprise one or more viral envelope protein(s). The presence of one or more viral envelope protein(s) may impart to the said virus-derived particle a more specific tropism for the cells which are targeted, as it is known in the art. The one or more viral envelope protein(s) may be selected in a group comprising envelope proteins from retroviruses, envelope proteins from non-retroviral viruses, and chimeras of these viral envelope proteins with other peptides or proteins. An example of a non-lentiviral envelope glycoprotein of interest is the lymphocytic choriomeningitis virus (LCMV) strain WE54 envelope glycoprotein. These envelope glycoproteins increase the range of cells that can be transduced with retroviral derived vectors. Pharmaceutical compositions

[0357] Other aspects of the present disclosure relate to pharmaceutical compositions comprising any of the various prime editing system described herein (e.g., including, but not limited to, the napDNAbps, reverse transcriptases, fusion proteins (e.g., comprising napDNAbps and reverse transcriptases), extended guide RNAs, and complexes comprising fusion proteins and extended guide RNAs, as well as accessory elements, such as second strand nicking components and 5´ endogenous DNA flap removal endonucleases for helping to drive the multi-flap prime editing process towards the edited product formation).

[0358] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g. for specific delivery, increasing half-life, or other therapeutic compounds).

[0359] Some examples of materials which can serve as pharmaceutically-acceptable carriers include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, 161 12954008Docket No.: B1195.70187WO00 sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.

[0360] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, e.g., for gene editing. Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration.

[0361] In some embodiments, the pharmaceutical composition described herein is administered locally to a diseased site (e.g., tumor site). In some embodiments, the pharmaceutical composition described herein is administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.

[0362] In other embodiments, the pharmaceutical composition described herein is delivered in a controlled release system. In one embodiment, a pump may be used (see, e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng.14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med.321:574). In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, 162 12954008Docket No.: B1195.70187WO00 New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem.23:61. See also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol.25:351; Howard et al., 1989, J. Neurosurg.71:105). Other controlled release systems are discussed, for example, in Langer, supra.

[0363] In some embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human. In some embodiments, pharmaceutical composition for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0364] A pharmaceutical composition for systemic administration may be a liquid, e.g., sterile saline, lactated Ringer’s or Hank’s solution. In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated.

[0365] The pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein. Compounds can be entrapped in “stabilized plasmid-lipid particles” (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et al., Gene Ther.1999, 6:1438-47). Positively charged lipids such as N-[1-(2,3-dioleoyloxi)propyl]-N,N,N- trimethyl-amoniummethylsulfate, or “DOTAP,” are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Patent Nos. 163 12954008Docket No.: B1195.70187WO00 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference.

[0366] The pharmaceutical composition described herein may be administered or packaged as a unit dose, for example. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

[0367] Further, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a compound of the invention in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized compound of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0368] In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierce- able by a hypodermic injection needle. The active agent in the composition is a compound of the invention. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. EXAMPLES 164 12954008Docket No.: B1195.70187WO00 Example 1. Mammals lack an endogenous mechanism to readthrough expanded codons or frameshift mutations.

[0369] To demonstrate that mammalian cells lack endogenous mechanisms to readthrough expanded codons or frameshift mutations, a reporter plasmid was designed with an expanded codon by inserting a single nucleotide at the permissive Y151 of EGFP and a silent mutation in D155, four amino acids upstream of the insertion (FIGs.4A-4E). This allows for an early PTC and protein truncation if translated by a tRNA or a frameshift suppression if translated by a qtRNA. As FIGs.4A-4E show, when Y151 is replaced with the expanded codons GGGG or TAGA (e.g., causing a frameshift mutation), EGFP fluorescence was not detected. In contrast, when Y151 was replaced with GGG or TCG, EGFP fluorescence was detected. Collectively, these results demonstrate that mammalian cells do not possess the machinary to readthrough frameshift mutations. Example 2. Generating endogenous suppressor qtRNA using prime editing to promote therapeutic readthrough of disease causing-frameshift mutations.

[0370] To demonstrate that qtRNAs can be used to readthrough the GGGG and TAGA frameshift mutations in the EGFP reporter plasmids described in Example 1, cells were treated with plasmids encoding either three isoforms of Homo Sapiens tRNA-Gly-CCC and four isoforms or Homo Sapiens tRNA-Ser-CGA that were converted to suppressor qtRNAs by expanding the anticodons to accommodate the frameshift mutation. As shown in FIGs.5A-5B, compared to cells exogenously expressing tRNAs with triplet anticodons, only cells exogenously expressing qtRNAs shows readthrough of the frameshifted EGFP reporter plasmids. Importantly, while others have shown that in bacteria, the efficiency of quadruplet decoding can be improved by accumulating additional mutations along the sides of the anticodon loop, however, these specific mutations did not improve read through efficiency in mammalian cells (see FIG.5B). REFERENCES

[0371] Riddle DL, Carbon J. Frameshift Suppression: a Nucleotide Addition in the Anticodon of a Glycine Transfer RNA. Nature New Biology.1973;242(121):230-4.

[0372] Riddle DL, Roth JR. Suppressors of frameshift mutations in Salmonella typhimurium. J Mol Biol.1970;54(1):131-44.

[0373] Riddle DL, Roth JR. Frameshift suppressors: II. Genetic mapping and dominance studies. Journal of Molecular Biology.1972;66(3):483-93. 165 12954008Docket No.: B1195.70187WO00

[0374] Roth JR. Frameshift suppression. Cell.1981;24(3):601-2.

[0375] Myerowitz R, Costigan FC. The major defect in Ashkenazi Jews with Tay- Sachs disease is an insertion in the gene for the alpha-chain of beta-hexosaminidase. J Biol Chem.1988;263(35):18587-9.

[0376] Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature. 2001;411(6837):603-6

[0377] Sanz J, von Känel T, Schneider M, Steiner B, Schaller A, Gallati S. The CFTR frameshift mutation 3905insT and its effect at transcript and protein level. European Journal of Human Genetics.2010;18(2):212-7

[0378] Kirby A, Gnirke A, Jaffe DB, Barešová V, Pochet N, Blumenstiel B, et al. Mutations causing medullary cystic kidney disease type 1 lie in a large VNTR in MUC1 missed by massively parallel sequencing. Nat Genet.2013;45(3):299-303

[0379] Kadiyska TK, Kaneva RP, Nedin DG, Alexandrova AB, Gegova AT, Lalchev SG, et al. Novel MLH1 frameshift mutation in an extended hereditary nonpolyposis colorectal cancer family. World J Gastroenterol.2006;12(48):7848-51

[0380] Spaanderman IT, Peters FS, Jongejan A, Redeker EJW, Punt CJA, Bins AD. Framing the potential of public frameshift peptides as immunotherapy targets in colon cancer. PLoS One.2021;16(6):e0251630

[0381] Giegé R, Eriani G. The tRNA identity landscape for aminoacylation and beyond. Nucleic Acids Res.2023;51(4):1528-70.

[0382] Giegé R, Sissler M, Florentz C. Universal rules and idiosyncratic features in tRNA identity. Nucleic Acids Res.1998;26(22):5017-35.

[0383] DeBenedictis EA, Söll D, Esvelt KM. Measuring the tolerance of the genetic code to altered codon size. eLife.2022;11:e76941 , citation = eLife 2022;11 e.

[0384] Hong S, Sunita S, Maehigashi T, Hoffer ED, Dunkle JA, Dunham CM. Mechanism of tRNA-mediated +1 ribosomal frameshifting. Proc Natl Acad Sci U S A. 2018;115(44):11226-31.

[0385] Lant JT, Berg MD, Heinemann IU, Brandl CJ, O'Donoghue P. Pathways to disease from natural variations in human cytoplasmic tRNAs. J Biol Chem. 2019;294(14):5294-308. 166 12954008Docket No.: B1195.70187WO00

[0386] Iannuzzi MC, Stern RC, Collins FS, Hon CT, Hidaka N, Strong T, et al. Two frameshift mutations in the cystic fibrosis gene. Am J Hum Genet.1991;48(2):227-31.

[0387] Roth JR. Frameshift suppression. Cell.1981;24(3):601-2.

[0388] Riddle DL, Carbon J. Frameshift Suppression: a Nucleotide Addition in the Anticodon of a Glycine Transfer RNA. Nature New Biology.1973;242(121):230-4.

[0389] DeBenedictis EA, Carver GD, Chung CZ, Söll D, Badran AH. Multiplex suppression of four quadruplet codons via tRNA directed evolution. Nature Communications. 2021;12(1):5706.

[0390] Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576(7785):149-57 EQUIVALENTS AND SCOPE

[0391] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0392] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim may be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) may be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements and / or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that 167 12954008Docket No.: B1195.70187WO00 the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub–range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0393] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention may be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0394] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims. 168 12954008

Claims

Docket No.: B1195.70187WO00 CLAIMS What is claimed is:

1. A method for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA comprising contacting the DNA sequence at a target site with a prime editor and one or more pegRNAs, wherein the prime editor installs one or more modifications in the DNA sequence at the target site, relative to the DNA sequence encoding the endogenous tRNA, thus converting the edited DNA sequence into a DNA sequence encoding a suppressor qtRNA.

2. The method of either claim 1, wherein the one or more pegRNA comprises a spacer sequence, a gRNA core, and an extension arm.

3. The method of claim 1 or 2, wherein the pegRNA sequence is any sequence listed in SEQ ID NOs: 431-2199724.

4. The method of any one of claims 1-3, wherein the DNA sequence encoding the endogenous tRNA is any sequence listed in SEQ ID NOs 1-430 or 2306989-2307418.

5. The method of any one of claims 1-4, wherein the suppressor qtRNA comprises an expanded anticodon sequence.

6. The method of any one of claims 1-5, wherein the target site in the DNA sequence encodes one or more domains of the endogenous tRNA.

7. The method of claim 6, wherein the one or more domains is a D-arm domain of the endogenous tRNA.

8. The method of claims 6 or 7, wherein the one or more domains is a T-arm domain of the endogenous tRNA. 169 12954008Docket No.: B1195.70187WO00 9. The method of any one of claims 6-8, wherein the one or more domains is a variable arm domain of the endogenous tRNA.

10. The method of any one of claims 6-9, wherein the one or more domains is an acceptor stem domain of the tRNA.

11. The method of any one of claims 6-10, wherein the one or more domains is an anticodon arm domain comprising an anticodon sequence of the tRNA.

12. The method of any one of claims 1-11, wherein installing the one or more modifications comprises installing one or more nucleotide insertions in the anticodon sequence of the anticodon arm domain.

13. The method of claim 12, wherein installing the one or more modification comprises installing a single nucleotide insertion in the anticodon sequence of the anticodon arm domain.

14. The method of claim 12, wherein installing the one or more modification comprises installing a two nucleotide insertions in the anticodon sequence of the anticodon arm domain.

15. The method of any one of claims 1-14, wherein installing the one or more modifications comprises installing one or more nucleotide insertions, deletions, or substitutions in an acceptor stem domain.

16. The method of any one of claims 1-15, wherein installing the one or more modifications comprises installing one or more nucleotide insertions, deletions, or substitutions in the D-arm domain.

17. The method of any one of claims 1-16, wherein installing the one or more modifications comprises installing one or more nucleotide insertions, deletions, or substitutions in the T-arm domain. 170 12954008Docket No.: B1195.70187WO00 18. The method of any one of claims 1-17, wherein installing the one or more modifications comprises installing one or more nucleotide insertions, deletions, or substitutions in the variable arm domain.

19. The method of any one of claims 1-18, wherein the expanded codon comprises 4 or more nucleotides.

20. The method of any one of claims 1-19, wherein the pegRNA further comprises a stabilizing 3′-tevopreQi motif.

21. The method of any one of claims 1-20, wherein the pegRNA directs the prime editor to install an edit at the target site located between positions +1 and +40 relative to a first editable base located 3′ of a pegRNA-directed nick.

22. The method of any one of claims 1-21, wherein the pegRNA directs the prime editor to install an edit at the target site located between positions +11 and +17, relative to a first editable base located 3′ of a pegRNA-directed nick.

23. The method of any one of claims 1-22, wherein the extension arm comprises a DNA synthesis template and a primer binding site (PBS).

24. The method of claim 23, wherein the DNA synthesis template encodes one or more nucleotide insertions to be installed at the target site of the DNA sequence encoding the anticodon arm domain of the tRNA.

25. The method of claim 23 or 24, wherein the DNA synthesis template encodes one or more nucleotide insertions, deletions, or substitutions to be installed at the target site of the DNA sequence encoding the D-arm domain of the tRNA. 171 12954008Docket No.: B1195.70187WO00 26. The method of any one of claims 23-25, wherein the DNA synthesis template encodes one or more nucleotide insertions, deletions, or substitutions to be installed at the target site of the DNA sequence encoding the T-arm domain of the tRNA.

27. The method of any one of claims 23-26, wherein the DNA synthesis template encodes one or more nucleotide insertions, deletions, or substitutions to be installed at the target site of the DNA sequence encoding the variable arm domain of the tRNA.

28. The method of any one of claims 23-27, wherein the DNA synthesis template encodes one or more nucleotide insertions, deletions, or substitutions to be installed at the target site of the DNA sequence encoding the acceptor stem arm domain of the tRNA.

29. The method of any one of claims 23-28, wherein the DNA synthesis template further encodes one or more PAM-disrupting mutations, MMR-evading mutations, or combinations thereof.

30. The method of any one of claims 1-29, further comprising an gRNA.

31. The method of any one of claims 1-30, further comprising a second pegRNA.

32. The method of any one of claims 1-31, wherein the prime editor comprises twinPE.

33. A composition for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA, comprising a prime editor and a pegRNAand wherein the pegRNA is configured to bind to a DNA sequence encoding an endogenous tRNA.

34. The composition of claim 33, wherein the pegRNA comprises a spacer sequence, a core gRNA, and an extension arm comprising a DNA synthesis template and a primer binding site (PBS). 172 12954008Docket No.: B1195.70187WO00 35. The composition of claim 34, wherein the DNA synthesis template encodes one or more edits to be installed at a target site in the DNA sequence encoding the endogenous tRNA.

36. The composition of claim 35, wherein the one or more edits is selected from the group consisting of insertions, deletions, and substitutions.

37. The composition of claim 35 or 36, wherein the DNA synthesis template encodes at least one nucleotide insertion to be installed at the target site of the DNA sequence encoding an anticodon sequence of the endogenous tRNA.

38. The composition of any one of claims 35-37, wherein the DNA synthesis template encodes one or more nucleotide insertions, deletions, or substitutions to be installed at the target site of the DNA sequence encoding the D-arm domain of the tRNA.

39. The composition of any one of claims 35-38, wherein the DNA synthesis template encodes one or more nucleotide insertions, deletions, or substitutions to be installed at the target site of the DNA sequence encoding the T-arm domain of the tRNA.

40. The composition of any one of claims 35-39, wherein the DNA synthesis template encodes one or more nucleotide insertions, deletions, or substitutions to be installed at the target site of the DNA sequence encoding the variable arm domain of the tRNA.

41. The composition of any one of claims 35-40, wherein the DNA synthesis template encodes one or more nucleotide insertions, deletions, or substitutions to be installed at the target site of the DNA sequence encoding the acceptor stem arm domain of the tRNA.

42. The composition of any one of claims 35-40, wherein the DNA synthesis template further encodes a PAM-disrupting mutation, an MMR-evading mutation, or any combination thereof, relative to the endogenous tRNA gene. 173 12954008Docket No.: B1195.70187WO00 43. The composition of any one of claims 33-42, further comprising an sgRNA.

44. The composition of any one of claims 33-43, further comprising a second pegRNA.

45. The composition of any one of claims 33-44, wherein the prime editor comprises twinPE.

46. A prime editing guide RNA (pegRNA) for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA, wherein the pegRNA comprises a spacer sequence, a gRNA core, and an extension arm comprising a DNA synthesis template and a primer binding site (PBS), wherein the spacer sequence is configured to bind to a DNA sequence encoding an endogenous tRNA.

47. The pegRNA of claim 46 further comprising a 3′-tevopreQi motif.

48. The pegRNA of claim 46 or 47, wherein the pegRNA sequence is any sequence listed in SEQ ID NOs: 431-2199724.

49. The pegRNA of any one of claims 46-48, wherein the spacer sequence comprises a PAM sequence.

50. The pegRNA of claim 49, wherein the PAM sequence is NGG.

51. The pegRNA of any one of claims 46-50, wherein the DNA synthesis template encodes a sequence that is complimentary to any one of the sequences listed SEQ ID NOs. 431-2199724.

52. The pegRNA of claim 51, wherein the DNA synthesis template encodes one or more edits to be installed at a target site in the DNA sequence encoding the endogenous 174 12954008Docket No.: B1195.70187WO00 tRNA, wherein the one or more edits is selected from the group consisting of insertions, deletions, and substitutions.

53. The pegRNA of any one of claims 46-52, wherein the DNA synthesis template encodes at least one nucleotide insertion to be installed at the target site of the DNA sequence encoding an anticodon sequence of the endogenous tRNA.

54. The pegRNA of claim 53, wherein the at least one nucleotide insertion converts the endogenous DNA sequence encoding the endogenous tRNA into a DNA sequence encoding the suppressor qtRNA.

55. The pegRNA of any one of claims 46-54, wherein the DNA synthesis template encodes one or more edits to be installed in the DNA sequence encoding an acceptor stem domain of the tRNA, relative to the endogenous tRNA.

56. The pegRNA of any one of claims 46-55, wherein the DNA synthesis template encodes one or more edits to be installed in the DNA sequence encoding a variable arm domain of the tRNA, relative to the endogenous tRNA.

57. The pegRNA of any one of claim 46-56, wherein the DNA synthesis template further encodes for one or more PAM-disrupting mutations, MMR-evading mutations, or combinations thereof.

58. A complex comprising a prime editor and a pegRNA for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA, wherein the pegRNA comprises a spacer sequence and an extension arm, wherein the spacer sequence is configured to bind to the DNA sequence encoding the endogenous tRNA.

59. A polynucleotide for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA, the polynucleotide comprising a first nucleic acid sequence encoding a prime editor and a second nucleic acid 175 12954008Docket No.: B1195.70187WO00 sequence encoding a pegRNA, wherein the pegRNA is configured to bind to a DNA sequence encoding an endogenous tRNA.

60. The polynucleotide of claim 59, wherein the pegRNA comprises a protospacer sequence with at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% sequence identity to any sequence listed in SEQ ID NOs: 431-2199724.

61. The polynucleotide of claim 59, wherein the pegRNA comprises an extension arm with at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% sequence identity to any sequence listed in SEQ ID NOs: 431-2199724.

62. A cell comprising a complex of claim 58 or a polynucleotide of any one of claims 59-61.

63. A pharmaceutical composition comprising a composition of any one of claims 33- 51, a pegRNA of any one of claims 46-57, a complex of claim 58, a polynucleotide of any one of claims 59-61, a cell of any one of claims 62, or any combination thereof, and a pharmaceutical excipient.

64. A kit comprising a composition of any one of claims 33-51, a pegRNA of any one of claims 46-57, a complex of claim 58, a polynucleotide of any one of claims 59-61, a cell of claim 62, or any combination thereof, a pharmaceutical excipient, and instructions for editing a one or more DNA sequences encoding one or more domains of a tRNA by prime editing.

65. A method for inserting a suppressor qtRNA gene into a target site in a host genome using prime editing, the method comprising contacting the target site with a prime editor and a pegRNA, wherein the prime editor and the pegRNA install the suppressor qtRNA gene sequence in the target site in the host genome.

66. The method of claim 65, wherein the prime editor comprises a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a DNA polymerase. 176 12954008Docket No.: B1195.70187WO00 67. The method of claim 64 or 65 wherein the pegRNA comprises a spacer sequence, a gRNA core, and an extension arm comprising a DNA synthesis template and a primer binding site (PBS).

68. The method of claim 67, wherein the spacer sequence comprises a region of complementarity to a target strand of a double stranded target tRNA gene sequence in the subject, wherein the gRNA core associates with the napDNAbp.

69. The method of claim 67 or 68, wherein the DNA synthesis template comprises a region of complementarity to the non-target strand of the double-stranded target tRNA gene sequence and encodes a suppressor qtRNA gene sequence to be installed within the target site.

70. The method of 67-69, wherein the primer binding site comprises a region of complementarity to a non-target strand of the double-stranded target tRNA gene sequence.

71. The method of any one of claims 65-70, wherein installation of the suppressor qtRNA gene results in the indefinite expression of the suppressor qtRNAs gene.

72. The method of any one of claims 65-71, wherein the suppressor qtRNA gene encodes a single nucleotide insertion in an anticodon sequence within an anticodon arm domain of the tRNA, relative to the endogenous tRNA.

73. The method of any one of claims 65-72, wherein the suppressor qtRNA gene sequence encodes for a qtRNA comprising and amino acid and an anticodon sequence, wherein the anticodon sequence comprises 4 or more nucleotides.

74. The method of any one of claims 73, wherein the suppressor qtRNA allows readthrough of frameshift mutations. 177 12954008Docket No.: B1195.70187WO00 75. The method of any one of claims 65-74, wherein the target site comprises a safe harbor locus site.

76. The method of claim 75, wherein the safe harbor site comprises a ROSA26 gene.

77. The method of claims 75 or 76, wherein the safe harbor site comprises a AAVS1 gene.

78. The method of any one of claims 75-77, wherein the safe harbor site comprises a CCR5 gene.

79. The method of any one of claims 65-78, wherein the target site comprises a general expression site.

80. The method of claim 79, wherein the general expression site comprises the ALB gene.

81. The method of any one of claims 65-80, wherein the polymerase is a reverse transcriptase.

82. The method of any one of claims 65-81, wherein the fusion protein further comprises a recombinase.

83. The method of claim 82, wherein the recombinase comprises an integrase.

84. The method of claims 83, wherein the integrase is a serine integrase.

85. The method of claim 84, wherein the serine integrase is Bxb1.

86. The method of any one of claims 65-85, wherein the suppressor qtRNA gene is derived from an endogenous tRNA of the host genome. 178 12954008Docket No.: B1195.70187WO00 87. The method of any one of claims 65-86, wherein the suppressor qtRNA gene is not derived from the endogenous tRNA of the host genome.

88. The method of any one of claims 65-87, wherein the host genome is a human genome.

89. A method for inserting a DNA sequence encoding a suppressor qtRNA gene into a target site in a host genome using prime editing, the method comprising contacting the target site with (i) a prime editor, (ii) a pegRNA, (iii) a sgRNA, and (iv) a nucleic acid, wherein the prime editor comprises a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp), a DNA polymerase, and a recombinase.

90. The method of claim 89, wherein the target site comprises a safe harbor locus site.

91. The method of claim 90, wherein the safe harbor site comprises a ROSA26 gene.

92. The method of claim 90 or 91, wherein the safe harbor site comprises a AAVS1 gene.

93. The method of any one of claims 90-92, wherein the safe harbor site comprises a CCR5 gene.

94. The method of any one of claims 89-93, wherein the target site comprises a general expression site.

95. The method of claim 94, wherein the general expression site comprises the ALB gene.

96. The method of any one of claims 89-95, wherein the polymerase is a reverse transcriptase.

97. The method of claim 89-96, wherein the recombinase comprises an integrase. 179 12954008Docket No.: B1195.70187WO00 98. The method of claim 97, wherein the integrase is a serine integrase.

99. The method of claim 98, wherein the serine integrase is Bxb1.

100. The method of any one of claims 89-99, wherein the nucleic acid encodes for the suppressor qtRNA comprising an anticodon sequence that is complementary to an extended codon.

101. The method of claim 100, wherein the nucleic acid is selected from the group consisting of a circular plasmid, a ssRNA, a ssDNA, and dsDNA.

102. The method of any one of claims 89-101, wherein the nucleic acid comprises a first recombinase recognition site.

103. The method of claim 102, wherein the first recombinase recognition site comprises an AttB sequence.

104. The method of claim 102, wherein the first recombinase recognition site comprises an AttP sequence.

105. The method of any one of claims 89-104, wherein the pegRNA comprises a spacer sequence, a gRNA core, and an extension arm comprising a DNA synthesis template and a primer binding site (PBS).

106. The method of claim 105, wherein the DNA synthesis template encodes a single stranded DNA sequence encoding a second recombinase recognition site.

107. The method of claim 106, wherein the second recombinase recognition site comprises an AttB sequence. 180 12954008Docket No.: B1195.70187WO00 108. The method of claim 106 or 107, wherein the second recombinase recognition site comprises an AttP sequence.

109. The method of claim 89-108, wherein the recombinase recombines the nucleic acid and the single strand DNA sequence via the first / second recognition recombination sites.

110. The method of any one of claims 89-109, further comprising a nicking guide RNA.

111. The method of any one of claims 89-110, wherein installation of the suppressor qtRNA gene at the target site results in the indefinite expression of the suppressor qtRNAs gene.

112. The method of any one of claims 89-111, wherein the DNA encoding the suppressor qtRNA is derived from the endogenous tRNA of the host genome.

113. The method of any one of claims 89-112, wherein the DNA encoding the suppressor qtRNA is not derived from the endogenous tRNA of the host genome.

114. The method of any one of claims 89-113, wherein the host genome is a human or mouse genome.

115. A method for treating a disease caused by frameshift mutations, the method comprising converting an endogenous tRNA gene into a suppressor qtRNA gene using prime editing, the method comprising administering to a subject (i) a prime editor and (ii) one or more pegRNAs, wherein the suppressor qtRNA gene encodes a suppressor qtRNA capable of binding to an expanded codon.

116. The method of claim 115, wherein the disease is selected from the group consisting of cancer, Crohn’s disease, cystic fibrosis, HIV, Tay-Sachs disease, Smith- Magenis syndrome, hypertrophic cardiomyopathy, thalassemia, Huntington’s disease, MUC1 kidney disease, and Alzheimer's disease. 181 12954008Docket No.: B1195.70187WO00 117. A method for treating a disease caused by frameshift mutations, the method comprising installing a suppressor qtRNA gene into a target site in a host genome using prime editing, the method comprising administering to a subject (i) a prime editor and (ii) one or more pegRNAs, wherein the suppressor qtRNA gene encodes a suppressor qtRNA capable of binding to an expanded codon.

118. The method of claim 117, wherein the disease is selected from the group consistin gof cancer, Crohn’s disease, cystic fibrosis, HIV, Tay-Sachs disease, Smith-Magenis syndrome, hypertrophic cardiomyopathy, thalassemia, Huntington’s disease, MUC1 kidney disease, and Alzheimer's disease.

119. The use of a prime editor and a pegRNA for the manufacture of a medicament for editing a DNA sequence encoding an endogenous tRNA to produce a DNA sequence encoding a suppressor qtRNA, comprising contacting the DNA sequence at a target site with the prime editor and one or more pegRNAs, wherein the prime editor installs one or more modifications in the DNA sequence at the target site, relative to the DNA sequence encoding the endogenous tRNA, thus converting the edited DNA sequence into a DNA sequence encoding a suppressor qtRNA.

120. The use of a prime editor and a pegRNA in the manufacture of a medicament for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA, wherein the pegRNA is configured to bind to a DNA sequence encoding an endogenous tRNA.

121. The use of a prime editing guide RNA (pegRNA) in the manufacture of a medicament for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA, wherein the pegRNA comprises a spacer sequence, a gRNA core, and an extension arm comprising a DNA synthesis template 182 12954008Docket No.: B1195.70187WO00 and a primer binding site (PBS), wherein the spacer sequence is configured to bind to a DNA sequence encoding an endogenous tRNA.

122. The use of a complex, comprising a prime editor and a pegRNA, in the manufacture of a medicament for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA, wherein the pegRNA comprises a spacer sequence and an extension arm, wherein the spacer sequence is configured to bind to the DNA sequence encoding the endogenous tRNA.

123. The use of a polynucleotide in the manufacture of a medicament for editing a DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor qtRNA, the polynucleotide comprising a first nucleic acid sequence encoding a prime editor and a second nucleic acid sequence encoding a pegRNA, wherein the pegRNA is configured to bind to a DNA sequence encoding an endogenous tRNA.

124. The use of a cell in the manufacture of a medicament for editing a DNA sequence, wherein the cell comprises the complex of claim 58 or the polynucleotide of any one of claims 59-61.

125. The use of a pharmaceutical composition in the manufacture of a medicament for editing a DNA sequence, wherein the pharmaceutical composition comprises the composition of any one of claims 33-45, the pegRNA of any one of claims 46-57, the complex of claim 58, the polynucleotide of any one of claims 59-61, the cell of claim 62, or any combination thereof, and a pharmaceutical excipient.

126. The use of a a prime editor and a pegRNA, in the manufacture of a medicament for inserting a suppressor qtRNA gene into a target site in a host genome, comprising contacting the target site with the prime editor and a pegRNA, wherein the prime editor and the pegRNA install the suppressor qtRNA gene sequence in the target site in the host genome. 183 12954008Docket No.: B1195.70187WO00 127. The use of a composition for inserting a DNA sequence encoding a suppressor qtRNA gene into a target site in a host genome using prime editing, comprising contacting the target site with the composition, wherein the composition comprises (i) a prime editor, (ii) a pegRNA, (iii) a sgRNA, and (iv) a nucleic acid, wherein the prime editor comprises a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp), a DNA polymerase, and a recombinase.

128. The use of a prime editor and one or more pegRNAs for treating a disease caused by frameshift mutations comprising converting an endogenous tRNA gene into a suppressor qtRNA gene using prime editing, the comprising administering to a subject the prime editor and the one or more pegRNAs, wherein the suppressor qtRNA gene encodes a suppressor qtRNA capable of binding to an expanded codon.

129. The use of a prime editor and one or more pegRNAs for treating a disease caused by frameshift mutations comprising installing a suppressor qtRNA gene into a target site in a host genome using prime editing, comprising administering to a subject the prime editor and the one or more pegRNAs, wherein the suppressor qtRNA gene encodes a suppressor qtRNA capable of binding to an expanded codon.

130. A suppressor qtRNA for reading through frameshift mutations in an mRNA, wherein the suppressor qtRNA comprises an expanded anticodon sequence comprising 4 or 5 amino acids.

131. A polynucleotide encoding the suppressor qtRNA of claim 130.

132. A vector encoding the polynucleotide of claim 131.

133. A cell comprising the suppressor qtRNA of claim 130, the polynucleotide of claim 131, or the vector of claim 132.

134. A cell comprising a DNA sequence encoding an endogenous tRNA, wherein the endogenous tRNA has been edited to a DNA sequence encoding a qtRNA. 184 12954008Docket No.: B1195.70187WO00 135. A prime editing guide RNA comprising: (i) a spacer sequence; (ii) a core sequence; and (iii) an extension arm sequence, wherein the spacer sequence comprises a region of complementarity to a target strand of a double stranded target tRNA gene sequence encoding a triplet anticodon sequence, wherein the extension arm sequences encodes a primer binding site sequence (PBS), an expanded anticodon sequence, and a 3′-flap sequence, wherein the PBS comprises a region of complementarity to a non-target strand of the double-stranded target tRNA gene sequence, the non-target strand comprising a 3´end generated from a nick site in the R-loop of the non-target strand, and wherein the PBS is between 8 to 16 nucleotides downstream of, and adjacent to, an expanded anticodon sequence, wherein the expanded anticodon sequence comprises 4 nucleotides having a first nucleotide position, a second nucleotide position, a third nucleotide position, and a fourth nucleotide position, and wherein the 3′-flap sequence is complementary to at least a portion of the target strand and is between 3 and 30 nucleotides upstream of, and adjacent to, the expanded anticodon sequence.

136. The pegRNA of claim 135, wherein the expanded anticodon sequence comprises an A, G, T, or C in the first nucleotide position, second nucleotide position, third nucleotide position and fourth nucleotide position.

137. A prime editing guide RNA comprising: (i) a spacer sequence; (ii) a core sequence; and (iii) an extension arm sequence, wherein the spacer sequence comprises a region of complementarity to a target strand of a double stranded target tRNA gene sequence encoding a triplet anticodon sequence, 185 12954008Docket No.: B1195.70187WO00 wherein the extension arm sequences encodes a primer binding site sequence (PBS), an expanded anticodon sequence, and a 3′-flap sequence, wherein the PBS comprises a region of complementarity to a non-target strand of the double-stranded target tRNA gene sequence, the non-target strand comprising a 3´end generated from a nick site in the R-loop of the non-target strand, and wherein the PBSis between 8 to 16 nucleotides downstream of, and adjacent to, the expanded anticodon sequence, wherein the expanded anticodon sequence comprises 5 nucleotides having a first nucleotide position, a second nucleotide position, a third nucleotide position, a fourth nucleotide position, and a fifth nucleotide position, and wherein the 3′-flap sequence is complementary to at least a portion of the target strand and is between 3 and 30 nucleotides upstream of, and adjacent to, the expanded anticodon sequence.

138. The pegRNA of claim 137, wherein the expanded anticodon sequence comprises an A, G, T, or C in the first nucleotide position, second nucleotide position, third nucleotide position, fourth nucleotide position, and fifth nucleotide position.

139. A prime editing guide RNA comprising: (i) a spacer sequence; (ii) a core sequence; and (iii) an extension arm sequence, wherein the spacer sequence comprises a region of complementarity to a non-target strand of a double stranded target tRNA gene sequence encoding a triplet anticodon sequence, wherein the extension arm sequences encodes a primer binding site sequence (PBS), an expanded anticodon sequence, and a 5′-flap sequence, wherein the PBS comprises a region of complementarity to a target strand of the double-stranded target tRNA gene sequence, the target strand comprising a 5´end generated from a nick site in R-loop of the target strand, and wherein the PBS is between 8 to 16 nucleotides upstream of, and adjacent to, the expanded anticodon sequence, 186 12954008Docket No.: B1195.70187WO00 wherein the expanded anticodon sequence comprises 4 nucleotides having a first nucleotide position, a second nucleotide position, a third nucleotide position, and a fourth nucleotide position, and wherein the 5′-flap sequence is complementary to at least a portion of the non-target strand and is between 3 and 30 nucleotides downstream of, and adjacent to, the expanded anticodon sequence.

140. The pegRNA of claim 139, wherein the expanded anticodon sequence comprises an A, G, T, or C in the first nucleotide position, second nucleotide position, third nucleotide position and fourth nucleotide position.

141. A prime editing guide RNA comprising: (i) a spacer sequence; (ii) a core sequence; and (iii) an extension arm sequence, wherein the spacer sequence comprises a region of complementarity to a non-target strand of a double stranded target tRNA gene sequence encoding a triplet anticodon sequence, wherein the extension arm sequences encodes a primer binding site sequence (PBS), an expanded anticodon sequence, and a 5′-flap sequence, wherein the PBS comprises a region of complementarity to a target strand of the double-stranded target tRNA gene sequence, the target strand comprising a 5´end generated from a nick site in R-loop of the target strand, and wherein the PBS is between 8 to 16 nucleotides upstream of, and adjacent to, the expanded anticodon sequence, wherein the expanded anticodon sequence comprises 5 nucleotides having a first nucleotide position, a second nucleotide position, a third nucleotide position, a fourth nucleotide position, and a fifth nucleotide position, and wherein the 5′-flap sequence is complementary to at least a portion of the non-target strand and is between 3 and 30 nucleotides downstream of, and adjacent to, the expanded anticodon sequence. 187 12954008Docket No.: B1195.70187WO00 142. The pegRNA of claim 141, wherein the expanded anticodon sequence comprises an A, G, T, or C in the first nucleotide position, second nucleotide position, third nucleotide position, fourth nucleotide position, and fifth nucleotide position. 188 12954008

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