Prime editing-mediated readthrough of premature termination codons (PERT)

Abstract

Aspects of the disclosure relate to methods, uses, compositions, kits, and systems for editing an endogenous tRNA into a suppressor tRNA. Additional aspects relate to compositions comprising the prime editing machinery, pegRNAs, and / or complexes comprising the prime editor and pegRNA that are capable of editing and / or replacing an endogenous tRNA to yield a suppressor tRNA. Polynucleotides, cells, vectors, and complexes are also contemplated.

Description

PRIME EDITING-MEDIATED READTHROUGH OF PREMATURE TERMINATION CODONS (PERT)RELATED APPLICATIONS

[0001] This application claims priority under 35 U. S. C. § 119(e) to U. S. Provisional Applications, U. S. S. N. 63 / 735,279, filed December 17, 2024, U. S. S. N. 63 / 842,686, filed July 11, 2025, and U. S. S. N. 63 / 921,020, filed November 19, 2025, each of which is incorporated herein by reference.GOVERNMENT SUPPORT

[0002] This invention was made with government support under 2RM1HG009490 awarded by the National Human Genome Research Institute (NHGRI) and 2R35GM118062 awarded by the National Institute of General Medical Sciences (NIGMS). The government has certain rights in the invention.BACKGROUND

[0003] Nonsense mutations in genomic DNA lead to premature termination codons (PTCs) in mRNAs, which in turn impede translation of full-length proteins. Diminished translation of full-length proteins due to PTCs can induce pathogenic effects in cells and organisms. Indeed, approximately 33% of known human genetic diseases and 11% of known pathogenic gene variants are caused by PTCs. Interestingly, many bacteria and viruses utilize suppressor tRNAs to enable translational stop codon readthrough (e.g., the ribosome goes past the stop codon and continues translating the mRNA into protein). However, suppressor tRNAs do not naturally occur in humans. Suppressor tRNAs were recently used to rescue a genetic disease in a mouse model carrying a nonsense mutation. While the therapy was found safe, the suppressor tRNA was delivered via an adeno-associated viral vector (herein “AAV”). It is generally known in the art that permanent expression of the suppressor tRNA is necessary for continued rescue from the disease, which is challenging to achieve using AAV and requires repeated administration of the suppressor tRNA vector. Prime editing allows for precise editing of the genomic DNA encoding tRNAs and may provide a platform for the treatment of diseases associated with PTCs.1 / 346Bl 195.70209 WOOO#14646633v2SUMMARY

[0004] Aspects of the disclosure relate to methods, uses, compositions, kits, and systems for editing an endogenous tRNA into a suppressor tRNA. Additional aspects relate to compositions comprising the prime editing machinery, pegRNAs, and / or complexes comprising the prime editor and pegRNA that are capable of editing and / or replacing an endogenous tRNA to yield a suppressor tRNA. Polynucleotides, cells, vectors, non-human organisms, kits, and complexes are also contemplated.

[0005] In some aspects, the disclosure relates to a prime editing guide RNA (pegRNA) for editing a target DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor tRNA, wherein the pegRNA includes a DNA synthesis template, (i) wherein the DNA synthesis template encodes a nonsense suppressor anticodon sequence to be inserted into the anticodon sequence of the endogenous tRNA, and (ii) wherein the DNA synthesis template further encodes one or more additional mutations to be inserted into an anticodon arm domain of the endogenous tRNA.

[0006] In some aspects, the disclosure relates to a prime editing guide RNA (pegRNA) for editing an endogenous tRNA-Leu-TAA gene by prime editing to produce a suppressor tRNA-Leu gene, wherein: (i) the endogenous tRNA-Leu-TAA gene includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 17-20 (tRNA-Leu-TAA- 1-1 to 4-1), and (ii) the pegRNA includes a DNA synthesis template, the DNA synthesis template encoding a 5'-CUA-3' or 5'-UCA-3' nonsense suppressor anticodon sequence to be inserted into the endogenous tRNA-Leu gene to produce the suppressor tRNA-Leu gene.

[0007] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Arg-CCT-3-1 gene (SEQ ID NO: 3) or a tRNA-Arg-CCT-4-1 gene (SEQ ID NO: 4) to produce a suppressor tRNA capable of reading through a TAG premature termination codon including: a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 116, and a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 39.

[0008] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Leu-TAA-4-1 gene (SEQ ID NO: 20) to produce a suppressor tRNA capable of reading through a TAG premature termination codon including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 40, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 117.2 / 346Bl 195.70209 WOOO#14646633v2

[0009] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Arg-CCT-5-1 gene (SEQ ID NO: 5) to produce a suppressor tRNA capable of reading through a TAG premature termination codon including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 41, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 116.

[0010] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Arg-CCT-5-1 gene (SEQ ID NO: 5), tRNA-Arg-CCT-1-1 gene (SEQ ID NO: 1), tRNA-Arg-CCT-3-1 gene (SEQ ID NO: 3), tRNA-Arg-CCT-4-1 gene (SEQ ID NO: 4), or a tRNA-Arg-CCT-2-1 gene (SEQ ID NO: 2) to produce a suppressor tRNA capable of reading through a TAG premature termination codon including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 42, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 116.

[0011] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Leu-AAG-2-1 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-3 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-2 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-4 gene (SEQ ID NO: 7), tRNA-Leu-AAG-3-1 gene (SEQ ID NO: 8), tRNA-Leu-AAG-1-2 gene (SEQ ID NO: 6), tRNA-Leu-AAG-1-3 gene (SEQ ID NO: 6), or a tRNA-Leu- A AG-1-1 gene (SEQ ID NO: 6) to produce a suppressor tRNA capable of reading through a TAG premature termination codon including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 43, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 118.

[0012] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Leu-TAA-1-1 gene (SEQ ID NO: 17) to produce a suppressor tRNA capable of reading through a TAG premature termination codon including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 44, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 110.

[0013] the disclosure relates to a pegRNA for editing an endogenous tRNA-Ser- AGA-4-1 gene (SEQ ID NO: 27) to produce a suppressor tRNA capable of reading through a TAG premature termination codon including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 45, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 119.

[0014] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Leu-AAG-2-1 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-3 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-2 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-4 gene (SEQ ID NO: 7),3 / 346Bl 195.70209 WOOO#14646633v2tRNA-Leu-AAG-3-1 gene (SEQ ID NO: 8), tRNA-Leu-AAG-1-2 gene (SEQ ID NO: 6), tRNA-Leu-AAG-1-3 gene (SEQ ID NO: 6), or a tRNA-Leu-AAG-1-1 gene (SEQ ID NO: 6) to produce a suppressor tRNA capable of reading through a TAG premature termination codon including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 46, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 118.

[0015] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Tyr-GTA-1-1 gene (SEQ ID NO: 134) to produce a suppressor tRNA capable of reading through a TAG premature termination codon including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 47, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 120.

[0016] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Leu-TAA-3-1 gene (SEQ ID NO: 19) to produce a suppressor tRNA capable of reading through a TAG premature termination codon including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 48, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 121.

[0017] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Tyr-GTA-2-1 gene (SEQ ID NO: 135) to produce a suppressor tRNA capable of reading through a TAG premature termination codon including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 49, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 122.

[0018] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Leu-TAG-1-1 gene (SEQ ID NO: 21), tRNA-Leu-AAG-2-1 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-3 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-2 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-4 gene (SEQ ID NO: 7), tRNA-Leu-TAG-2- 1 gene (SEQ ID NO: 22), tRNA-Leu-AAG-3-1 gene (SEQ ID NO: 8), tRNA-Leu-AAG-1-2 gene (SEQ ID NO: 6), tRNA-Leu-AAG-1-3 gene (SEQ ID NO: 6), or a tRNA-Leu-AAG-1-1 gene (SEQ ID NO: 6) to produce a suppressor tRNA capable of reading through a TAG premature termination codon including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 50, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 123.

[0019] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Tyr-GTA-5-5 gene (SEQ ID NO: 142) to produce a suppressor tRNA capable of reading through a TAG premature termination codon including: a DNA synthesis template 4 / 346Bl 195.70209 WOOO#14646633v2sequence having a nucleic acid sequence identical to SEQ ID NO: 51, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 124.

[0020] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Leu-TAG-1-1 gene (SEQ ID NO: 21), tRNA-Leu-AAG-2-1 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-3 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-2 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-4 gene (SEQ ID NO: 7), tRNA-Leu-TAG-2- 1 gene (SEQ ID NO: 22), tRNA-Leu-AAG-3-1 gene (SEQ ID NO: 8), tRNA-Leu-AAG-1-2 gene (SEQ ID NO: 6), tRNA-Leu-AAG-1-3 gene (SEQ ID NO: 6), or a tRNA-Leu- A AG-1-1 gene (SEQ ID NO: 6) to produce a suppressor tRNA capable of reading through a TAG premature termination codon including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 50, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 123.

[0021] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Arg-TCG-6-1 gene (SEQ ID NO: 133) to produce a suppressor tRNA capable of reading through a TGA premature termination codon including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 54, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 126.

[0022] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Arg-TCG-1-1 gene (SEQ ID NO: 128), tRNA-Arg-TCG-2-1 gene (SEQ ID NO: 129), tRNA-Arg-TCG-3-1 gene (SEQ ID NO: 130), tRNA-Arg-TCG-4-1 gene (SEQ ID NO: 131), or a tRNA-Arg-TCG-5-1 gene (SEQ ID NO: 132), to produce a suppressor tRNA capable of reading through a TGA premature termination codon including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 55, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 126.

[0023] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Arg-CCT-1-1 gene (SEQ ID NO: 1) or a tRNA-Arg-CCT-2-1 gene (SEQ ID NO: 2) to produce a suppressor tRNA capable of reading through a TGA premature termination codon including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 56, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 116.

[0024] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Arg-TCG-6-1 gene (SEQ ID NO: 133) to produce a suppressor tRNA capable of reading through a TGA premature termination codon, including: a DNA synthesis template5 / 346Bl 195.70209 WOOO#14646633v2sequence having a nucleic acid sequence identical to SEQ ID NO: 54, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 126.

[0025] In some aspects, the disclosure relates to a pegRNA for editing an endogenous tRNA-Arg-TCG-1-1 gene (SEQ ID NO: 128), tRNA-Arg-TCG-2-1 gene (SEQ ID NO: 129), tRNA-Arg-TCG-3-1 gene (SEQ ID NO: 130), tRNA-Arg-TCG-4-1 gene (SEQ ID NO: 131), or a tRNA-Arg-TCG-5-1 gene (SEQ ID NO: 132), to produce a suppressor tRNA capable of reading through a TGA premature termination codon, including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 57, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 126.

[0026] In some aspects, the disclosure provides a pegRNA (or a nucleic acid sequence encoding the pegRNA) for editing an endogenous tRNA-Leu-TAA-1-1 gene (SEQ ID NO: 17) to produce a suppressor tRNA capable of reading through a TGA premature termination codon, including: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 58, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 110.

[0027] In some aspects, the disclosure relates to a composition including: (i) a first nucleotide sequence encoding a N-terminal portion of a prime editor fused at its C-terminus to an intein-N; and (ii) a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the prime editor, wherein the second nucleotide sequence further encodes for any one of the pegRNAs disclosed herein, the pegRNA operably linked to a promoter.

[0028] This Summary 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.

[0029] Further, the strategies described in this Summary are not intended to be limiting in any way. As such, any one of the herein disclosed strategies may be used independently or in combination with one or more of the other strategies. For example, in some embodiments, the strategy may comprise using prime editing to edit one or more domains of the tRNA molecule (e.g., anticodon domain and the acceptor stem domain). In some embodiments, the strategy may comprise editing a DNA sequence encoding endogenous tRNA to produce a suppressor tRNA comprising an anticodon that is complimentary to a premature termination codon (PTC) and charged with a non-cognate amino acid. In some embodiments, an endogenous tRNA isodecoder gene is replaced with a suppressor tRNA gene charged with 6 / 346Bl 195.70209 WOOO#14646633v2non-cognate amino acid using prime editing. Other embodiments are also envisioned and are discussed in detail elsewhere herein.BRIEF DESCRIPTION OF DRAWINGS

[0030] 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.

[0031] FIG. 1 shows a schematic illustrating the creation of suppressor tRNAs from endogenous tRNA genes using prime editing.

[0032] FIG. 2 shows the editing efficiency of 11 endogenous tRNAs following prime editing conversion to suppressor tRNAs in HEK293T cells. Edits include: Arg-CCT-5-1 (CCT> TCA), Arg-CCT-5-1 (CCT> CTA), Arg-CCG-2-1 (CCG> CTA), Arg-CCG-2-1 (CCG> TCA), Arg-TCT-4-1 (TCT> TCA); Arg-TCT-1-1 (TCT> TCA), Lys-CTT-3-1 (CTT> TCA), Lys-CTT-15-1 (CTT> TCA), Lys-CTT-15-1 (CTT> CTA), Leu-CAA-6-1 (CAA> TCA), Leu-CAA-6-1 (CAA> CTA), Leu-TAA-2-1 (TAA> TCA), Leu-TAA-2-1 (TAA> CTA), Leu-TAG-3-1 (TAG> TCA), Leu-TAG-3-1 (TAG> CTA), Gln-CTG-5-1 (CTG> TCA), Gln-CTG-5-1 (CTG> CTA), Ala-AGC-5-1 (AGOCTA), and Ala-AGC-5-1 (AGOTCA).

[0033] FIG. 3A shows an illustration of a generalized reporter assay used to determine the readthrough efficiency following prime editing conversion of endogenous tRNAs into suppressor tRNAs.

[0034] FIG. 3B shows a plot of the percent of sequencing reads with the specified edit or indels for Arg-CCG-2-1 (CCG> CTA) and Leu-TAA-2-1 (TAA> TCA) using prime editing in HEK293T cells.

[0035] FIG. 3C shows a plot of the percentage of fluorescent cells obtained using the reporter assay shown in FIGs. 3A and 3B. The eGFP reporter plasmid was edited to contain a single premature termination codon (PTC) located at R109X or L42X. Fluorescent cells are the result of PTC readthrough.

[0036] FIG. 4 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., T C loop), variable arm domain (e.g., variable loop), and the anticodon arm domain encoding the anticodon sequence (e.g., anticodon loop) (SEQ ID NO: 230).7 / 346Bl 195.70209 WO00#14646633v2

[0037] FIGs. 5A-5C show that editing tRNA-Leu-TAA-2-1 (FIG. 5A) (from top to bottom, SEQ ID NOs: 281-282) leads to detectable read through at mRNA (FIG. 5B) but not protein levels (FIG. 5C).

[0038] FIGs. 6A-6D show that each Leu-TAA tRNA gene (FIG. 6A) (from top to bottom, SEQ ID NOs: 283-286) can be specifically targeted using prime editing (FIGs. 6B-6D). FIG.6B shows editing efficiency of uniquely targeted Leu-TAA tRNA gene family members. FIG. 6C shows readthrough efficiency measured by percentage of GFP positive singleintegrant HEK293T reporter cells following Prime Editing-mediated Readthrough of premature Termination codons (PERT) treatment. FIG. 6D shows exemplary quantification of GFP positive cells using flow cytometry.

[0039] FIGs. 7A-7B illustrate that converting endogenous Leu-TAA tRNA genes into suppressors using PERT rescues protein expression at two different disease loci (Niemann-Pick disease type C) in HEK293 cells.

[0040] FIGs. 8A-8B show that endogenous tRNAs are expressed at different levels (FIG.8A) and with different number of isodecoders (FIG. 8B)

[0041] FIGs. 9A-9B illustrate that overwriting endogenous tRNA genes (SER-GCT-3-1 or Cys-GCA-3-1) with suppressor tRNA sequences (Leu-TAA-3-1) elicits readthrough of PTCs. Editing efficiencies of tRNA genes are shown in FIG. 9A. Readthrough efficiency measured by median fluorescent signal from single-integrant HEK293T reporter cell populations subject to twinPE-mediated tRNA gene replacement are shown in FIG. 9B.

[0042] FIGs. 10A-10B illustrate that overwriting endogenous tRNA genes (Ser-GCT-3-1) with suppressor tRNA sequences (Leu-TAA-4-1) elicits readthrough of PTCs. Editing efficiencies of tRNA genes are shown in FIG. 10A. Readthrough efficiency measured by median fluorescent signal from single-integrant HEK293T reporter cell populations subject to twinPE-mediated tRNA gene replacement are shown in FIG. 10B.

[0043] FIG. 11 outlines the reporter construct used to monitor PTC-containing protein translation readthrough. The lentiviral reporter construct contains an mCherry fluorescent protein followed by a premature termination codon (PTC), a ribosomal skipping element (2a), and a GFP fluorescent protein.

[0044] FIGs. 12A-12D show validating sup-tRNA (suppressor-tRNA) reporter constructs for reading through nonsense mutations. FIG. 12A shows a schematic of reporter system with an Efl alpha promoter driving expression of an mCherry fluorescent protein, followed by a 2a ribosomal skipping element, followed by a GFP fluorescent protein. Premature termination codons were installed at one of three positions, indicated by black arrows. Amino acids at 8 / 346Bl 195.70209 WOOO#14646633v2these positions were switched to all 20 possible amino acids. FIGs. 12B-12D show % GFP+ cells following transfection of plasmids with indicated positions switched to any of the 20 indicated amino acids, for PTC location 1 (FIG. 12B), PTC location 2 (FIG. 12C), or PTC location 3 (FIG. 12D).

[0045] FIG. 13 shows the screening strategy used to compare the ability of different suppressor tRNA variants to enable PTC readthrough. The lentiviral tRNA screening construct containing a library of suppressor tRNA variants and one of three promoters: a human U6 promoter, a minimal U6 promoter, or no exogenous promoter beyond the endogenous promoter elements embedded within the tRNA.

[0046] FIGs. 14A-14C show results of quality control experiments performed of candidate suppressor tRNA screening plasmid pools. FIG. 14A shows a plot of the percentage of individually miniprepped colonies as a function of promoter backbone that contain the correct versus incorrect sequence. FIG. 14B shows a plot of the number of alignments as a function of the promoter backbone. FIG. 14C shows a plot of the perfect match as a function of the promoter backbone.

[0047] FIG. 15 shows flow cytometry data representing the 10-fold loss in mCherry protein expression observed when a PTC is included in the reporter system. Frequency of events normalized to the mode indicated on the y-axis. Values and error bars reflect mean+s.d. of n=2 independent biological replicates. FIG. 15 demonstrates that when a premature termination codon is installed before GFP in an mCherry-2a-GFP mRNA construct, a 10-fold lower protein expression of mCherry is observed. Without wishing to be bound by any particular theory, it is hypothesized that this is due to nonsense-mediated mRNA decay. Typically, nonsense-mediated decay is initiated by factors that are recruited to splice sites that are not present in the lentivirus construct. Therefore, it is suspected that this effect is being induced by the lack of a polyA tail in the lentiviral construct, which results in a long 3'-UTR that can also be a substrate for the proteins required to initiate nonsense-mediated decay.

[0048] FIG. 16 shows exemplary flow cytometry results illustrating readthrough with hU6 and min-hU6 promoter sup-tRNA pools and TAG reporter systems.

[0049] FIGs. 17A-17B show results after sorting the top 5% (FIG. 17A) and 0.5% GFP+ cells that exhibited readthrough with the reporter construct following transduction with the lentiviral library of suppressor tRNAs. The theoretical maximum enrichment value is 200-fold. Results from suppressor tRNAs preceded by a human U6 promoter (top), a minimal U6 promoter (middle), and no exogenous promoter (bottom) are shown.9 / 346Bl 195.70209 WOOO#14646633v2

[0050] FIGs. 18A-18B show that 40-bp leader sequences that precede endogenous tRNAs are important for regulating suppressor tRNA function. FIG. 18A illustrates a pool of lentiviral constructs containing a suppressor tRNA (Leu-TAA-4-1 with anticodon switched to CTA) or control tRNA (Leu-TAA-4-1 with native anticodon) as well as the 40-bp leader sequences that precede every endogenous tRNA in the genome. FIG. 18B demonstrates enrichment of leader sequences that precede the control construct (left) or the suppressor tRNA construct (right) in the GFP+ population. Bulk GFP+ cells represent all cells exhibiting readthrough (-44% of cells, with a theoretical maximum enrichment value of 2.27-fold), whereas the [0-25%], [25-50%], [50-75%], and [75-100%] populations represent quadrants of GFP+ cells (25% of the -44% of GFP+ cells, with a theoretical maximum enrichment value of 9-fold).

[0051] FIG. 19 shows that termination sequences are required for activity of hU6-Leu-TAA-3 and hU6-Leu-TAA-4 suppressor tRNAs.

[0052] FIGs. 20A-20D shows the results from a pegRNA screen to identify optimal suppressor tRNA sequences installed using PERT. Schematic of pegRNA screen. 22,177 PE2 epegRNAs targeting tRNAs and converting their anticodons to CTA as well as 1,616 control epegRNAs were packaged into lentivirus and transduced into 293T reporter cells. Cells were transfected with PEmax prime editor to initiate prime editing and cells exhibiting GFP+ readthrough were sorted and processed for next generation sequencing (FIG. 20A). Fold enrichment of pegRNAs targeting each of the amino acid families. Amino acid and anticodon sequence are indicated on the x-axis (FIG. 20B). Example of the performance of pegRNAs targeting one tRNA in the screen (Leu-AAG-2-1) with various RTT homology and PBS lengths (FIG. 20C). Fold enrichment of pegRNAs targeting each of the amino acid families introducing a TGA suppressor anticodon with amino acid and anticodon sequences as indicated on the x-axis (FIG. 20D).

[0053] FIG. 21 shows the results from saturation mutagenesis of the Leu-TAA-4-1 suppressor tRNA (SEQ ID NO: 348).

[0054] FIG. 22 shows the results of a study in which tRNA expression with native leader sequences was determined. The results indicate that the best leader sequences seem to precede highly expressed tRNAs in 293T cells.

[0055] FIGs. 23A-23C shows that tRNA sequences can be installed into safe harbor loci with high frequency using twinPE. FIG. 23A shows results for experiments employing a 20 bp overlap, while FIG. 23B shows results for experiments using a 30 bp overlap. FIG. 23C10 / 346Bl 195.70209 WOOO#14646633v2shows data demonstrating successful twinPE installation of a mature suppressor tRNA sequence.

[0056] FIGs. 24A-24D show variants identified in a saturation mutagenesis screen enhance readthrough in Leu-TAA-1-1 and Leu-TAA-3-1. Editing efficiency of Leu-TAA-1-1 to introduce an anticodon edit, or an anticodon edit and the variants indicated on the x-axis (FIG. 24A). Readthrough efficiency measured by percentage of GFP positive single-integrant HEK293T reporter cells following PERT treatment introducing additional sequence variants in Leu-TAA-1-1 (FIG. 24B). Editing efficiency of Leu-TAA-3-1 to introduce an anticodon edit, or an anticodon edit and the variants indicated on the x-axis (FIG. 24C). Readthrough efficiency measured by percentage of GFP positive single-integrant HEK293T reporter cells following PERT treatment introducing additional sequence variants in Leu-TAA-3-1 (FIG.24D)

[0057] FIGs. 25A-25C shows that the introduction of variant sequences identified in saturation mutagenesis screen enhances readthrough. FIG. 25A shows readthrough efficiency by western blot following delivery of epegRNA and ngRNA pairs capable of introducing a change of hpl3 from G*C to T*A (a top hit from the validation in the reporter cell line described in FIG. 24) to HEK293T Niemann-Pick disease type C cell models. The introduction of the hairpin change alongside the anticodon edit led to a marked increase in full-length NPC1 protein production, reaching approximately 1% of wildtype control expression. FIG. 25B shows prime editing efficiency achieved for the indicated suppressor mutations in a mouse Neuro-2a cell model of Hurler syndrome. FIG. 25C shows the editing efficiency and corresponding readthrough in a TGA reporter cell line.

[0058] FIGs. 26A-26B illustrate an exemplary prime editor (FIG. 26A) and a plot of known pathogenic gene variants as a function of recessive genetic diseases (FIG. 26B).

[0059] FIG. 27 illustrates an exemplary method of using prime editing to install a target gene within a host genome at a safe harbor site.

[0060] FIGs. 28A-28B illustrate a mutation- agnostic and disease-agnostic editing strategy to address nonsense mutations that create premature termination codons (PTCs). Nonsense mutations arise from single base pair substitutions and account for 11% (>11,000) of known pathogenic alleles. Approximately 30% of genetic diseases are caused by mutations that include nonsense mutations. Suppressor tRNAs rescue PTCs, but challenges arise with their exogenous expression.

[0061] FIG. 29 shows why cells may tolerate an editing strategy disclosed herein.Suppressor tRNAs — either natural or introduced via AAV or LNPs — are well-tolerated in 11 / 346Bl 195.70209 WOOO#14646633v2eukaryotes. Safeguard mechanisms minimize the risk of natural stop codon readthrough. These mechanisms include representation differences between natural stop codons and PTCs, redundancy in natural stop codons, recruitment of release factor machinery at polyadenylation sequences, non-stop mediated decay initiation, and recognition and degradation of proteins extending into the 3'-UTR. Widespread variation exists among human tRNA genes, with examples such as the deletion of Lys (CUU) tRNA in -50% of humans and the presence of miscoding tRNAs in healthy individuals.

[0062] FIGs. 30A-30C illustrate previous approaches for reading through stop codons using AAVs and / or lipid nanoparticles.

[0063] FIG. 31 shows a schematic illustrating a strategy using prime editing to convert a dispensable endogenous tRNA gene into a suppressor tRNA. This approach enables one prime editor to potentially treat multiple genetic diseases caused by nonsense mutations.

[0064] FIG. 32 illustrates an exemplary workflow for identifying and creating effective suppressor tRNAs using prime editing.

[0065] FIGs. 33A-33C illustrate an exemplary reporter assay used to screen for suppressor tRNA function. FIG. 33A is a schematic of readthrough quantification from flow cytometry data. MFI, mean fluorescence intensity. Values and error bars reflect mean+s.d. of n=3 independent biological replicates. FIG. 33B shows % GFP+ cells (top) and relative protein yield (bottom) for two sup-tRNAs delivered via transfection or as a single-copy PE-edited tRNA, paired with either an overexpressed reporter or a single copy reporter. FIG. 33C shows sequencing reads with the specified edit for two endogenous tRNAs that were converted to sup-tRNAs with prime editing.

[0066] FIG. 34 is a schematic demonstrating an approach to reveal the potential of each human tRNA to function as an effective suppressor tRNA. Prime editing with 17,579 epegRNAs was used to convert 418 high-confidence human endogenous tRNA genes into suppressor tRNAs by editing the anticodon only in human reporter cells.

[0067] FIGs. 35A-35D illustrate the experimental results from the screen described in FIG. 34. FIG. 35A shows an example flow gating strategy for the PE2 screens. Cells are gated by size, singlets, transduced cells, and GFP+ readthrough. FIGs. 35B-35C show epegRNA design and NGS library generation for PE2 screens. FIG. 35B shows PCR amplification strategy for PE2 screens. PCR1 involves attaching sequencing handles with primers that have the Nextera Readl and Nextera Read2 sequences attached. PCR2 involves attaching sample indices and flow cell adapters for Illumina-based sequencing. Sequencing is performed with a custom primer that reads directly into a diverse barcode representation of 12 / 346Bl 195.70209 WOOO#14646633v2each epegRNA. FIG. 35C shows distribution of tRNA-targeting epegRNAs across the genome, with spacers targeted indicated by black lines across each chromosome. FIG. 35D shows example recording of flow cytometry data for the indicated gates from FIG. 35A.

[0068] FIG. 36 shows graphs depicting saturation mutagenesis results for various pegRNAs. The left graph shows fold enrichment of epegRNAs generating sup-tRNAs compared to controls in GFP sorted cells compared to the plasmid pool in the TAG screen. The right graph shows fold enrichment of epegRNAs, color-coded by the amino acids of the isoacceptor tRNA family that they target. These results identify multiple endogenous human tRNAs that can function as suppressor tRNAs following anticodon conversion through prime editing. Arg, Leu, Ser, and Tyr tRNA backbones demonstrated the greatest effectiveness as suppressor tRNAs.

[0069] FIGs. 37A-37C illustrate the effect of RTT homology and PBS length on the readthrough efficiency of suppressor tRNAs. Endogenous tRNA genes can be converted to TAG sup-tRNAs with prime editing. FIGs. 37A-37B show fold enrichment in GFP-sorted cells compared to plasmid pool for epegRNAs targeting the endogenous tRNA-Leu-TAA-1-1 (FIG. 37 A) and tRNA-Ser-AGA-4-1 (FIG. 37B) genes with the indicated spacers, RTT homology lengths, and PBS lengths. Values and error bars reflect mean+s.d. of n=2 independent biological replicates. FIG. 37C shows sequencing reads with the specified edit for 16 epegRNAs validated individually from the TAG PE2 screen.

[0070] FIG. 38 shows a comparison of fold enrichment between TAG and TGA stop codon reporters and illustrates validation studies of top hits from PE2 screening assays. Values and error bars reflect mean+s.d. of n=2 independent biological replicates.

[0071] FIG. 39 illustrates that pegRNAs that only target a single tRNA are effective at generating functional suppressor tRNAs. Fold enrichment in GFP-sorted cells compared to plasmid pool for arginine, serine, leucine, and tyrosine tRNA isoacceptors is shown.

[0072] FIG. 40 shows validation of top-performing epegRNAs in an arrayed transfection format and illustrates that suppressor tRNA backbones that work for “TAG” readthrough are not always the same as for “TGA” readthrough.

[0073] FIG. 41 illustrates an exemplary iterative pooled lentiviral screening assay used to optimize the leader sequence, suppressor tRNA sequence, and terminator sequence.

[0074] FIGs. 42A-42C illustrate the results from the screen illustrated in FIG. 41. FIG.42A shows the effect of exogenous promoters, hU6 and minU6, on the fold enrichment in suppresser tRNAs capable of reading through CTA mutations. FIG. 42A, left, shows fold enrichment of sup-tRNA variants in GFP sorted cells compared to the plasmid pool 13 / 346Bl 195.70209 WOOO#14646633v2representation when driven by a hU6, minU6, or endogenous promoter with no specified leader sequence (no exogenous promoter). Variants are separated by controls (with a non-sup-tRNA anticodon) or sup-tRNAs with a CUA anticodon. FIG. 42A, right, shows a comparison of Pol III promoters and their respective sizes for sup-tRNA expression. FIG. 42B illustrates fold enrichment in GFP sorted cells compared to bulk transduced cells for a single sup-tRNA (tRNA-Leu-TAA-3-1, UAA> CUA) downstream of 418 distinct tRNA leader sequences. Top and bottom hits that were validated independently are indicated in blue and green.. FIG. 42C shows arrayed lentiviral validation of a single sup-tRNA (tRNA-Leu-TAA-4-1, UAA> CUA) downstream of the top and bottom hits from the screen in FIG. 42B compared to the endogenous leader sequence for the indicated tRNA as well as randomly designed synthetic 40-bp sequences.

[0075] FIGs. 43A-43B show fold enrichment of sup-tRNA variants in GFP sorted cells compared to plasmid pool separated based on upstream (FIG. 43 A) or downstream (FIG. 43G) sequence.

[0076] FIG. 44 shows Log2 fold change of sup-tRNA saturation mutagenesis variants compared to a wild-type sup-tRNA for tRNA-Arg-CCT-4-1 (CCU > CUA), tRNA-Tyr-GTA-2-1 (GUA > CUA), tRNA-Leu-TAA-4-1 (UAA > CUA), or mouse tRNA-Leu-TAA-2-1 (UAA > CUA). Values and error bars reflect mean+s.d. of n=2 independent biological replicates FIG.44 also illustrates that suppressor tRNAs can be mutagenized to further improve readthrough function.

[0077] FIGs. 45A-45C shows the various mutable bases for an exemplary tRNA (Leu). FIG. 45A, left, shows results of saturation mutagenesis of tRNA-Leu-TAA-4-1, with mutations that are comparable or better than an anticodon-only sup-tRNA shown in black. Bases that would easily be accessible with PE at the same time as changing the anticodon are circled with a dashed line. Many of the mutable bases are located within the anticodon stem loop that is PE-accessible. FIG. 45A, right, is a heatmap showing the relative fold change in activity of hairpin mutations compared to the anticodon-only sup-tRNA. FIG. 45B illustrates the five mutations of Leu-TAA suppressor-tRNAs that are PE accessible for a total of 19 different combinations. FIG. 45C shows relative protein yield after readthrough with an endogenously converted tRNA-Leu-TAA-1-1 sup-tRNA, a single-copy lentiviral tRNA-Leu-TAA-1-1 sup-tRNA with the indicated mutations, or with alternative engineered sup-tRNAs. The number of bases from the nearest endogenous tRNA are indicated. Values and error bars reflect mean+s.d. of n=2 independent biological replicates. FIG. 45C also shows that tRNA variants significantly improve suppression efficiency. Optimized suppressor tRNA variants produce 14 / 346Bl 195.70209 WOOO#14646633v25-fold more full-length protein compared to anticodon-only edited tRNAs, yielding -35% of wild- type GFP protein levels.

[0078] FIGs. 46A-46D illustrate an exemplary experimental screening assay for a twinPE-based installation of suppressor-tRNAs (FIG. 46A). FIG. 46B illustrates that suppressor-tRNAs are more effective when expressed at tRNA-specific loci. FIGs. 46C-46D illustrate that twinPE installed suppressor tRNAs are not as effective as PE3-installed suppressor-tRNAs.

[0079] FIGs. 47A-47D illustrates an exemplary method for optimizing a PE-based installation strategy via replacement of an endogenous tRNA with a suppressor-tRNA. FIG.47A shows a schematic illustrating a pooled screening strategy and prime editing design variants. FIG 47B shows percent desired editing at a synthetic target site for each of the indicated editors in HeLas (top) and HEK293Ts (bottom). FIG. 47B also shows that PE6c performed the best for converting an endogenous tRNA into a suppressor-tRNA. FIG. 47C shows percent editing at a synthetic target site in the pooled screen plotted against the percent editing at the endogenous locus in arrayed validation in both HEK293T cells, left, and HeLa cells, right. Pearson correlation is indicated in the top right. FIG. 47C also illustrates that editing levels at endogenous locus differ between 293T cells and HeLa cells. FIG. 47D is an illustration of editing efficiencies at a synthetic target site across PBS length and RTT length combinations for the hairpin (hp)12ta>cg + hpl3gc>cg variant combination. FIG. 47D also shows that prime editing efficiently converts an endogenous human tRNA into an optimized suppressor tRNA. Results show pegRNA with a nicking guide can achieve -80% editing efficiency in 293T cells.

[0080] FIG. 48 shows that one prime editor can rescue protein production and activity in multiple human cell models of unrelated genetic diseases. Readthrough efficiency achieved with a prime editor- installed suppressor tRNA varies between <1% to >60% relative protein levels, depending on the specific mutation. Results are demonstrated in human cell models of Batten disease, Tay-Sachs disease, and Niemann-Pick disease type Cl. FIG. 48, left, shows TPP1 enzyme activity in treated versus untreated human cell models of Batten disease relative to wild- type controls. FIG. 48, middle, shows HEXA enzyme activity in treated versus untreated human cell models of Tay-Sachs disease relative to wild-type controls. FIG.48, right, shows measurement of NPC1 protein expression via Western blot in treated versus untreated human cell models of Niemann-Pick disease type Cl relative to wild- type controls, wild-type protein lysate was loaded at l / 10th the quantity of all other samples.15 / 346Bl 195.70209 WOOO#14646633v2

[0081] FIGs. 49A-49C illustrate an exemplary assay for investigating the differences in readthrough efficiencies based on sequence context. FIG. 49A shows a schematic of screening strategy to identify the impact of sequence context on readthrough efficacy. FIG.49B shows mCherry as a function of frequency normalized to mode. FIG. 49C shows an RNA score of each sequence context library subgroup.

[0082] FIG. 50 shows that readthrough scores assigned in the sequence context screen correlate well with full-length cDNA readthrough. FIG. 50, left, shows RNA scores for each variant in the CFTR gene measured in the sequence context library screen. FIG. 50, right, shows sequence context RNA score plotted against relative CFTR protein yield from arrayed expression of full-length CFTR mutants. Values and error bars reflect mean+s.d. of n=2 independent biological replicates.

[0083] FIGs. 51A-51B show that a prime editor delivered in vivo can rescue protein production in a GFP reporter of nonsense suppression. Results show a suppressor tRNA installed using a prime editor and delivered via AAV-expressed reporter results in -25% protein rescue. FIG. 51A shows a schematic of experimental procedure for in vivo assessment of readthrough of exogenously supplied reporter constructs. FIG. 5 IB shows in vivo readthrough efficacy of an exogenously supplied amber or opal premature stop codon reporter construct. n=4 mice per condition.

[0084] FIGs. 52A-52B show that the same prime editor delivered in vivo (FIG. 51 A) can partially rescue protein production in a mouse model of Hurler syndrome. FIG. 52 A shows in vivo prime editing of an endogenous tRNA into a suppressor tRNA, partially restoring iduronidase enzyme activity in disease-relevant tissues in a mouse model of mucopolysaccharidosis type I. Additional mutations do not alter readthrough efficiency in the mouse-specific context. FIG. 52A, top, shows a schematic of experimental procedure for in vivo treatment of Idua-W392X mouse model. FIG. 52A, bottom, shows desired editing efficiencies in treated homozygous Idua-W392X mice (left); Idua enzyme activity in treated homozygous Idua-W392X mice (middle); and Idua enzyme activity in untreated homozygous Idua-W392X mice (right). N=3 mice per PE condition and N=2 mice per wild-type condition. FIG. 52B demonstrates that in vivo prime editing of an endogenous tRNA alleviates Hurler syndrome pathology, including vacuolization, foam cell accumulation, and glycosaminoglycan (GAG) buildup across all examined tissues. FIG. 52B, left, shows hematoxylin and eosin staining of the brain and liver. Arrows show vacuolated cells and inset is a high-power magnification of a Purkinje cell. Scale bars are 20 pM, and arrow heads16 / 346Bl 195.70209 WOOO#14646633v2are pointing to areas of marked vacuolization. FIG. 52B, right, shows tissue pathology score based on GAG storage evaluated by microscopy. Values and error bars reflect mean+s.d.

[0085] FIGs. 53A-53D show endogenous tRNA genes can be converted to TGA sup-tRNAs with prime editing. FIG. 53A shows fold enrichment of epegRNAs generating sup-tRNAs compared to controls in GFP sorted cells compared to the plasmid pool in the TGA screen. FIG. 53B shows fold enrichment of epegRNAs, color-coded by the amino acids of the isoacceptor tRNA family that they target in the TGA screen. FIG. 53C shows validation of top-performing epegRNAs in an arrayed transfection format in the TGA screen. FIG. 53D shows sequencing reads with the specified edit for 8 epegRNAs validated individually from the TGA PE2 screen. Values and error bars reflect mean+s.d. of n=2 independent biological replicates.

[0086] FIG. 54 shows sup-tRNA backbones for reading through TAG and TGA stop codons are differentially enriched. Fold enrichment of epegRNAs in the GFP-sorted population in the TAG screen (y-axis) compared to the TGA screen (x-axis).

[0087] FIGs. 55A-55C show endogenous tRNA genes could not be converted to efficient TAA sup-tRNAs with prime editing. FIG. 55A shows example flow cytometry data for TAA screen for GFP+ cells. Only 0.18% cells had readthrough and expression of GFP in these cells was low. FIGs. 55B-55C show fold enrichment in GFP+ cells compared to the plasmid pool for both control epegRNAs and sup-tRNA generating epegRNAs (FIG. 55B), as well as broken down by amino acid family (FIG. 55C). Compared to the TAG and TGA screens, the signal-to-noise ratio is much smaller.

[0088] FIGs. 56A-56B show engineering the upstream sequences of sup-tRNAs. FIG. 56A shows fold enrichment in GFP-positive cells versus the plasmid pool of sup-tRNA sequences expressed using a hU6 promoter (top), minU6 promoter (middle), or with no exogenous promoter (bottom). The anticodon of the sup-tRNA is indicated. FIG. 56B shows a summary of how the delivery mechanism of a sup-tRNA coupled with its surrounding sequence influences its effectiveness to read through a GFP stop codon reporter. Check marks indicate successful ability to identify a sup-tRNA capable of reading through the indicated stop codon.

[0089] FIGs. 57A-57E show engineering the downstream sequences of sup-tRNAs. FIGs.57A-57B show Logio distance in bp to a naturally occurring 4T (FIG. 57A) or 5T (FIG. 57B) sequence in the genome for each endogenous human tRNA gene. FIG. 57C shows sequencing reads with the specified edit (%) following transfection with prime editing reagents designed to target each of the endogenous Leu-TAA tRNAs independently. FIG. 57D 17 / 346Bl 195.70209 WOOO#14646633v2shows percent GFP-positive cells (left) and relative protein yield relative to wild-type GFP (right) with a GFP stop codon readthrough reporter following prime editing of each of the four Leu-TAA tRNAs into sup-tRNAs. FIG. 57E shows normalized counts of each Leu-TAA tRNA from a HEK293T mim-tRNA-seq dataset. Values and error bars reflect mean+s.d. of n=2 independent biological replicates.

[0090] FIGs. 58A-58D shows engineering the tRNA body of sup-tRNAs. FIG. 58 A shows Log2fold change of mutant sup-tRNAs compared to a wild-type sup-tRNA for tRNA-Tyr-GTA-2-1 with its anticodon changed to CUA. Mutation position is indicated on the x-axis. FIG. 58B shows a heatmap representing log2fold change of mutant sup-tRNAs compared to a wild-type sup-tRNA for each single nucleotide variant (SNV) in the four Leu-TAA sup-tRNA genes. Mutation position is indicated on the x-axis. FIG. 58C shows Log2fold change of mutant sup-tRNAs compared to a wild-type sup-tRNA for all naturally occurring human variants of Leu-TAA-1-1 documented in tRNAdb. FIG. 58D shows sequencing reads with the specified edit (%) for epegRNAs used to install each of the indicated combinations of mutations in the endogenous tRNA-Leu-TAA-1-1 gene alongside changing its anticodon to CUA. Values and error bars reflect mean+s.d. of n=2 independent biological replicates.

[0091] FIGs. 59A-59C shows an effect of deletions and variable loop replacement in Leu-TAA sup-tRNAs. FIG. 59A shows a heatmap of the log2fold change of each of the indicated single nt deletion mutants compared to the wild-type sup-tRNA sequence for each of the 4 Leu-TAA sup-tRNAs. FIGs. 59B-59C show log2fold change of each of the indicated single nt deletion mutants compared to the wild-type sup-tRNA sequence when swapping each tRNA’s native variable loop with the variable loop of the indicated Leucine tRNAs.

[0092] FIGs. 60A-60D show optimization of a prime editing strategy for engineered sup-tRNA installation. FIG. 60A shows percent editing efficiency across all epegRNAs with the indicated spacer from the PE6c screen in HeLa (top) and HEK293T (bottom) cells. Trends across the given mutation combinations are shown. FIG. 60B shows epegRNA spacerdependent percent editing in HeLa cells transfected with the PE6c prime editor. Each point indicates a unique epegRNA sequence. FIG. 60C shows fold change in desired editing efficiency when cells were transfected with MLHldn and PEmax (PE4) compared to being transfected with PEmax alone (PE2) in HeLa (top) and HEK293T (bottom) cells. epegRNAs are split up based on the intended edit being made, indicated by the mutations being introduced. FIG. 60D shows sequencing reads with specified edit in HEK293Ts (y-axis) and HeLas (x-axis) transfected with the PE6c prime editor with the indicated epegRNA spacers. Pearson correlation is shown in the top right.18 / 346Bl 195.70209 WOOO#14646633v2

[0093] FIGs. 61A-61F show off-target analysis for optimized epegRNA. FIG. 61 A shows computational analysis of number of sites in the hgl9 genome with the indicated number of mismatches from our epegRNA spacer. FIG. 6 IB shows percent of sequencing reads containing single nucleotide polymorphisms (SNPs) (top) or insertions or deletions (indels) (bottom) at each of the indicated locations in the genome, either on-target at the Leu-TAA-1-1 locus or at 18 potential off-target sites. The number of mismatches from the spacer is indicated below. FIG. 61C shows percent prime editing at each of the indicated target site types in the off-target screen after subtracting background frequencies observed in cells transfected with epegRNA only and no prime editor. FIG. 6 ID shows percent prime editing marker frequency (top) or percent indel frequency after background subtraction (bottom) for individual validation of nominated off-targets from the off-target screen. FIG. 6 IE shows RNA-seq comparing cells transfected with PE6c and an epegRNA / ngRNA designed to convert Leu-TAA-1-1 into a sup-tRNA compared to an unrelated epegRNA / ngRNA pair designed to edit the HEK3 locus. Each data point represents the expression of a gene. No genes had an adjusted p-value < 0.05 and a |log2fold change| >1. FIG. 6 IF shows quantitative PCR analysis of the expression levels of 28 different tRNA families in cells expressing a PE-installed anticodon-only (“ac-only”) sup-tRNA, engineered sup-tRNA, or wild-type cells, n.s. = not significant.

[0094] FIGs. 62A-62C show editing outcomes and validation of functional assays in human cells models of disease. FIG. 62A shows measured editing outcomes in six HEK293T cell models of disease for each specified edit. FIG. 62B shows standard curve showing accuracy of measured TPP1 activity across various concentrations of wild-type protein (AU = arbitrary units). FIG. 62C shows standard curve showing accuracy of measured HEXA activity (normalized to HEXB activity) across various concentrations of wild-type protein. Values and error bars reflect mean+s.d. of n=2 independent biological replicates.

[0095] FIGs. 63A-63F show converting mouse tRNA-Leu-TAA-2-1 to a sup-tRNA with prime editing. FIGs. 63A-63B show heatmaps of sequencing reads (%) with the desired edit (left) and undesired edit (right) for epegRNAs targeting mouse tRNA-Leu-TAA-2-1 with the anticodon-only edit (FIG. 63A) or the edit additionally encoding the mutations hpl3gc>ta and mut38a>t (FIG. 63B). FIGs. 63C-63D show sequencing reads with the specified edit for epegRNAs targeting mouse tRNA-Leu-TAA-2-1 with the anticodon-only edit incorporated using the indicated ngRNAs (FIG. 63C) or prime editors (FIG. 63D). FIGs. 63E-63F show sequencing reads with the specified edit for epegRNAs targeting mouse tRNA-Leu-TAA-2-1 with the edit additionally encoding the mutations hp!3gc>ta and mut38a>t using the 19 / 346Bl 195.70209 WOOO#14646633v2indicated ngRNAs (FIG. 63E) or prime editors (FIG. 63F). Values and error bars reflect mean+s.d. of n=2 independent biological replicates.

[0096] FIGs. 64A-64E shows prime editing installed suppressor tRNAs that readthrough premature stop codons in GFP reporters in vivo. FIG. 64A shows sequencing reads with the specified edit (%) for the brain cortex of BL / 6 mice treated with prime editing agents to convert tRNA-Leu-TAA-2-1 into CUA or UCA suppressors alongside a wild-type GFP reporter (WT) or a GFP reporter containing a TAG or TGA stop codon. FIG. 64B shows sequencing reads with the specified edit (%) for the brain cortex of BL / 6 mice treated with prime editing agents to convert tRNA-Leu-TAA-2-1 into a CUA suppressor or to introduce a benign Dnmtl edit. FIGs. 64C and 64D show body weight in grams (g) over time following treatment with prime editing agents, corresponding to editing shown in FIG. 64B. N = 3 female sup-tRNA mice, 5 female Dnmtl mice, 4 male sup-tRNA mice, 4 male Dnmtl mice. FIG. 64E shows Idua enzymatic activity in Hurler syndrome mice without prime editing treatment.

[0097] FIGs. 65A-65B show uncropped original Western blot data. Both FIGs. 65A and 65B show NPC1 stained (top), and GAPDH stained (bottom).

[0098] FIG. 66 shows a schematic of standard tRNA nomenclature. For example, in indicating “tRNA-Gln-CTG-6-1”: Gin indicates the amino acid that the tRNA normally carries (here, glutamine); CTG is the anticodon sequence that recognizes the corresponding codon in mRNA; 6 is the isodecoder index, which distinguishes different tRNA genes that share the same amino acid and anticodon but differ slightly in sequence elsewhere; and 1 is the locus copy number, identifying which genomic copy of this isodecoder is being referenced.

[0099] FIG. 67A-67D illustrates the abundance levels of PE-installed sup-tRNAs compared to wild-type tRNAs. FIG. 67A shows the targeted tRNA-seq of tRNA-Leu-TAA-1-1 measures the abundance of engineered sup-tRNA, ac-only sup-tRNA, or wild-type tRNA at the DNA and RNA levels. FIG. 67B shows the sequence logo of targeted tRNA-seq for reads corresponding to the wild-type tRNA, ac-only sup-tRNA, and engineered sup-tRNA at the RNA and DNA levels. FIG. 67C shows the probability of a G base at position 26 (left figure) or a G base at position 37 (right figure) for reads corresponding to the engineered sup-tRNA, ac-only sup-tRNA, or wild-type tRNA. FIG. 67D shows a schematic of a Leu-TAA tRNA and the position of its modified bases. The Induro RT used for cDNA generation will result in an “N” nucleotide at any position that is modified in the RNA.20 / 346Bl 195.70209 WOOO#14646633v2

[0100] FIGs. 68A-68G shows mass spectrometry on PE-installed sup-tRNAs to evaluate readthrough of PTCs and natural TAG termination codons (NTCs). (FIG. 68A shows the evaluation by mass spectrometry of the frequency of amino acid installation in a Flag-tagged murine IDUA cDNA at position 405 with a wild-type cDNA sequence (W405) or with a cDNA sequence containing a PTC (W405X) and with a PE-installed sup-tRNA sequence. FIGs. 68B-68D show protein-level analysis of whole proteome mass spectrometry data from cells prime edited to install an ac-only sup-tRNA (FIG. 68B), engineered sup-tRNA (FIG. 68C), or unedited control cells (FIG. 68D). Eog2 fold-change in protein abundance compared to unedited control cells. Dashed lines indicate an adjusted p-value of 0.05 on the y-axis and log2 fold change in abundance of ±1 compared to unedited control cells. FIG. 68E shows normalized abundance of peptides identified past a premature termination codon (PTC) (top) or past natural TAG termination codons (NTCs) (bottom) for non-prime edited control HEK293T cells. Dotted lines correspond to trypsin cleavage sites. FIGs. 68F-68g shows log2 fold change in abundance of peptides identified in cells expressing an ac-only sup-tRNA (FIG. 68F) or an engineered sup-tRNA (FIG. 68G) compared to control cells. All peptides identified post-PTC (GFP) are significant (adjusted p < 0.05). Peptides for pre-NTC controls and for one post-NTC peptide identified are not significant (adjusted p > 0.05).

[0101] FIGs. 69A-69B shows that prime editing-installed sup-tRNAs can rescue protein expression across diverse disease contexts. Normalized abundance of peptides identified past a premature termination codon (PTC) (top) or past natural TAG termination codons (NTCs) (bottom) for cells prime edited to express an ac-only sup-tRNA (FIG. 69A) or an engineered sup-tRNA (FIG. 69B). Dotted lines correspond to predicted potential trypsin cleavage sites.

[0102] FIG. 70A and 70B shows readthrough with a sup-tRNA inhibits nonsense-mediated decay. FIG. 70A shows a histogram showing mCherry and GFP fluorescence of cells expressing the pSEP0201 sup-tRNA reporter (mCherry- STOP-GFP) treated with NMD inhibitors (NMDi, KVS0001)108 and / or treated with prime editing agents to install a sup-tRNA. FIG. 70B shows the median fluorescence intensity of mCherry followed by a PTC for reporter cells (mCherry- STOP-GFP) treated with NMD inhibitors or treated with prime editing agents to install a sup-tRNA.

[0103] FIG. 71 illustrates representative images of H& E stained brain and liver tissues of Idua - / -, + / - ICV AAV vector. In panels A-C (untreated mouse #9), there is moderate intracellular vacuolization of cerebellar Purkinje cells (black arrows, A) and thalamic neurons (white arrows, B) and the hepatic foam cells (gray arrows, C). In contrast, in panels D-F21 / 346Bl 195.70209 WOOO#14646633v2(treated mouse #7), there is minimal cerebellar Purkinje cell vacuolization (black arrow, inset, D), a lack of thalamic neuronal vacuolization (E), and a lack of hepatic foam cells (F).

[0104] FIG. 72 shows representative images of anti-GFP stained brain tissues from animals (Idua - / -, + / - ICV AAV vector, GFP-IHC). In panels A-D (untreated mouse #9), there is the complete lack of anti-GFP immuno staining, including within the cerebellum (A), Purkinje cells (B), hippocampus (C), and thalamus (D). In contrast, in the intracerebroventricular AAV vector treated mouse (panels E-H, mouse #7), there is widespread anti-GFP immuno staining in the same sites.DEFINITIONS

[0105] 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.Anticodon arm domain

[0106] 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.Cas9

[0107] The term “Cas9” or “Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 domain, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and / or the gRNA binding domain of Cas9). A “Cas9 domain” as used herein, is a protein fragment comprising an active or inactive cleavage domain of Cas9 and / or the gRNA binding domain of Cas9. A “Cas9 protein” is a full length Cas9 protein. A Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II 22 / 346Bl 195.70209 WO00#14646633v2CRISPR systems, correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (me), 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., 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 Ml strain of Streptococcus pyogenes.” Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y„ Jia H. G., Najar F. Z., Ren Q„ Zhu H„ Song L„ White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U. S. A.98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes 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.

[0108] 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.,23 / 346Bl 195.70209 WOOO#14646633v2Science. 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 RuvC 1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvCl subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the 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: 147). 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: 147). In some embodiments, the Cas9 variant comprises a fragment of SEQ ID NO: 147 (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: 147). 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%, 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: 147).

[0109] The skilled artisan will appreciate that any wild type Cas9 or derivative thereof known to the skilled artisan may be used as disclosed herein, such as, for example:24 / 346Bl 195.70209 WOOO#14646633v2SpCas9, Streptococcus pyogenes Ml, SwissProt Accession No. Q99ZW2, Wild type MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHE RHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFT VYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECF DSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVL SMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVL VVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS LFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLF VEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTN LGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 147)Cognate Amino Acid

[0110] 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

[0111] 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 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 25 / 346Bl 195.70209 WOOO#14646633v2CRISPR-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 (me) 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 Ml strain of Streptococcus pyogenes.” Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U. S. A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes 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.26 / 346Bl 195.70209 WOOO#14646633v2

[0112] 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 (me), 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.

[0113] 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.D-arm domain

[0114] 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.DNA synthesis template

[0115] 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 3' 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 27 / 346Bl 195.70209 WOOO#14646633v2polymerase 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 “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 3' extension arm, the DNA synthesis template can include the portion of the extension arm that spans from the 5' end of the primer binding site (PBS) to 3' 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 5' extension arm, the DNA synthesis template can include the portion of the extension arm that spans from the 5' end of the pegRNA molecule to the 3' end of the edit template. Preferably, the DNA synthesis template excludes the primer binding site (PBS) of pegRNAs either having a 3' extension arm or a 5' 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

[0116] 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 nonedited 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.Edit template28 / 346Bl 195.70209 WOOO#14646633v2

[0117] The term “edit template” refers to a portion of the extension arm that encodes the desired edit in the single strand 3' 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

[0118] 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 3' end of the guide RNA. In other embodiments, the extension arm is located at the 5' 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 5' to 3' direction: the homology arm, the edit template, and the primer binding site. Since polymerization activity of the reverse transcriptase is in the 5' to 3' direction, the preferred arrangement of the homology arm, edit template, and primer binding site is in the 5' to 3' direction such that the reverse transcriptase, once primed by an annealed primer sequence, polymerizes a single strand of DNA using the edit template as a complementary template strand. Further details, such as the length of the extension arm, are described elsewhere herein.

[0119] 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 3' 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 3' end (i.e., the 3' of the primer sequence), which then provides a substrate for a polymerase to begin polymerizing a single strand of DNA from the exposed 3' end along the length of the DNA synthesis template. The sequence of the single strand DNA product is the complement of the DNA synthesis template. Polymerization continues towards the 5' of the 29 / 346Bl 195.70209 WOOO#14646633v2DNA synthesis template (or extension arm) until polymerization terminates. Thus, the DNA synthesis template represents the portion of the extension arm that is encoded into a single strand DNA product (i.e., the 3' single strand DNA flap containing the desired genetic edit information) by the polymerase of the prime editor complex and which ultimately replaces the corresponding endogenous DNA strand of the target site that sits immediately downstream of the PE-induced nick site. Without being bound by theory, polymerization of the DNA synthesis template continues towards the 5' end of the extension arm until a termination event. Polymerization may terminate in a variety of ways, including, but not limited to (a) reaching a 5' terminus of the pegRNA (e.g., in the case of the 5' extension arm wherein the DNA polymerase simply runs out of template), (b) reaching an impassable RNA secondary structure (e.g., hairpin or stem / loop), or (c) reaching a replication termination signal, e.g., a specific nucleotide sequence that blocks or inhibits the polymerase, or a nucleic acid topological signal, such as, supercoiled DNA or RNA.Fusion protein

[0120] 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 (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2012)), the entire contents of which are incorporated herein by reference.General expression site

[0121] 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")30 / 346Bl 195.70209 WOOO#14646633v2

[0122] 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 Cpfl (a type-V CRISPR-Cas systems), C2cl (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”).

[0123] Guide RNAs or pegRNAs may comprise various structural elements that include, but are not limited to:

[0124] 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.

[0125] 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.

[0126] Extension arm-a single strand extension at the 3' end or the 5' 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 genetic change of interest, which then integrates into the endogenous DNA by replacing the corresponding endogenous strand, thereby installing the desired genetic change.

[0127] Transcription terminator - the guide RNA or pegRNA may comprise a transcriptional termination sequence at the 3' of the molecule.Host cell31 / 346Bl 195.70209 WOOO#14646633v2

[0128] 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

[0129] 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

[0130] 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.

[0131] 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- 32 / 346Bl 195.70209 WOOO#14646633v2target 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

[0132] The term “nickase” refers to a Cas9 with one of the two nuclease domains inactivated (e.g., via a mutation such as D10A and / or H840A). This enzyme is capable of cleaving only one strand of a target DNA.Non-cognate amino acid

[0133] The term “non-cognate amino acid” refers to an amino acid that pairs with a tRNA molecule that does not include an anticodon sequence encoding said amino acid.Nonsense mutation

[0134] 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

[0135] 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

[0136] 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, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxy cytidine), 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, 0(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.,33 / 346Bl 195.70209 WOOO#14646633v22 '-fluororibose, ribose, 2 '-deoxyribose, 2'-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5' N phosphoramidite linkages). Nuclear localization sequence

[0137] 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

[0138] 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 to aptamers, stem loops, hairpins, toe loops (e.g., a 3' toe loop), or an RNA-protein recruitment domain (e.g., MS2 hairpin).

[0139] 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.

[0140] In certain embodiments, the pegRNAs have a 5' extension arm, a spacer, and a gRNA core. The 5' extension further comprises in the 5' to 3' 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.34 / 346Bl 195.70209 WOOO#14646633v2

[0141] In certain other embodiments, the pegRNAs have a 5' extension arm, a spacer, and a gRNA core. The 5' extension further comprises in the 5' to 3' 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.

[0142] In still other embodiments, the pegRNAs have in the 5' to 3' direction a spacer (1), a gRNA core (2), and an extension arm (3). The extension arm (3) is at the 3' end of the pegRNA. The extension arm (3) further comprises in the 5' to 3' 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 3' and 5' ends, which may be the same sequences or different sequences. In addition, the 3' end of the pegRNA may comprise a transcriptional terminator sequence. These sequence elements of the pegRNAs are further described and defined herein.

[0143] In still other embodiments, the pegRNAs have in the 5' to 3' direction an extension arm (3), a spacer (1), and a gRNA core (2). The extension arm (3) is at the 5' end of the pegRNA. The extension arm (3) further comprises in the 3' to 5' 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 3' and 5' ends, which may be the same sequences or different sequences. The pegRNAs may also comprise a transcriptional terminator sequence at the 3' end. These sequence elements of the pegRNAs are further described and defined herein.PEI

[0144] As used herein, “PEI” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a wild type MMLV RT having the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(wt)] + a desired pegRNA, wherein the PE fusion has the amino acid sequence of SEQ ID NO: 231, which is shown as follows;MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNT DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDS FFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEH HQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKIL35 / 346Bl 195.70209 WOOO#14646633v2TFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVP QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRD KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETR IDLSQLGGD5GG55GG55G5ErPGr5E5ArPE'55GG55GG55TLNIEDEYRLHETSKEPDV SLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPH IQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYN LLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQG FKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLG NLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREF LGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVA AIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDR VQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYT DSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKG HSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV(SEQ ID NO: 231)

[0145] KEY:

[0146] NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO: 232), BOTTOM: (SEQ ID NO: 233)

[0147] CAS9IH840A) (SEQ ID NO: 177)36 / 346Bl 195.70209 WO00#14646633v2

[0148] 33-AMINO ACID LINKER (SEQ ID NO: 234)

[0149] M-MLV reverse transcriptase (SEQ ID NO: 235).PE 2

[0150] As used herein, “PE2” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a variant MMLV RT having the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] + a desired pegRNA, wherein the PE fusion has the amino acid sequence of SEQ ID NO: 236, which is shown as follows:

[0151] MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNT DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDS FFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEH HQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKIL TFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVP QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRD KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETR IDLSQLGGD5GG55GG55G5ErPGr5E5ArPE'55GG55GG55TLNIEDEYRLHETSKEPDV 37 / 346 Bl 195.70209 WOOO #14646633v2SLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPH IQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYN LLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQG FKNSPTEFNEAEHRDEADFRIQHPDEIEEQYVDDEEEAATSEEDCQQGTRAEEQTEG NLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREF LGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLP DLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAA IAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRV QFGPVVAENPATEEPEPEEGEQHNCEDIEAEAHGTRPDETDQPEPDADHTWYTDGSS LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTD SRYAFATAHIHGEIYRRRGWETSEGKEIKNKDEIEAEEKAEFEPKRESIIHCPGHQKGH SAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV(SEQ ID NO: 236)

[0152] KEY:

[0153] NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO: 232), BOTTOM: (SEQ ID NO: 233)

[0154] CAS9IH840A) (SEQ ID NO: 177)

[0155] 33-AMINO ACID LINKER (SEQ ID NO: 234)

[0156] M-MLV reverse transcriptase (SEQ ID NO: 237).PE3

[0157] As used herein, “PE3” refers to PE2 plus a second-strand nicking guide RNA that complexes with the PE2 and introduces a nick in the non-edited DNA strand in order to induce preferential replacement of the edited strand.PE3b

[0158] As used herein, “PE3b” refers to PE3 but wherein the second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing a gRNA with a spacer sequence that matches only the edited strand, but not the original allele. Using this strategy, referred to hereafter as PE3b, mismatches between the protospacer and the unedited allele should disfavor nicking by the sgRNA until after the editing event on the PAM strand takes place.PE438 / 346Bl 195.70209 WOOO#14646633v2

[0159] As used herein, “PE4” refers to a system comprising PE2 plus an MLH1 dominant negative protein (i.e., wild-type MLH1 with amino acids 754-756 truncated as described further herein) expressed in trans.PE5

[0160] As used herein, “PE5” refers to a system comprising PE3 plus an MLH1 dominant negative protein (i.e., wild-type MLH1 with amino acids 754-756 truncated as described further herein, which may be referred to as “MLH1 A754-756” or “MLHldn”) expressed in trans. “PE5b” refers to a prime editing composition comprising a PE3 and an MLH1 dominant negative protein, wherein the second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing the second strand nicking guide RNA with a spacer sequence that comprise complementarity to, and hybridizes with, only the edited strand after installation of the desired nucleotide edit(s), but not the endogenous target DNA sequence.PE-short

[0161] As used herein, “PE-short” refers to a PE construct that is fused to a C-terminally truncated reverse transcriptase, and has the following amino acid sequence:

[0162] MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKF KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAK LQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPI LEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNR EKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDN EENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKL INGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHI ANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRER MKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYD VDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLIT QRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI39 / 346Bl 195.70209 WO00#14646633v2REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESE FVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIET NGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFL YLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAY NKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSIT GLYETRIDLSQLGGD5GG55GG55G5ErPGr5E'5ArPE55GG55GG55TLNIEDEYRLHET SKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEA RLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPT VPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTW TRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPR QLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAP ALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLR MVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLD TDRVQFGPVVALNPATLLPLPEEGLQHNCLDNSRLINSGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 238)KEY:NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO: 232), BOTTOM: (SEQ ID NO: 233)CAS9IH840A) (SEQ ID NO: 177)33-AMINO ACID LINKER 1 (SEQ ID NO: 234)M-MLV TRUNCATED REVERSE TRANSCRIPTASE (SEQ ID NO: 239)PEmax

[0163] As used herein, “PEmax” refers to a prime editing composition comprising 1) a fusion protein comprising a Cas9 protein variant Cas9(R221K N39K H840A) and a variant MMLV RT having the following structure: [bipartite NLS]- [Cas9(R221K)(N394K)(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)]-[bipartite NLS]-[NLS] and 2) a desired PEgRNA, wherein the fusion protein (referred to as the PEmax protein) has the amino acid sequence of SEQ ID NO: 240, which is shown as follows:

[0164] MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKF KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD40 / 346Bl 195.70209 WOOO#14646633v2KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASG VDAKAILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAK LQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPI LEKMDGTEELLVKLKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNR EKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDN EENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKL INGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHI ANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRER MKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYD VDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLIT QRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESE FVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIET NGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFL YLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAY NKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSIT GLYETRIDLSOLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSTLNIEDEYR LHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMS QEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDI HPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQ LTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTP KTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQAL LTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWP PCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQA LLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADH TWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEG KKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIH41 / 346Bl 195.70209 WOOO#14646633v2CPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFES PKKKRKNGSGPAAKRVKLD (SEQ ID NO: 240)key:Bipartite sv40 Nuclear localization sequence (NLS) Top: (SEQ ID NO: 232), Cas9(R221K N39K H840A) (SEQ ID NO: 241)SGGSx2-bipartite sv40NLS-SGGSx2 linker (SEQ ID NO: 242)M-MLV reverse transcriptase(D200N T306K W313F T330P L603W) (SEQ ID NO: 243) Other linker sequence (SEQ ID NO: 244)bipartite sv40NLS (SEQ ID NO: 245)Other linker sequencec-Myc NLS (SEQ ID NO: 246)PE6 series

[0165] The term “PE6” refers to a suite of prime editors (PE6a, PE6b, PE6c, PE6d, PE6e, PE6f, and PE6g) comprising improved reverse transcriptase and / or Cas9 variants which are published in Doman et al., “Phage-assisted evolution and protein engineering yield compact, efficient prime editors,” Cell, 2003, 186, pp. 3983-4002, the contents of which are incorporated herein by reference. The improved reverse transcriptase and Cas9 domains of the PE6 variants can also be combined with each other to offer cumulative benefits. For example, a PE6 prime editor comprising an improved reverse transcriptase variant of PE6a and an improved Cas9 variant of PE6e is referred to herein as the prime editor “PE6a-e” (or “PE6e-a”). Any possible combination of PE6 prime editors is contemplated by the present disclosure including, for example, PE6a-e, PE6a-f, PE6a-g, PE6b-e, PE6b-f, PE6b-g, PE6c-e, PE6c-f, PE6c-g, PE6d-e, PE6d-f, and PE6d-g.

[0166] Any of the PE6 prime editors may also comprise the architecture of the PEmax protein as provided herein. In some embodiments, any of the PE6 prime editors provided herein may further comprise additional amino acid mutations, e.g., any of those included in

[0167] In some embodiments, a PE6 protein comprises a reverse transcriptase of the following amino acid sequence (the RT domain of “PE6a”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence:

[0168] GRPYVTLNLNGMFMDKFKPYSKSNAPITTLEKLSKALSISVEELKAIAELS LDEKYTLKKIPKIDGSKRIVYSLHPKMRLLQSRINERIFKELVVFPSFLFGSVPSKNDV LNSNVKRDYVSCAKAHCGAKTVLKVDISNFFDNIHRDLVRSVFEEILHIKDEALDYL VDICTKDDFVVQGALTSSYIATLCLFAVEGDVVRRAQRKGLVYTRLVDDITVSSKIS42 / 346Bl 195.70209 WO00#14646633v2NYDFSQMQSHIERMLSEHNLPINKHKTKIFHCSSEPIKVHGLIVDYDSPRLPSDKVKRI RASIHNLKLLAAKNNTKTSVAYRKEFNRCMGRVNELGRVGHEKYESFKKQLQAIKP MPSNRDVAVIDAAIKSLELSYSKGNQNKHWYKRKYDLTRYKMIILTRSESFKEKLEC FKSRLASLKPL (SEQ ID NO: 247).

[0169] In some embodiments, a PE6 protein comprises a reverse transcriptase of the following amino acid sequence (the RT domain of “PE6b”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence:

[0170] ISSSKHTLSQMNKVSNIVKEPELPDIYKEFKDITADTNTEKLPKPIKGLEFE VELTQENYRLPIRNYPLTPVKMQAMNDEINQGLKGGIIRESKAINACPVIFVPRKEGT LRMVVDYRPLNKYVKPNVYPLPLIEQLLAKIQGSTIFTKLDLKSAYHQIRVRKGDEH KLAFRCPRGVFEYLVMPYGISTAPAHFQYFINTILGEAKESHVVCYMDDILIHSKSESE HVKHVKDVLQKLKNANLIINQAKCEFHQSQVKFIGYHISEKGLTPCQENIDKVLQWK QPKNRKELRQFLGSVNYLRKFIPKTSQLTHPLNKLLKKDVRWKWTPTQTQAIENIKQ CLVSPPVLRHFDFSKKILLETDVSDVAVGAVLSQKHDDDKYYPVGYYSAKMSKAQL NYSVSDKEMLAIIKSLEHWRHYLESTIEPFKILTDHRNLIGRITNESEPENKRLARWQL FLQDFNFEINYRPGSANHIADALSRIVDETEPIPKDNEDNSINFVNQISI (SEQ ID NO: 248).

[0171] In some embodiments, a PE6 protein comprises a reverse transcriptase of the following amino acid sequence (the RT domain of “PE6c”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence:

[0172] ISSSKHTLSQMNKVSNIVKEPELPDIYKEFKDITADTNTEKLPKPIKGLEFE VELTQENYRLPIRNYPLTPVKMQAMNDEINQGLKGGIIRESKAINACPVIFVPRKEGT LRMVVDYRPLNKYVKPNVYPLPLIEQLLAKIQGSTIFTKLDLKSAYHQIRVRKGDEH KLAFRCPRGVFEYLVMPYGIKTAPAHFQYFINTILGEAKESHVVCYMDDILIHSKSES EHVKHVKDVLQKLKNANLIINQAKCEFHQSQVKFLGYHISEKGLTPCQENIDKVLQ WKQPKNQKELRQFLGQVNYLRKFIPKTSQLTHPLNKLLKKDVRWKWTPTQTQAIEN IKQCLVSPPVLRHFDFSKKILLETDVSDVAVGAVLSQKHDDDKYYPVGYYSAKMSK AQLNYSVSDKEMLAIIKSLEHWRHYLESTIEPFKILTDHRNLIGRITNESEPENKRLAR WQLFLQDFNFEINYRPGSANHIADALSRIVDETEPIPKDNEDNSINFVNQISI (SEQ ID NO: 249).

[0173] In some embodiments, a PE6 protein comprises a reverse transcriptase comprising the following amino acid sequence (the RT domain of “PE6d”), or an amino acid sequence at 43 / 346Bl 195.70209 WO00#14646633v2least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence:

[0174] TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIP LKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDY RPVQDLREVNKRVEDIHPNVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQP LFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFCEALHRDLADFRIQHPDLILLQYYD DLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNW GPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPV AYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPP DRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLD (SEQ ID NO: 250).

[0175] In some embodiments, a PE6 protein comprises a Cas9 protein of the following amino acid sequence (the Cas9 domain of “PE6e”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence:

[0176] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKAL VRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED YFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFE DREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFL KSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTV KVVDELVKVMGRHKPENIVIEMARENQTTQKGQRNSRERMKRIEEGIKELGSQILKE HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLS ELDKAGFIARQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR KDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI44 / 346Bl 195.70209 WOOO#14646633v2AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSP TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(SEQ ID NO: 251).

[0177] In some embodiments, a PE6 protein comprises a Cas9 protein of the following amino acid sequence (the Cas9 domain of “PE6f ’), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence:

[0178] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFRRLEESFLVE EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKAL VRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL ARGNSRFAWMTRKSEKTITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED YFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFE DREMVEERLKTYAHLFDNKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFL KSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLYEHIANLAGSPAIKKGILQTV KVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLS ELDKAGFIARQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR KDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSP TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH45 / 346Bl 195.70209 WOOO#14646633v2LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 252).

[0179] In some embodiments, a PE6 protein comprises a Cas9 protein of the following amino acid sequence (the Cas9 domain of “PE6g”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence:

[0180] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFRRLEESFLVE EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKAL VRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL ARGNSRFAWMTRKSEKTITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED YFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFE DREMVEERLKTYAHLFDNKVMKQLKRCRYTGWGRLSRKLINGIRDKQSGKTILDFL KSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLYEHIANLAGSPAIKKGILQTV KVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLS ELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR KDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSP TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(SEQ ID NO: 253).Polymerase

[0181] 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 46 / 346Bl 195.70209 WO00#14646633v2herein. 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

[0182] 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 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.47 / 346Bl 195.70209 WOOO#14646633v2Prime editing

[0183] 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.

[0184] 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 5' or 3' 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 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 48 / 346Bl 195.70209 WOOO#14646633v2RNA. 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 desired nucleotide change (e.g., a single nucleotide change, a deletion, 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 49 / 346Bl 195.70209 WOOO#14646633v2changes 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 5' 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.

[0185] 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 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 50 / 346Bl 195.70209 WOOO#14646633v2to 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: trans versions, transitions, deletions, and insertions.

[0186] 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 endonucleases (e.g., FEN1) for helping to drive the prime editing process towards the edited product formation.

[0187] 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 5' or 3' 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 editor51 / 346Bl 195.70209 WOOO#14646633v2

[0188] 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”). Prime editor complex

[0189] 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.Primer binding site

[0190] 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 3' 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 3'-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

[0191] 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 most commonly used 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.52 / 346Bl 195.70209 WOOO#14646633v2Protospacer adjacent motif (PAM)

[0192] 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 5' to 3' 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 5'-NGG-3' 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.

[0193] For example, with reference to the canonical SpCas9 amino acid sequence is SEQ ID NO: 147, the PAM sequence can be modified by introducing one or more mutations, including (a) DI 135V, 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) DI 135V, G1218R, R1335E, and T1337R “the VRER variant”, which alters the PAM specificity to NGCG. In addition, the DI 135E variant of canonical SpCas9 still recognizes NGG, but it is more selective compared to the wild type SpCas9 protein.

[0194] 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).Recombinase53 / 346Bl 195.70209 WOOO#14646633v2

[0195] 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 Bxbl). In some embodiments, the recombinase binds to a recognition site and cleaves the DNA at the recognition site.Reverse transcriptase

[0196] 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 5'-3' RNA-directed DNA polymerase activity, 5'-3' DNA-directed DNA polymerase activity, and RNase H activity. RNase H is a processive 5' and 3' ribonuclease specific for the RNA strand for RNA-DNA hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)). Errors in transcription cannot be corrected by reverse transcriptase because known viral reverse transcriptases lack the 3 '-5' 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.

[0197] 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 54 / 346Bl 195.70209 WOOO#14646633v2more 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 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

[0198] 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

[0199] In other embodiments, e.g., installing a suppressor tRNA gene into a safe harbor site in the genome, may involve a prime editing system referred to herein and in the literature as PASSIGE (an acronym for prime-editing-assisted site- specific integrase gene editing) and PASSIGE variants evoPASSIGE and eePASSIGE, see Pandey, S., Gao, X. D., Krasnow, N. A. et al, “Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing,” Nat. Biomed. Eng. (June 10, 2024), / / doi.org / 10.1038 / s41551-024-01227-l, the entire contents of which are incorporated herein by reference.Pharmaceutically acceptable carrier

[0200] 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

[0201] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) 55 / 346Bl 195.70209 WOOO#14646633v2bonds. 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 famesyl 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 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

[0202] 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

[0203] 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

[0204] 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 56 / 346Bl 195.70209 WOOO#14646633v2codon) 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.T-arm domain

[0205] 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 T C arm due to the presence of thymidine, pseudouridine, and cytidine.Target site

[0206] 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

[0207] 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.

[0208] This disclosure may refer in parts to ‘tRNA isoacceptors’ and ‘tRNA isodecoders,’ which are terms known in the art. In summary, a tRNA isoacceptor is a tRNA that carries the same amino acid as another tRNA but contains a different anticodon, whereas a tRNA isodecoder is a tRNA that contains the same anticodon and is charged with the same amino acid, but differs in nucleotide sequence outside of the anticodon. Variable arm domain

[0209] As used herein, the term “variable arm domain” refers to a loop that is present between the anticodon arm and the T C arm. The length of the variable arm domain is57 / 346Bl 195.70209 WOOO#14646633v2important in the recognition of the aminoacyl-tRNA synthetase for the tRNA. In some embodiments, the tRNA lacks the variable arm domain.Variant

[0210] As used herein the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant Cas9 is a Cas9 comprising one or more changes in amino acid residues as compared to a wild type Cas9 amino acid sequence. The term “variant” encompasses homologous proteins having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% percent identity with a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence, and which display the same or substantially the same functional activity or activities as the reference sequence.Vector

[0211] 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.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

[0212] Aspects of the disclosure relate to methods, compositions, kits, and systems for editing an endogenous tRNA into a suppressor tRNA using prime editing (e.g., to treat diseases caused by premature termination codons). Other aspects of the disclosure relate to methods, compositions, and systems for editing an endogenous indispensable tRNA into a suppressor tRNA using prime editing (e.g., to treat diseases caused by premature termination codons). 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 tRNA. 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,58 / 346Bl 195.70209 WOOO#14646633v2and / or delivery systems for administering any one of the compositions, complexes, polynucleotides, or vectors disclosed herein to a subject in need thereof (e.g., lipid nanoparticles). Additional aspects relate to methods for inserting a new suppressor tRNA gene into a target site in a genome (e.g., a safe harbor locus site) using prime editing.Installing a suppressor tRNA at an endogenous tRNA locus using prime editing

[0213] Certain aspects of the present disclosure relate to using prime editing to edit a target DNA sequence encoding a tRNA gene at its endogenous locus. In some embodiments, the target DNA sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% identical to anyone of SEQ ID NOs: 1-5 (Arg-CCT), SEQ ID NOs: 6-9 (Leu-AAG), SEQ ID NOs: 10-16 (Leu-CAA), SEQ ID NOs: 17-20 (Leu-TAA), SEQ ID NOs: 21-23 (Leu-TAG), SEQ ID NOs: 24-27 (Ser-AGA), SEQ ID NOs: 128- 133 (Arg-TCG), SEQ ID NOs: HJ-HL (Leu-TAG), and / or SEQ ID NOs: 134-146 (Tyr-GTA).

[0214] Exemplary tRNA genes are shown below:tRNA gene Genomic Sequence SEQ ID name coordinates NO: Arg-CCTHomo_sapiens chrl7:75033906- GCCCCAGTGGCCTAATGGATAAGG 1 _tRNA- Arg- 75033978 (+) CACTGGCCTCCTAAGCCAGGGATTGCCT- 1-1 TGGGTTCGAGTCCCACCTGG GGTA Homo_sapiens chrl7:75034431- GCCCCAGTGGCCTAATGGATAAGG 2 _tRNA- Arg- 75034503 (-) CACTGGCCTCCTAAGCCAGGGATTGCCT-2-1 TGGGTTCGAGTCCCACCTGG GGTG Homo_sapiens chrl6:3152900- GCCCCGGTGGCCTAATGGATAAGG 3 _tRNA- Arg- 3152972 (+) CATTGGCCTCCTAAGCCAGGGATTGCCT-3-1 TGGGTTCGAGTCCCACCCGG GGTA Homo_sapiens chr7: 139340700- GCCCCAGTGGCCTAATGGATAAGG 4 _tRNA- 139340772 (+) CATTGGCCTCCTAAGCCAGGGATTGArg-CCT-4-1 TGGGTTCGAGTCCCATCTGGGGTGHomo_sapiens chrl6:3193918- GCCCCAGTGGCCTGATGGATAAGG 5 _tRNA- 3193990 (+) TACTGGCCTCCTAAGCCAGGGATTGArg-CCT-5-159 / 346Bl 195.70209 WOOO#14646633v2TGGGTTCGAGTTCCACCTGGGGTALeu-AAGHomo_sapiens chr5: 181097474- GGTAGCGTGGCCGAGCGGTCTAAG 6 _tRNA- 181097555 (-) GCGCTGGATTAAGGCTCCAGTCTCT Leu-AAG-1-1 TCGGAGGCGTGGGTTCGAAT CCCACCGCTGCCAHomo_sapiens chr5:181101840- GGTAGCGTGGCCGAGCGGTCTAAG 6 _tRNA- Leu- 181101921 (+) GCGCTGGATTAAGGCTCCAGTCTCT AAG- 1-2 TCGGAGGCGTGGGTTCGAAT CCCACCGCTGCCAHomo_sapiens chr5: 181174044- GGTAGCGTGGCCGAGCGGTCTAAG 6 _tRNA- Leu- 181174125 (-) GCGCTGGATTAAGGCTCCAGTCTCT AAG- 1-3 TCGGAGGCGTGGGTTCGAAT CCCACCGCTGCCAHomo_sapiens chr5:181187701- GGTAGCGTGGCCGAGCGGTCTAAG 7 _tRNA- Leu- 181187782 (+) GCGCTGGATTAAGGCTCCAGTCTCT AAG-2-1 TCGGGGGCGTGGGTTCGAAT CCCACCGCTGCCAHomo_sapiens chr6:28943622- GGTAGCGTGGCCGAGCGGTCTAAG 7 _tRNA- Leu- 28943703 (-) GCGCTGGATTAAGGCTCCAGTCTCT AAG-2-2 TCGGGGGCGTGGGTTCGAAT CCCACCGCTGCCAHomo_sapiens chrl4:20610132- GGTAGCGTGGCCGAGCGGTCTAAG 7 _tRNA- Leu- 20610213 (+) GCGCTGGATTAAGGCTCCAGTCTCT AAG-2-3 TCGGGGGCGTGGGTTCGAAT CCCACCGCTGCCAHomo_sapiens chrl6:22297140- GGTAGCGTGGCCGAGCGGTCTAAG 7 _tRNA- 22297221 (+) GCGCTGGATTAAGGCTCCAGTCTCT Leu-AAG-2-4 TCGGGGGCGTGGGTTCGAATCCCACCGCTGCCA60 / 346Bl 195.70209 WOOO#14646633v2Homo_sapiens chr6:28989002- GGTAGCGTGGCCGAGCGGTCTAAG 8 _tRNA- 28989083 (+) GCGCTGGATTAAGGCTCCAGTCTCT Leu-AAG-3-1 TCGGGGGCGTGGGTTCAAAT CCCACCGCTGCCAHomo_sapiens chr6:28478623- GGTAGCGTGGCCGAGTGGTCTAAG 9 _tRNA- 28478704 (-) ACGCTGGATTAAGGCTCCAGTCTCT Leu-AAG-4-1 TCGGGGGCGTGGGTTTGAAT CCCACCGCTGCCALeu-CAAHomo_sapiens chr6:28896223- GTCAGGATGGCCGAGTGGTCTAAG 10 _tRNA- Leu- 28896328 (-) GCGCCAGACTCAAGCTAAGCTTCCT CAA- 1-1 CCGCGGTGGGGATTCTGGTC TCCAATGGAGGCGTGGGTTCGAAT CCCACTTCTGACAHomo_sapiens chr6:28941053- GTCAGGATGGCCGAGTGGTCTAAG 11 _tRNA- Leu- 28941157 (+) GCGCCAGACTCAAGCTTGGCTTCCT CAA- 1-2 CGTGTTGAGGATTCTGGTCTC CAATGGAGGCGTGGGTTCGAATCC CACTTCTGACAHomo_sapiens chr6:27605638- GTCAGGATGGCCGAGTGGTCTAAG 12 _tRNA- Leu- 27605745 (-) GCGCCAGACTCAAGCTTACTGCTTC CAA-2-1 CTGTGTTCGGGTCTTCTGGTC TCCGTATGGAGGCGTGGGTTCGAAT CCCACTTCTGACAHomo_sapiens chr6:27602569- GTCAGGATGGCCGAGTGGTCTAAG 13 _tRNA- Leu- 27602675 (-) GCGCCAGACTCAAGTTGCTACTTCC CAA-3-1 CAGGTTTGGGGCTTCTGGTCT CCGCATGGAGGCGTGGGTTCGAAT CCCACTTCTGACAHomo_sapiens chr 1:248873855- GTCAGGATGGCCGAGTGGTCTAAG 14 _tRNA- Leu- 248873960 (+) GCGCCAGACTCAAGGTAAGCACCT CAA-4-1 TGCCTGCGGGCTTTCTGGTCTC61 / 346Bl 195.70209 WOOO#14646633v2CGGATGGAGGCGTGGGTTCGAATC CCACTTCTGACAHomo_sapiens chrl 1:9275243- GCCTCCTTAGTGCAGTAGGTAGCGC 15 _tRNA- Leu- 9275316 (+) ATCAGTCTCAAAATCTGAATGGTCC CAA-5-1 TGAGTTCAAGCCTCAGAGGG GGCA Homo_sapiens chrl:161611946- GTCAGGATGGCCGAGCAGTCTTAA 16 _tRNA- 161612029 (-) GGCGCTGCGTTCAAATCGCACCCTC Leu-CAA-6-1 CGCTGGAGGCGTGGGTTCGA ATCCCACTTTTGACALeu-TAAHomo_sapiens chr6: 144216547- ACCAGGATGGCCGAGTGGTTAAGG 17 _tRNA- 144216629 (+) CGTTGGACTTAAGATCCAATGGAC Leu-TAA-1-1 ATATGTCCGCGTGGGTTCGAAC CCCACTCCTGGTAHomo_sapiens chr6:27721119- ACCGGGATGGCCGAGTGGTTAAGG 18 _tRNA- 27721201 (-) CGTTGGACTTAAGATCCAATGGGCT Leu-TAA-2-1 GGTGCCCGCGTGGGTTCGAA CCCCACTCTCGGTAHomo_sapiens chrl 1:59551755- ACCAGAATGGCCGAGTGGTTAAGG 19 _tRNA- 59551837 (+) CGTTGGACTTAAGATCCAATGGATT Leu-TAA-3-1 CATATCCGCGTGGGTTCGAAC CCCACTTCTGGTAHomo_sapiens chr6:27230555- ACCGGGATGGCTGAGTGGTTAAGG 20 _tRNA- Leu- 27230637 (-) CGTTGGACTTAAGATCCAATGGAC TAA-4-1 AGGTGTCCGCGTGGGTTCGAG CCCCACTCCCGGTALeu-TAGHomo_sapiens chrl7:8120314- GGTAGCGTGGCCGAGCGGTCTAAG 21 _tRNA- Leu- 8120395 (-) GCGCTGGATTTAGGCTCCAGTCTCT TAG- 1-1 TCGGAGGCGTGGGTTCGAATCCCACCGCTGCCA62 / 346Bl 195.70209 WOOO#14646633v2Homo_sapiens chrl4:20625370- GGTAGTGTGGCCGAGCGGTCTAAG 22 _tRNA- Leu- 20625451 (+) GCGCTGGATTTAGGCTCCAGTCTCT TAG-2-1 TCGGGGGCGTGGGTTCGAAT CCCACCACTGCCAHomo_sapiens chrl6:22195711- GGTAGCGTGGCCGAGTGGTCTAAG 23 _tRNA- Leu- 22195792 (-) GCGCTGGATTTAGGCTCCAGTCATT TAG-3-1 TCGATGGCGTGGGTTCGAATC CCACCGCTGCCASer-AGAHomo_sapiens chr6:27541775- GTAGTCGTGGCCGAGTGGTTAAGG 24 _tRNA- Ser- 27541856 (-) CGATGGACTAGAAATCCATTGGGG AGA- 1-1 TTTCCCCGCGCAGGTTCGAATC CTGCCGACTACGHomo_sapiens chr6:26327589- GTAGTCGTGGCCGAGTGGTTAAGG 25 _tRNA- Ser- 26327670 (+) CGATGGACTAGAAATCCATTGGGG AGA-2-1 TCTCCCCGCGCAGGTTCGAATC CTGCCGACTACGHomo_sapiens chr6:27478812- GTAGTCGTGGCCGAGTGGTTAAGG 25 _tRNA- Ser- 27478893 (+) CGATGGACTAGAAATCCATTGGGG AGA-2-2 TCTCCCCGCGCAGGTTCGAATC CTGCCGACTACGHomo_sapiens chr6:27495814- GTAGTCGTGGCCGAGTGGTTAAGG 25 _tRNA- Ser- 27495895 (+) CGATGGACTAGAAATCCATTGGGG AGA-2-3 TCTCCCCGCGCAGGTTCGAATC CTGCCGACTACGHomo_sapiens chr6:27503039- GTAGTCGTGGCCGAGTGGTTAAGG 25 _tRNA- Ser- 27503120 (+) CGATGGACTAGAAATCCATTGGGG AGA-2-4 TCTCCCCGCGCAGGTTCGAATC CTGCCGACTACGHomo_sapiens chr8:95269657- GTAGTCGTGGCCGAGTGGTTAAGG 25 _tRNA- 95269738 (-) CGATGGACTAGAAATCCATTGGGG Ser-AGA-2-5 TCTCCCCGCGCAGGTTCGAATCCTGCCGACTACG63 / 346Bl 195.70209 WOOO#14646633v2Homo_sapiens chrl7:8226610- GTAGTCGTGGCCGAGTGGTTAAGG 25 _tRNA- 8226691 (-) CGATGGACTAGAAATCCATTGGGG Ser-AGA-2-6 TCTCCCCGCGCAGGTTCGAATC CTGCCGACTACGHomo_sapiens chr6:27532208- GTAGTCGTGGCCGAGTGGTTAAGG 26 _tRNA- 27532289 (+) CGATGGACTAGAAATCCATTGGGG Ser- AGA-3-1 TTTCCCCACGCAGGTTCGAATC CTGCCGACTACGHomo_sapiens chr6:27553413- GTAGTCGTGGCCGAGTGGTTAAGG 27 _tRNA- Ser- 27553494 (-) TGATGGACTAGAAACCCATTGGGG AGA-4-1 TCTCCCCGCGCAGGTTCGAATC CTGCCGACTACGArg-TCGHomo_sapiens chrl5:89335073- GGCCGCGTGGCCTAATGGATAAGG _tRNA-Arg- 89335145 (+) CGTCTGACTTCGGATCAGAAGATTG 128 TCG-1-1 CAGGTTCGAGTCCTGCCGCGGTCGHomo_sapiens chr6:26322818- GACCACGTGGCCTAATGGATAAGG _tRNA- Arg- 26322890 (+) CGTCTGACTTCGGATCAGAAGATTG 129 TCG-2-1 AGGGTTCGAATCCCTCCGTGG TTA Homo_sapiens chrl7:75035113- GACCGCGTGGCCTAATGGATAAGG _tRNA- Arg- 75035185 (+) CGTCTGACTTCGGATCAGAAGATTG 130 TCG-3-1 AGGGTTCGAGTCCCTTCGTGG TCG Homo_sapiens chr6:26299677- GACCACGTGGCCTAATGGATAAGG _tRNA- Arg- 26299749 (+) CGTCTGACTTCGGATCAGAAGATTG 131 TCG-4-1 AGGGTTCGAATCCCTTCGTGG TTA Homo_sapiens chr6:28543114- GACCACGTGGCCTAATGGATAAGG _tRNA- Arg- 28543186 (-) CGTCTGACTTCGGATCAGAAGATTG 132 TCG-5-1 AGGGTTCGAATCCCTTCGTGG TTG Homo_sapiens chr9: 110198523- GGCCGTGTGGCCTAATGGATAAGG _tRNA- Arg- 110198595 (+) CGTCTGACTTCGGATCAAAAGATTG 133 TCG-6-1 CAGGTTTGAGTTCTGCCACGG TCG Leu-TAG64 / 346Bl 195.70209 WOOO#14646633v2Homo_sapiens chrl7:8120314- GGTAGCGTGGCCGAGCGGTCTAAG _tRNA- Leu- 8120395 (-) GCGCTGGATTTAGGCTCCAGTCTCT 21 TAG-1-1 TCGGAGGCGTGGGTTCGAAT CCCACCGCTGCCAHomo_sapiens chrl4:20625370- GGTAGTGTGGCCGAGCGGTCTAAG _tRNA- Leu- 20625451 (+) GCGCTGGATTTAGGCTCCAGTCTCT 22 TAG-2-1 TCGGGGGCGTGGGTTCGAAT CCCACCACTGCCAHomo_sapiens chrl6:22195711- GGTAGCGTGGCCGAGTGGTCTAAG _tRNA- Leu- 22195792 (-) GCGCTGGATTTAGGCTCCAGTCATT 23 TAG-3-1 TCGATGGCGTGGGTTCGAATC CCACCGCTGCCATyr-GTAHomo_sapiens chr6:26568858- CCTTCGATAGCTCAGTTGGTAGAGC _tRNA- Tyr- 26568948 (+) GGAGGACTGTAGTTGGCTGTGTCCT 134 GTA- 1-1 TAGACATCCTTAGGTCGCTG GTTCGAATCCGGCTCGAAGGAHomo_sapiens chr2:27050782- CCTTCGATAGCTCAGTTGGTAGAGC _tRNA- Tyr- 27050870 (+) GGAGGACTGTAGTGGATAGGGCGT 135 GTA-2-1 GGCAATCCTTAGGTCGCTGGT TCGATTCCGGCTCGAAGGAHomo_sapiens chr6:26577104- CCTTCGATAGCTCAGTTGGTAGAGC _tRNA- Tyr- 26577192 (+) GGAGGACTGTAGGCTCATTAAGCA 136 GTA-3-1 AGGTATCCTTAGGTCGCTGGT TCGAATCCGGCTCGGAGGAHomo_sapiens chrl4:20657464- CCTTCGATAGCTCAGCTGGTAGAGC _tRNA- Tyr- 20657557 (-) GGAGGACTGTAGATTGTATAGACA 137 GTA-4-1 TTTGCGGACATCCTTAGGTCG CTGGTTCGATTCCAGCTCGAAGGAHomo_sapiens chr8:66113367- CCTTCGATAGCTCAGCTGGTAGAGC _tRNA- Tyr- 66113459 (+) GGAGGACTGTAGCTACTTCCTCAGC 138 GTA-5-1 AGGAGACATCCTTAGGTCGCTGGTTCGATTCCGGCTCGAAGGA65 / 346Bl 195.70209 WOOO#14646633v2Homo_sapiens chr8:66113988- CCTTCGATAGCTCAGCTGGTAGAGC _tRNA- 66114076 (+) GGAGGACTGTAGGCGCGCGCCCGT 139 Tyr-GTA-5-2 GGCCATCCTTAGGTCGCTGG TTCGATTCCGGCTCGAAGGAHomo_sapiens chrl4:20653099- CCTTCGATAGCTCAGCTGGTAGAGC _tRNA- 20653192 (-) GGAGGACTGTAGCCTGTAGAAACA 140 Tyr-GTA-5-3 TTTGTGGACATCCTTAGGTCG CTGGTTCGATTCCGGCTCGAAGGAHomo_sapiens chrl4:20663192- CCTTCGATAGCTCAGCTGGTAGAGC _tRNA- 20663285 (-) GGAGGACTGTAGATTGTACAGACA 141 Tyr-GTA-5-4 TTTGCGGACATCCTTAGGTCG CTGGTTCGATTCCGGCTCGAAGGAHomo_sapiens chrl4:20683273- CCTTCGATAGCTCAGCTGGTAGAGC _tRNA- Tyr- 20683361 (+) GGAGGACTGTAGTACTTAATGTGTG 142 GTA-5-5 GTCATCCTTAGGTCGCTGGTT CGATTCCGGCTCGAAGGAHomo_sapiens chr6:26594874- CCTTCGATAGCTCAGCTGGTAGAGC _tRNA- Tyr- 26594962 (+) GGAGGACTGTAGGGGTTTGAATGT 143 GTA-6-1 GGTCATCCTTAGGTCGCTGGT TCGAATCCGGCTCGGAGGAHomo_sapiens chrl4:20659958- CCTTCGATAGCTCAGCTGGTAGAGC _tRNA- Tyr- 20660051 (-) GGAGGACTGTAGACTGCGGAAACG 144 GTA-7-1 TTTGTGGACATCCTTAGGTCG CTGGTTCAATTCCGGCTCGAAGGAHomo_sapiens chr6:26575570- CTTTCGATAGCTCAGTTGGTAGAGC _tRNA- Tyr- 26575659 (+) GGAGGACTGTAGGTTCATTAAACT 145 GTA-8-1 AAGGCATCCTTAGGTCGCTGGT TCGAATCCGGCTCGAAGGAHomo_sapiens chr8:65697297- TCTTCAATAGCTCAGCTGGTAGAGC _tRNA- Tyr- 65697384 (-) GGAGGACTGTAGGTGCACGCCCGT 146 GTA-9-1 GGCCATTCTTAGGTGCTGGTTTGATTCCGACTTGGAGAG66 / 346Bl 195.70209 WOOO#14646633v2

[0215] For any of the disclosed nucleotide sequences herein (e.g. tRNA sequences), the disclosure contemplates variants of said sequences which are defined in terms of degree of percent identity. For example, the disclosure contemplates any nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or up to 100% sequence identity with any of the disclosed nucleotide sequences herein (e.g., any tRNA sequence).

[0216] In addition, for any of the disclosed nucleotide sequences herein (e.g. tRNA sequences), the disclosure contemplates variants of said sequences which are defined in terms of numbers of nucleotide changes. For example, the disclosure contemplates any nucleotide sequence having or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 nucleotide changes relative to any one of the herein disclosed nucleotide sequences (e.g., any tRNA sequence disclosed herein).

[0217] For any of the disclosed amino acid sequences herein (e.g. Cas9 sequences), the disclosure contemplates variants of said sequences which are defined in terms of degree of percent identity. For example, the disclosure contemplates any nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or up to 100% sequence identity with any of the disclosed amino acid sequences herein (e.g., any Cas9 sequence).

[0218] In addition, for any of the disclosed amino acid sequences herein (e.g. Cas9 sequences), the disclosure contemplates variants of said sequences which are defined in terms of numbers of amino acid substitutions. For example, the disclosure contemplates any amino acid sequence having or having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 amino acid substitutions relative to any one of the herein disclosed amino acid sequences (e.g., a Cas9 disclosed herein).

[0219] As disclosed elsewhere herein, some aspects of the current disclosure relate to pegRNAs for editing a tRNA gene at its endogenous locus into a suppressor tRNA gene via prime editing. In some embodiments, the pegRNA is for editing an endogenous tRNA-Leu-TAA gene comprising a nucleic acid sequence having at least 80%, at least 85%, at least 67 / 346Bl 195.70209 WOOO#14646633v290%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to any one of SEQ ID NOs: 17-20 (tRNA-Leu-TAA-1-1 to 4-1). In some embodiments, the endogenous tRNA-Leu gene comprises a nucleic acid sequence that is identical to SEQ ID NO: 17.

[0220] In some embodiments, the pegRNA comprises a DNA synthesis template encoding a 5'-CUA-3' or 5'-UCA-3' nonsense suppressor anticodon sequence to be inserted into the endogenous tRNA-Leu gene to produce the suppressor tRNA-Leu gene (e.g., to read through TGA or TAG premature stop codons).

[0221] In some embodiments, the DNA synthesis template (e.g. RTT) further encodes one or more mutations to be inserted into an anticodon loop of an endogenous tRNA-Leu gene (e.g., SEQ ID NOs: 17-20). In some embodiments, the one or more mutations to be inserted into an anticodon loop of the endogenous tRNA comprises a TA> CG mutation at hpl2, a GOCG mutation at hpl3, a GOTA mutation at hpl4, and / or mut38 A> T.

[0222] In some embodiments, the one or more mutations to be inserted into an anticodon loop of the endogenous tRNA comprises a TA> CG mutation at hpl2, a GOTA mutation at hpl3, a GOTA mutation at hpl4, and / or mut38 A> T.

[0223] In some embodiments, the one or more mutations to be inserted into an anticodon loop of the endogenous tRNA comprises a TA> CG mutation at hpl2, a GOAT at hp 13, a GOTA mutation at hpl4, and / or mut38 A> T.

[0224] The DNA synthesis template (e.g. RTT) may further encode one or more single nucleotide deletions, e.g., in a D-loop, an anti-codon loop, a variable loop, or a T-loop of the endogenous tRNA-Leu gene, relative to any one of SEQ ID NOs: 17-20, according to some embodiments.

[0225] The DNA synthesis template (e.g., RTT), according to some embodiments, further encodes a variable loop having a nucleic acid sequence that is different than a variable loop of the endogenous tRNA gene (e.g., tRNA-Leu). For example, in some embodiments, the DNA synthesis template encodes for a variable loop of tRNA-Leu-TAA-3-1 (SEQ ID NO: 19) to be inserted into a variable loop domain of the endogenous tRNA-Leu-TAA-1-1 (SEQ ID NO: 17).

[0226] In some embodiments, a pegRNA guides a prime editor (PE) to a tRNA gene locus, such as a tRNA-Leu-TAA-1-1 (SEQ ID NO: 17) locus. The skilled artisan may use any suitable PE known in the art to edit the endogenous tRNA gene into a suppressor tRNA gene. Exemplary embodiments include, but are not limited to, PE6a, PE6b, PE6c, PE6d,68 / 346Bl 195.70209 WOOO#14646633v2PE6d+MLHldn, PE6e, PE6f, PE6g, PEmax, PEmax+MLHldn, and PEmaxA. In some embodiments, the PE is PE6c.

[0227] As described elsewhere herein, the pegRNA comprises an extension arm (e.g., 3' or 5') comprising a DNA synthesis template (RTT) and a PBS domain.

[0228] In some embodiments, the PBS is between 8 and 16 nucleotides. In some embodiments, the PBS is 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. In some embodiments, the PBS is 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. Combinations are also possible in some embodiments. For example, in some embodiments, the PBS is greater than or equal to 8 nucleotides and less than or equal to 16 nucleotides. Combinations of other ranges are also possible (e.g., greater than or equal to 8 nucleotides and less than or equal to 16 nucleotides). Other ranges are also possible.

[0229] In some embodiments, the PBS has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to any one of SEQ ID NOs: 61-86.

[0230] In some embodiments, the DNA synthesis template (e.g., RTT) is between 25 and 35 nucleotides. In some embodiments, the DNA synthesis is 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, greater than or equal to 30 nucleotides, greater than or equal to 31 nucleotides, greater than or equal to 32 nucleotides, greater than or equal to 33 nucleotides, greater than or equal to 34 nucleotides or greater than or equal to 35 nucleotides. In some embodiments, the DNA synthesis is of less than or equal to 35 nucleotides, less than or equal to 34 nucleotides, less than or equal to 33 nucleotides, less than or equal to 32 nucleotides, less than or equal to 31 nucleotides, 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 or less than or equal to 25 nucleotides. Combinations are also possible in some embodiments. For example, in some embodiments, the DNA synthesis is greater than or equal to 25. nucleotides69 / 346Bl 195.70209 WOOO#14646633v2and less than or equal to 35. nucleotides. Combinations of other ranges are also possible (e.g., greater than or equal to 25. nucleotides and less than or equal to 35. nucleotides). Other ranges are also possible. In some embodiments, the DNA synthesis template is between 33 and 35 nucleotides.

[0231] In some embodiments, the DNA synthesis template has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to any one of SEQ ID NOs: 29-60.

[0232] The pegRNA also comprises a spacer sequence and pegRNA scaffold, according to some embodiments. In some embodiments, the spacer sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to any one of SEQ ID NOs: 108-127.

[0233] In some embodiments, the pegRNA comprises a pegRNA scaffold having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 28.

[0234] In some embodiments, a pegRNA is for editing an endogenous tRNA-gene (SEQ ID NOs: 1-27 and 128-146) to produce a suppressor tRNA capable of reading through a premature termination codon. In some embodiments, the pegRNA comprises a DNA synthesis template that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to any one of SEQ ID NOs: 29-60. In some embodiments, the pegRNA comprises a PBS having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to any one of SEQ ID NOs: 61-86. In some embodiments, the pegRNA comprises a spacer sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to any one of SEQ ID NOs: 108-127. In some embodiments, the pegRNA comprises a pegRNA scaffold having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 28.

[0235] In some embodiments, a pegRNA is for editing an endogenous tRNA-Arg-CCT-3-1 gene (SEQ ID NO: 3) or a tRNA-Arg-CCT-4-1 gene (SEQ ID NO: 4) to produce a suppressor tRNA capable of reading through a TAG premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 71, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 39, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 116.70 / 346Bl 195.70209 WOOO#14646633v2

[0236] In some embodiments, a pegRNA is for editing an endogenous tRNA-Leu-TAA- 4-1 gene (SEQ ID NO: 20) to produce a suppressor tRNA capable of reading through a TAG premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 72, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 40, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 117.

[0237] In some embodiments, a pegRNA is for editing an endogenous tRNA-Arg-CCT- 5-1 gene (SEQ ID NO: 5) to produce a suppressor tRNA capable of reading through a TAG premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 73, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 41, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 116.

[0238] In some embodiments, a pegRNA is for editing an endogenous tRNA-Arg-CCT-5-1 gene (SEQ ID NO: 5), tRNA-Arg-CCT-1-1 gene (SEQ ID NO: 1), tRNA-Arg-CCT-3-1 gene (SEQ ID NO: 3), tRNA-Arg-CCT-4-1 gene (SEQ ID NO: 4), or tRNA-Arg-CCT-2- 1 gene (SEQ ID NO: 2) to produce a suppressor tRNA capable of reading through a TAG premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 71, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 42, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 116.

[0239] In some embodiments, a pegRNA is for editing an endogenous tRNA-Leu-AAG-2-1 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-3 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-2 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-4 gene (SEQ ID NO: 7), tRNA-Leu-AAG-3-1 gene (SEQ ID NO: 8), tRNA-Leu-AAG-1-2 gene (SEQ ID NO: 6), tRNA-Leu-AAG-1-3 gene (SEQ ID NO: 6), or tRNA-Leu-AAG-1-1 gene (SEQ ID NO: 6) to produce a suppressor tRNA capable of reading through a TAG premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 74, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 43, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 118.71 / 346Bl 195.70209 WOOO#14646633v2

[0240] In some embodiments, a pegRNA is for editing an endogenous tRNA-Leu-TAA- 1-1 gene (SEQ ID NO: 17) to produce a suppressor tRNA capable of reading through a TAG premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 75, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 44, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 110.

[0241] In some embodiments, a pegRNA is for editing an endogenous tRNA-Ser- AGA-4-1 gene (SEQ ID NO: 27) to produce a suppressor tRNA capable of reading through a TAG premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 76, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 45, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 119.

[0242] In some embodiments, a pegRNA is for editing an endogenous tRNA-Leu-AAG- 2-1 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-3 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-2 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-4 gene (SEQ ID NO: 7), tRNA-Leu-AAG-3-1 gene (SEQ ID NO: 8), tRNA-Leu-AAG-1-2 gene (SEQ ID NO: 6), tRNA-Leu-AAG-1-3 gene (SEQ ID NO: 6), or tRNA-Leu-AAG-1-1 gene (SEQ ID NO: 6) to produce a suppressor tRNA capable of reading through a TAG premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 77, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 46, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 118.

[0243] In some embodiments, a pegRNA is for editing an endogenous tRNA-Tyr-GTA-1-1 gene (SEQ ID NO: 134) to produce a suppressor tRNA capable of reading through a TAG premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 78, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 47, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 120.

[0244] In some embodiments, a pegRNA is for editing an endogenous tRNA-Leu-TAA- 3-1 gene (SEQ ID NO: 19) to produce a suppressor tRNA capable of reading through a TAG 72 / 346Bl 195.70209 WOOO#14646633v2premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 79, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 48, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 121.

[0245] In some embodiments, a pegRNA is for editing an endogenous tRNA-Tyr-GTA-2-1 gene (SEQ ID NO: 135) to produce a suppressor tRNA capable of reading through a TAG premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 80, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 49, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 122.

[0246] In some embodiments, a pegRNA is for editing an endogenous tRNA-Leu-TAG-1-1 gene (SEQ ID NO: 21), tRNA-Leu-AAG-2-1 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-3 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-2 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-4 gene (SEQ ID NO: 7), tRNA-Leu-TAG-2-1 gene (SEQ ID NO: 22), tRNA-Leu-AAG-3-1 gene (SEQ ID NO: 8), tRNA-Leu-AAG-1-2 gene (SEQ ID NO: 6), tRNA-Leu-AAG-1-3 gene (SEQ ID NO: 6), or a tRNA-Leu-AAG-1-1 gene (SEQ ID NO: 6) to produce a suppressor tRNA capable of reading through a TAG premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 81, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 50, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 123.

[0247] In some embodiments, a pegRNA is for editing an endogenous tRNA-Tyr-GTA-5-5 gene (SEQ ID NO: 142) to produce a suppressor tRNA capable of reading through a TAG premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 82, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 51, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 124.

[0248] In some embodiments, a pegRNA is for editing an endogenous tRNA-Leu-TAG-1-1 gene (SEQ ID NO: 21), tRNA-Leu-AAG-2-1 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-3 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-2 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-473 / 346Bl 195.70209 WOOO#14646633v2gene (SEQ ID NO: 7), tRNA-Leu-TAG-2-1 gene (SEQ ID NO: 22), tRNA-Leu-AAG-3-1 gene (SEQ ID NO: 8), tRNA-Leu-AAG-1-2 gene (SEQ ID NO: 6), tRNA-Leu-AAG-1-3 gene (SEQ ID NO: 6), or a tRNA-Leu-AAG-1-1 gene (SEQ ID NO: 6) to produce a suppressor tRNA capable of reading through a TAG premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 83, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 50, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 123.

[0249] In some embodiments, a pegRNA is for editing an endogenous tRNA-Arg-TCG-6-1 gene (SEQ ID NO: 133) to produce a suppressor tRNA capable of reading through a TGA premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 85, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 54, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 126.

[0250] In some embodiments, a pegRNA is for editing an endogenous tRNA-Arg-TCG-1-1 gene (SEQ ID NO: 128), tRNA-Arg-TCG-2- 1 gene (SEQ ID NO: 129), tRNA-Arg-TCG-3-1 gene (SEQ ID NO: 130), tRNA-Arg-TCG-4- 1 gene (SEQ ID NO: 131), or tRNA- Arg -TCG-5-1 gene (SEQ ID NO: 132), to produce a suppressor tRNA capable of reading through a TGA premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 85, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 55, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 126.

[0251] In some embodiments, a pegRNA is for editing an endogenous tRNA-Arg-CCT-1-1 gene (SEQ ID NO: 1) or a tRNA-Arg-CCT-2-1 gene (SEQ ID NO: 2) to produce a suppressor tRNA capable of reading through a TGA premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 73, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 56, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 116.74 / 346Bl 195.70209 WOOO#14646633v2

[0252] In some embodiments, a pegRNA is for editing an endogenous tRNA-Arg-TCG-6-1 gene (SEQ ID NO: 133) to produce a suppressor tRNA capable of reading through a TGA premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 85, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 54, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 126.

[0253] In some embodiments, a pegRNA is for editing an endogenous tRNA-Arg-TCG-1-1 gene (SEQ ID NO: 128), tRNA-Arg-TCG-2- 1 gene (SEQ ID NO: 129), tRNA-Arg-TCG-3-1 gene (SEQ ID NO: 130), tRNA-Arg-TCG-4- 1 gene (SEQ ID NO: 131), or tRNA- Arg -TCG-5-1 gene (SEQ ID NO: 132), to produce a suppressor tRNA capable of reading through a TGA premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 85, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 57, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 126.

[0254] In some embodiments, a pegRNA is for editing an endogenous tRNA-Leu-TAA-1-1 gene (SEQ ID NO: 17) to produce a suppressor tRNA capable of reading through a TGA premature termination codon. In some embodiments, the pegRNA comprises a PBS sequence having a nucleic acid sequence identical to SEQ ID NO: 75, a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 58, a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 110.

[0255] In some embodiments, the DNA synthesis template further encodes one or more mutations to be inserted into an anticodon loop of the endogenous tRNA gene. In some embodiments, the one or more mutations to be inserted into an anticodon loop of the endogenous tRNA comprises a TA> CG mutation at hpl2, a GOCG mutation at hpl3, a GOTA mutation at hpl3, a GOAT mutation at hpl3, a GOTA mutation at hpl4, and / or a A> T mutation at amino acid position 38.

[0256] Some aspects of the disclosure relate to compositions comprising any one of the pegRNAs disclosed herein, any one of the complexes disclosed herein, any one of the polynucleotides disclosed herein, and / or any one of the cells disclosed herein.

[0257] In some embodiments, the composition further comprises a nicking guide RNA (ngRNA). In some embodiments, the nicking guide RNA (ngRNA) comprises a nucleic acid 75 / 346Bl 195.70209 WOOO#14646633v2sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% identical to any one of SEQ ID NOs: 91-100.

[0258] Other compositions are directed to other embodiments. For example, in some embodiments, the composition comprises (i) a first nucleotide sequence encoding a N-terminal portion of a prime editor fused at its C-terminus to an intein-N, and (ii) a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the prime editor. In some embodiments, the second nucleotide sequence further encodes for any one of the pegRNAs disclosed herein. In some embodiments, the pegRNA is operably linked to a promoter.

[0259] In some embodiments, a N-terminal portion of the prime editor comprises a portion of any one of SEQ ID NOs: 147-207 that corresponds to amino acids 1-844 or 1-1024 of SEQ ID NO: 147. In some embodiments, the C-terminal portion of the prime editor comprises a portion of any one of SEQ ID NOs: 147-207 that correspond to amino acids 845-1368 or 1025-1368 of SEQ ID NO: 147.

[0260] In some embodiments, a first nucleotide sequence further comprises a nucleotide sequence encoding a sgRNA sequence operably linked to a promoter. In some embodiments, the second nucleotide sequence further encodes for a nicking guide RNA (ngRNA) sequence.

[0261] In some case, the ngRNA sequence is operably linked to a promoter, although this is not a requirement of the invention. In some embodiments, the ngRNA 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%, or at least 100% identical to any one of SEQ ID NOs: 91-100.

[0262] The compositions disclosed herein may further comprise one or more additional embodiments. For example, in some embodiments, the three terminal amino acids at the N-terminus of the C-terminal portion are SEQ, SFQ, SFN, SEN, or CFN. Additionally, in some embodiments, the pegRNA comprises a evopreQl motif at its 3' end. In some embodiments, the second nucleotide sequence further comprises a nucleotide encoding a MMLV RT ARNaseH codon optimized for expression in a mammalian cell. In some embodiments, the first nucleotide sequence and / or second nucleotide sequence are operably linked to a promoter selected from the group consisting of EFS, Cbh, sCAG, hCMV, mPGK, hSYN, and U6. Lastly, in some embodiments, the first nucleotide sequence and / or second nucleotide sequence further encode for a SV40 late polyadenylation signal (e.g., SV40 late poly A).

[0263] Aspects of the disclosure are directed to editing a DNA sequence encoding a tRNA at a target site. The target site in the DNA sequence, according to some embodiments, encodes for one or more domains of the tRNA. In some embodiments, the domain is a D-arm 76 / 346Bl 195.70209 WOOO#14646633v2domain, a T-arm domain, a variable arm domain, an acceptor stem domain, and an anticodon arm domain comprising an anticodon sequence.

[0264] In some embodiments, the methods comprise editing a DNA sequence encoding an endogenous tRNA at a target site, comprising contacting the DNA sequence at the target site with a prime editor and a pegRNA, 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 encoded tRNA into an encoded suppressor tRNA, wherein the pegRNA comprises a spacer sequence, a gRNA core, and an extension arm

[0265] In some embodiments, the methods comprise editing a editing a DNA sequence encoding an endogenous tRNA at a target site, comprising contacting the DNA sequence at the target site with a prime editor and a pegRNA, 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 encoded tRNA into an encoded suppressor tRNA, wherein the pegRNA comprises a spacer sequence, a gRNA core, and an extension arm, wherein the spacer sequence and extension arms are any sequences listed in Table 2 of PCT / US2024 / 011892 wherein the DNA sequence is any sequence listed in Table 1 of PCT / US2024 / 011892.

[0266] In some embodiments, the methods comprise contacting the DNA sequence at a target site with a prime editor and a pegRNA. The prime editor may install one or more modifications at the target site (e.g., insertion, deletion, or substitution), relative to the endogenous tRNA, thus converting said tRNA into a suppressor tRNA. In some embodiments, the one or more modifications comprise installing a single base nucleotide in the variable arm domain of the tRNA. In some embodiments, installing the single base nucleotide in the variable arm results in replacement of a cognate amino acid with a noncognate amino acid. In some embodiments, the non-cognate amino acid is serine.

[0267] In some embodiments, the one or more edits (e.g., modifications) are selected from the group consisting of insertions, deletions, and substitutions. In some embodiments, the edit is an insertion. In some embodiments, the edits are deletions. In some embodiments, the edits are substitutions.

[0268] In some embodiments, the one or more modifications comprise installing a C70U mutation in the acceptor stem domain. In some embodiments, installing the C70U mutation creates a G3: U70 base pair in the acceptor stem domain and results in the replacement of the cognate amino acid with a non-cognate amino acid. In some embodiments, the non-cognate amino acid is alanine.77 / 346Bl 195.70209 WOOO#14646633v2

[0269] In some embodiments, the one or more modifications comprise installing one or more edits (e.g., insertions, deletion, substitution, etc.) in the anticodon sequence of the anticodon arm domain, thus converting the anticodon sequence into a nonsense suppressor anticodon sequence. In some embodiments, the one or more modifications comprises substituting the DNA sequence encoding the anticodon sequence with a nonsense suppressor anticodon sequence. The nonsense suppressor sequence, in some embodiments, is selected from the group consisting of 5'-UUA-3', 5'-UCA-3', and 5'-CUA-3'.

[0270] In some embodiments, an edited tRNA comprising a nonsense suppressor anticodon is configured to bind to a PTC sequence. In some embodiments, the PTC is an ochre stop codon with sequence 5'-UAA-3'. In some embodiments, the PTC is an opal stop codon with sequence 5'-UGA-3'. In some embodiments, the PTC is an amber stop codon with sequence 5'-UAG-3'.

[0271] In some embodiments, the anticodon sequence is a single transition mutation away from a nonsense suppressor anticodon. As defined elsewhere herein, a nonsense suppressor anticodon is the complementary sequence to a premature termination codon or PTC. There are currently 3 known PTCs, each of which, comprises a different sequence. The ochre stop codon has sequence 5 -UAA-3 ' and corresponds to nonsense suppressor anticodon with sequence 5 -UUA-3 '. The opal stop codon has sequence 5 -UGA-3 ' and corresponds to the nonsense suppressor anticodon with sequence 5 -UCA-3 '. The amber stop codon has sequence 5 -UAG-3 ' and corresponds to nonsense suppressor anticodon with sequence 5 CUA-3'.

[0272] The single transition mutation may be any transition mutation known in the art. For example, in some embodiments, the single transition mutation consists of a OT (e.g., C-to-T) mutation, a T> C mutation (e.g., T-to-C) mutation, an A> G (e.g., A-to-G) mutation, or a G> A (G-to-A) mutation.

[0273] In some embodiments, the anticodon sequence is a single transversion mutation away from a nonsense suppressor anticodon. The single trans version mutation may be any transversion mutation known in the art. For example, in some embodiments, the single transversion mutation is selected from the group consisting of an A> C (e.g., A-to-C) mutation, T> G (T-to-G) mutation, G> T (G-to-T) mutation, OA (C-to-A) mutation, OG (C-to-G) mutation, G> C (G-to-C) mutation, A> T (A-to-T) mutation, and T> A (T-to-A) mutation.

[0274] In some embodiments, the endogenous tRNA comprises an anticodon sequence that is 3'-Xl-X2-X3-5'. In some embodiments, the prime editor installs the mutation (e.g.,78 / 346Bl 195.70209 WOOO#14646633v2transition or transversion) at position XI. In some embodiments, the mutation is selected from the group consisting of G> A, OA, and U> A, relative to the endogenous tRNA. In some embodiments, the anticodon sequence comprises a N> A mutation at XI, C at X2, and U at X3, wherein N is G, C, or U (e.g., which is configured to bind to the PTC 5'-UGA-3'). In some embodiments, the anticodon sequence comprises a N> A mutation at XI, U at X2, and C at X3, wherein N is G, C, or U (e.g., which is configured to bind to the PTC 5'-UAG-3'). In some embodiments, the anticodon sequence comprises a N> A mutation at XI, U at X2, and U at X3, wherein N is G, C, or U (e.g., which is configured to bind to the PTC 5'-UAA-3').

[0275] In some embodiments, the prime editor installs the mutation (e.g., transition or transversion) at position X2. In some embodiments, the mutation is selected from the group consisting of A> C, G> C, and U> C, relative to the endogenous tRNA. In some embodiments, the anticodon sequence comprises an A at XI, an N> C mutation at X2, and a U at X3, wherein N is A, G, or U (e.g., which is configured to bind to PTC 5'-UGA-3').

[0276] In some embodiments, the mutation is selected from the group consisting of A> U, G> U, or OU at position X2, relative to the endogenous tRNA. In some embodiments, the anticodon sequence comprises an A at XI, an N> U mutation at X2, and a C at X3, wherein N is A, G, or C (e.g., which is configured to bind to PTC 5'-UAG-3'). In some embodiments, the anticodon sequence comprises an A at XI, a N> U mutation at X2, and C at X3, wherein N is A, G, or C (e.g., which is configured to bind to PTC 5'-UAG-3'). In some embodiments, the anticodon sequence comprises an A at XI, a N> U mutation at X2, and a U at X3, wherein N is A, G, or C (e.g., which is configured to bind to PTC 5'-UAA-3').

[0277] In some embodiments, the prime editor installs the mutation (e.g., transition or transversion) at position X3. In some embodiments, the mutation is selected from the group consisting of A> U, G> U, and OU, relative to the endogenous tRNA. In some embodiments, the anticodon sequence comprises an A at XI, a C at X2, and a N> U at X3, wherein N is an A, G, or C (e.g., which is configured to bind to PTC 5'-UGA-3'). In some embodiments, the anticodon sequence comprises an A at XI, a U at X2 and a N> U at X3, wherein N is an A, G, or C (e.g., which is configured to bind to PTC 5'-UAA-3').

[0278] In some embodiments, the mutation is selected from the group consisting of U> C, A> C, and G> C at position X3, relative to the endogenous tRNA. In some embodiments, the anticodon sequence comprises an A at XI, a U at X2 and a N> C at X3, wherein N is U, A, or G (e.g., which is configured to bind to PTC 5'-UAG-3').79 / 346Bl 195.70209 WOOO#14646633v2

[0279] In some embodiments, the methods comprise a 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 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.

[0280] In some embodiments, an extension arm of a 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., nonsense suppressor anticodon sequence), which then integrates into the endogenous DNA sequence by replacing the corresponding endogenous strand, thereby installing the desired genetic change.

[0281] Accordingly, in some embodiments, the DNA synthesis template encodes an opal PTC (e.g., 5'-UGA-3'), an ochre PTC (e.g., 5'-UAA-3'), or an amber PTC (e.g., 5'-UAG-3') (e.g., when the extension arm is at a 3' end of the pegRNA). In some embodiments, the DNA synthesis template encodes a sequence that is complementary to an opal PTC (e.g., 5'-UCA-3'), an ochre PTC (e.g., 5'-UUA-3'), or an amber PTC (e.g., 5'-CUA-3') (e.g., when the extension arm is at a 5' end of the pegRNA). Likewise, in some embodiments, the DNA synthesis template encodes the C70U mutation to be installed at the target site of the DNA sequence encoding the acceptor stem domain of the endogenous tRNA. In some embodiments, the DNA synthesis template encodes the single base nucleotide to be installed at the target site of the DNA sequence encoding the variable arm domain of the endogenous tRNA.

[0282] Additionally, in some embodiments, the DNA synthesis template further encodes one or more PAM-disrupting mutation 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 incorporated herein by reference in their entirety.

[0283] In other embodiments, the methods disclosed herein may further require a gRNA and / or a second pegRNA.80 / 346Bl 195.70209 WOOO#14646633v2

[0284] In some embodiments, the pegRNA comprises any one of the spacer sequences and an extension arms listed in Table 2 of PCT / US2024 / 011892. In some embodiments, the endogenous tRNA is any tRNA listed in Table 1 of PCT / US2024 / 011892.

[0285] Aspects of the disclosure relate to compositions comprising a prime editor and a pegRNA that are capable of editing an endogenous tRNA into a suppressor tRNA. Any prime editor known in the art may be used to edit the endogenous tRNA into a suppressor tRNA. 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. In some embodiments, the pegRNA is tailored to maximize editing efficiency. In some embodiments, the DNA synthesis template encodes a nonsense suppressor anticodon to be installed at a target site of the DNA sequence encoding the anticodon sequence of the endogenous tRNA. In some embodiments, the nonsense suppressor anticodon is selected from the group consisting of 5'-UUA-3', 5'-UCA-3', and 5'-CUA-3'.

[0286] The DNA synthesis template may encode other edits in other embodiments. For example, in some embodiments, the DNA synthesis template encodes a C70U mutation to be installed at the target site of the DNA sequence encoding an acceptor stem domain of the endogenous tRNA. Alternatively, or additionally, the DNA synthesis template may encode for a single base nucleotide insertion into the DNA sequence encoding a variable arm domain of the endogenous tRNA molecule. The DNA synthesis template further encodes a PAM-disrupting mutation and / or an MMR-evading mutation, relative to the endogenous tRNA, in some embodiments.

[0287] 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.

[0288] Some aspects of the present disclosure relate to methods using twinPE prime editors to edit one or more domains of the endogenous tRNA. 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 81 / 346Bl 195.70209 WOOO#14646633v2comprising a spacer sequence, gRNA core, an extension arm comprising a DNA synthesis template and a primer binding site.

[0289] 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 a nonsense suppressor anticodon sequence 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 encode a premature termination sequence 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 encode a C70U mutation to be installed at the target site of the DNA sequence encoding the acceptor stem domain of the endogenous tRNA. In some embodiments, the first single strand DNA sequence and the second single stranded DNA sequence encode a single nucleotide insertion to be installed at the target site of the DNA sequence encoding the variable 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.

[0290] 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, 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 nonsense suppressor anticodon at the target site of the DNA sequence encoding the anticodon sequence of the endogenous tRNA. In some embodiments, the nonsense suppressor anticodon has the sequence 5 '-UUA-3 ' and is configured to bind to an ochre stop codon having sequence 5'-UAA-3'. In some embodiments, the nonsense suppressor anticodon has the sequence 5'-UCA-3' configured to bind to an opal stop codon having sequence 5'-UGA-3'. In some embodiments, the nonsense suppressor anticodon has the sequence 5'-CUA-3' configured to bind to an amber stop codon having sequence 5'-UAG-3'.

[0291] In some embodiments, integrating the duplex into the target site installs a C70U mutation in the DNA sequence encoding an acceptor stem domain of the endogenous tRNA.82 / 346Bl 195.70209 WOOO#14646633v2In some embodiments, installing the C70U mutation creates a G3: U70 base pair in the acceptor stem domain of the tRNA. In some embodiments, having the G3: U70 base pair in the acceptor stem domain of the tRNA causes the tRNA to be charged with the non-cognate amino acid alanine via the alanine-aminoacyl-tRNA synthetase. Other edits within the acceptor stem domain, leading to the incorporation of other non-cognate amino acids, is also possible in other embodiments.

[0292] In some embodiments, integrating the duplex into the target site installs a single base nucleotide insertion in the DNA sequence encoding a variable arm domain of the endogenous tRNA. In some cases, installing the additional nucleotide base, relative to the unedited endogenous tRNA, causes the tRNA to be charged with a noncognate amino acid serine via the serine-aminoacyl-tRNA synthetase.

[0293] 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.

[0294] In some embodiments, the pegRNAs comprise any protospacer sequence and pegRNA extension sequence listed in Table 2 of PCT / US2024 / 011892. In some embodiments, the endogenous tRNA is any tRNA listed in Table 1 of PCT / US2024 / 011892.

[0295] Other aspects of the disclosure relate to compositions comprising twinPEs for editing endogenous tRNAs into suppressor tRNAs. 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 singlestranded 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 singlestranded 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.

[0296] In some embodiments, the pegRNAs comprise any spacer sequence and pegRNA extension sequence listed in Table 2 of PCT / US2024 / 011892. In some embodiments, the endogenous tRNA is any tRNA listed in Table 1 of PCT / US2024 / 011892.83 / 346Bl 195.70209 WOOO#14646633v2

[0297] Aspects of the disclosure relate to pegRNAs for editing a DNA sequence encoding an endogenous tRNA by prime editing into a suppressor tRNA. 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.

[0298] In some embodiments, the DNA synthesis template encodes an opal PTC (e.g., 5'-UGA-3'), an ochre PTC (e.g., 5'-UAA-3'), or an amber PTC (e.g., 5'-UAG-3') (e.g., when the extension arm is at a 3' end of the pegRNA). In some embodiments, the DNA synthesis template encodes a sequence that is complementary to an opal PTC (e.g., 5'-UCA-3'), an ochre PTC (e.g., 5'-UUA-3'), or an amber PTC (e.g., 5 -CUA-3') (e.g., when the extension arm is at a 5' endo of the pegRNA). Likewise, in some embodiments, the DNA synthesis template encodes the C70U mutation to be installed at the target site of the DNA sequence encoding the acceptor stem domain of the endogenous tRNA. In some embodiments, the DNA synthesis template encodes the single base nucleotide to be installed at the target site of the DNA sequence encoding the variable arm domain of the endogenous tRNA.

[0299] Additionally, in some embodiments, the DNA synthesis template further encodes one or more PAM-disrupting mutation and / or MMR-evading mutations. In some embodiments, the pegRNA comprises any one of the spacer sequences and an extension arm listed in Table 2 of PCT / US2024 / 011892. In some embodiments, the endogenous tRNA is any tRNA listed in Table 1 of PCT / US2024 / 011892.

[0300] Other aspects of the disclosure relate to a complex comprising a prime editor (e.g., PEI, PE2, PE3, and / or twinPE) and a pegRNA for editing a DNA sequence encoding an endogenous tRNA by prime editing into a suppressor tRNA. In some embodiments, the pegRNA comprises a spacer sequence and an extension arm. In some embodiments, the pegRNAs comprise any protospacer sequence and pegRNA extension sequence listed in Table 2 of PCT / US2024 / 011892. In some embodiments, the endogenous tRNA is any tRNA listed in Table 1 of PCT / US2024 / 011892.

[0301] Aspects of the disclosure relate to polynucleotides comprising a first nucleic acid sequence encoding a prime editor and a second nucleic acid sequence encoding a pegRNA. In some embodiments, the pegRNAs comprise any protospacer sequence and pegRNA extension sequence listed in Table 2 of PCT / US2024 / 011892. In some embodiments, 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 protospacer sequence 84 / 346Bl 195.70209 WOOO#14646633v2listed in Table 2 of PCT / US2024 / 011892. In other embodiments, 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 extension arm listed in Table 2 of PCT / US2024 / 011892.

[0302] In some embodiments, the pegRNAs comprise any spacer sequence, pegRNA scaffold sequence, DNA synthesis template sequence (RTT), and / or PBS sequence listed in Tables 8A-8B (see Example 7). In some embodiments, the pegRNA comprises a spacer sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 100% sequence identity to any spacer sequence listed in Table 8A. In some embodiments, the pegRNA comprises an pegRNA scaffold sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 100% sequence identity to any pegRNA scaffold sequence listed in Table 8A. In some embodiments, the pegRNA comprises a DNA synthesis template (e.g., RTT) sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 100% sequence identity to any DNA synthesis template sequence (e.g., RTT) listed in Table 8A. In some embodiments, the pegRNA comprises an PBS sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 100% sequence identity to any PBS sequence listed in Table 8 A. In some embodiments, the pegRNA is configured to bind to a DNA sequence encoding an endogenous tRNA.Exemplary endogenous tRNAs may be found in Table 1 of PCT / US2024 / 011892 and in Table 7 (see Example 7).

[0303] Additional aspects of the disclosure relate to methods for changing the amino acid that is charged onto a tRNA in a subject in need thereof. In some embodiments, the methods comprise administering to the subject: (i) a prime editor and (ii) a pegRNA, wherein the prime editor and gRNA form a prime editing complex. In some embodiments, the prime editing complex binds to a DNA sequence encoding an acceptor stem domain of the tRNA. In some embodiments, the prime editing complex installs a mutation in the acceptor stem domain. In some embodiments, mutation results in the replacement of a cognate amino acid with a non-cognate amino acid.

[0304] In some embodiments, the cognate amino acid is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, pyrrolysine, and selenocysteine.85 / 346Bl 195.70209 WOOO#14646633v2

[0305] In some embodiments, the non-cognate amino acid is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, pyrrolysine, and selenocysteine.

[0306] In some embodiments, the tRNA comprises an anticodon sequence that encodes for the cognate amino acid but is charged with the non-cognate amino acid. For example, in some embodiments, the cognate amino acid is lysine, and the non-cognate amino acid is alanine. In other embodiments, the cognate amino acid is lysine, and the non-cognate amino acid is serine.

[0307] In some embodiments, the act of editing an anticodon sequence of an endogenous tRNA to create a suppressor tRNA configured to bind to a premature termination codon may alter the aminoacylation of the endogenous tRNA, as described by Wang et al., “AAV-delivered suppressor tRNA overcomes a nonsense mutations in mice” Nature, 2022; 604 (7905):348. For example, in some embodiments, an endogenous tRNA-Trp edited into a suppressor tRNA-Trp with an anticodon designed to bind to an amber stop codon (5'-UAG-3') is charged with a lysine. In some embodiments, endogenous tRNA-Gln edited into a suppressor tRNA-Gln with an anticodon designed to bind to an amber stop codon (5'-UAG-3') is charged with a lysine.

[0308] In some embodiments, the pegRNA comprises any one of the spacer sequences and an extension arm listed in Table 2 of PCT / US2024 / 011892. In some embodiments, the endogenous tRNA is any tRNA listed in Table 1 of PCT / US2024 / 011892.Installing suppressor tRNA via replacing endogenous tRNAs

[0309] Aspects of the disclosure relate to methods using prime editing to replace endogenous tRNAs with suppressor tRNAs. As such, certain embodiments relate to overwriting an existing RNA with a suppressor tRNA. The RNA gene to be overwritten is, according to some embodiments, highly expressed, relative to the desired endogenous tRNA to be edited. The level of expression is easily determined using known techniques in the art, such as, for example, high throughput gene expression profiling.

[0310] In some embodiments, the highly expressed RNA gene to be edited may be any suitable RNA gene known to the skilled artisan. In some embodiments, the highly expressed RNA gene is an endogenous tRNA gene.

[0311] 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 86 / 346Bl 195.70209 WOOO#14646633v2to 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 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 tRNA 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.Installing a suppressor tRNA via gene insertion

[0312] Aspects of the disclosure relate to methods for inserting a new suppressor tRNA 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 tRNA 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 tRNA or a suppressor tRNA expression cassette into the human genome is likewise also envisioned.

[0313] Accordingly, in some embodiments, the methods comprise inserting a suppressor tRNA gene into a target site in a genome (e.g., human genome) using prime editing (e.g., twinPE). In some embodiments, the methods comprise 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 napDNAbp and a polymerase.87 / 346Bl 195.70209 WOOO#14646633v2

[0314] 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 tRNA 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.

[0315] In some embodiments, the prime editor and the pegRNA install the suppressor tRNA gene sequence in the target site in the genome (e.g., human genome). In some embodiments, installation of the suppressor tRNA gene results in the indefinite expression of the suppressor tRNAs gene. Any suitable pegRNA, or pairs of pegRNAs, may be used to replace an endogenous tRNA with a suppressor tRNA, such the exemplary pegRNAs provided in Table 5 of PCT / US2024 / 011892, the contents of which are incorporated herein by reference.

[0316] In some embodiments, the suppressor tRNA gene encodes for a suppressor tRNA charged with an amino acid. In some embodiments, the amino acid is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, pyrrolysine, and selenocysteine.

[0317] In some embodiments, the suppressor tRNA gene further encodes for a suppressor tRNA with a nonsense suppressor anticodon that is complementary to a premature termination codon. For example, in some embodiments, the nonsense suppressor anticodon is 5 -UCA-3' and binds to an opal premature termination codon having sequence 5'-UGA-3'. In some embodiments, the nonsense suppressor anticodon is 5 -UUA-3' and binds to an ochre premature termination codon having sequence 5'-UAA-3'. In other embodiments, the nonsense suppressor anticodon is 5 -CUA-3 ' and binds to an amber premature termination codon having sequence 5'-UAG-3'.

[0318] In some embodiments, the suppressor tRNA encodes for one or more mutations, relative to an endogenous tRNA. In some embodiments, the suppressor tRNA encodes for a tRNA and its cognate amino acid, but includes one or more mutations in one or more domains that results in the suppressor tRNA being charged with a non-cognate amino acid.88 / 346Bl 195.70209 WOOO#14646633v2Any mutation known in the art that results in amino acid misincorporation is envisioned herein. For example, in some embodiments, the suppressor tRNA gene encodes a Lys-tRNA-CUU comprising a C70U mutation in the acceptor stem domain. In this embodiment, expression of the suppressor tRNA 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 tRNA gene encodes a single base nucleotide insertion in a variable arm domain, relative to an endogenous tRNA.

[0319] In some embodiments, the suppressor tRNA 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.

[0320] 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.

[0321] 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).

[0322] Other aspects of the disclosure relate to inserting a suppressor tRNA 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., Bxbl) 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).89 / 346Bl 195.70209 WOOO#14646633v2

[0323] In some embodiments, the method comprises a circular DNA plasmid that encodes the suppressor tRNA gene to be inserted into the target site. In some embodiments, the suppressor tRNA gene comprises an anticodon sequence that is complementary to a premature termination sequence (e.g., complementary the following PTCs: 5'-UUA-3', 5'-UCA-3', 5'-CUA-3'). In some embodiments, the circular DNA plasmid encodes for the suppressor tRNA molecule comprising a C70U mutation in the acceptor stem domain of the tRNA. In some embodiments, the circular DNA plasmid encodes for the suppressor tRNA molecule comprising a single nucleotide insertion (e.g., mutation) in a variable arm domain of the suppressor tRNA, relative to an endogenous tRNA.

[0324] In some embodiments, the methods and compositions comprise a DNA plasmid (e.g., a circular plasmid) that encodes a suppressor tRNA comprising an anticodon sequence that is complementary to a PTC (e.g., 5'-UUA-3', 5 -UCA-3', 5 -CUA-3'). The DNA plasmid (e.g., circular plasmid) may further comprise a first recombinase recognition site (e.g., AttP). The recombination site may be any suitable recombination site known in the art. For example, in some cases, the first recombinase recognition site comprises an AttB sequence with a sequence identity of at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% to SEQ ID NOs: 208-218. In other embodiments, the first recombinase recognition site comprises an AttP sequence with a sequence identity of at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% to SEQ ID NOs: 219-229. Other first recombinase recognition sites are also possible, according to other embodiments.

[0325] Exemplary attB and attP sites for phage integrase C31 (cpC31 attB) are shown below and are described in Groth et al., A phage integrase directs efficient site- specific integration in human cells” Proceedings of the National Academy of Science USA. May 23, 2000, vol. 97, no. 11, pgs. 5995-6000, and Anzalone et al., “Programmable deletion, replacement, integration, and inversion of large DNA sequences with twin prime editing” Nature Biotechnology. May 2022, 40(5): 731-740, both of which are incorporated herein by reference in their entirety. However, the skilled artisan will appreciate that the invention is not limited to phage integrases, and that any integrase with known attB and attP sites known by the skilled artisan may be used in the current disclosure.

[0326] attB sites:CTCGA AGCCG CGGTG CGGGT GCCAG GGCGT GCCCTTGGGC TCCCC GGGCG CGTAC TCCAC CTCAC CCATC (SEQ ID NO: 208);90 / 346Bl 195.70209 WOOO#14646633v2CCG CGGTG CGGGT GCCAG GGCGT GCCCTTGGGC TCCCC GGGCG CGTAC TCCACC (SEQ ID NO: 209);CGGGT GCCAG GGCGT GCCCTTGGGC TCCCC GGGCG CGTAC (SEQ ID NO: 210); GGT GCCAG GGCGT GCCCTTGGGC TCCCC GGGCG CG (SEQ ID NO: 211);GT GCCAG GGCGT GCCCTTGGGC TCCCC GGGCG CG (SEQ ID NO: 212);GT GCCAG GGCGT GCCCTTGGGC TCCCC GGGCG C (SEQ ID NO: 213);T GCCAG GGCGT GCCCTTGGGC TCCCC GGGCG CG (SEQ ID NO: 214);T GCCAG GGCGT GCCCTTGGGC TCCCC GGGCG C (SEQ ID NO: 215);GGCGT GCCCTTGGGC TCCCC (SEQ ID NO: 216);GGCTTGTCGACGACGGCGGACTCCGTCGTCAGGATCAT (SEQ ID NO: 217); and GGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 218).

[0327] attP sites:CGGGA GTAGT GCCCC AACTG GGGTA ACCTTTGAGT TCTCT CAGTT GGGGG CGTAG GGTCG (SEQ ID NO: 219);GTAGT GCCCC AACTG GGGTA ACCTTTGAGT TCTCT CAGTT GGGGG CGTAG(SEQ ID NO: 220);GCCCC AACTG GGGTA ACCTTTGAGT TCTCT CAGTT GGGGG (SEQ ID NO: 221); GCCCC AACTG GGGTA ACCTTTGAGT TCTCT CAGTT GGGG (SEQ ID NO: 222); CCCC AACTG GGGTA ACCTTTGAGT TCTCT CAGTT GGGGG (SEQ ID NO: 223); CCCC AACTG GGGTA ACCTTTGAGT TCTCT CAGTT GGGG (SEQ ID NO: 224); CCC AACTG GGGTA ACCTTTGAGT TCTCT CAGTT GGG (SEQ ID NO: 225);CC AACTG GGGTA ACCTTTGAGT TCTCT CAGTT GG (SEQ ID NO: 226);GGTTTGTCTGGTCAACCACCGCGGACTCAGTGGTGTACGGTACAAACC (SEQ ID NO: 227);GGTTTGTCTGGTCAACCACCGCGGTCTCAGTGGTGTACGGTACAAACC (SEQ ID NO: 228); and AGGTTTGTCTGGTCAACCACCGCGGTCTCAGTGGTGTACGGTACAAACCT (SEQ ID NO: 229).

[0328] In some embodiments, the circular DNA plasmid comprises a first recombinase recognition site (e.g., AttP). In some embodiments, the first recombinase recognition site comprises an AttB sequence with a sequence identity of at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% of SEQ ID NOs: 208-218. In some embodiments, the first recombinase recognition site comprises an AttP sequence with a91 / 346Bl 195.70209 WOOO#14646633v2sequence identity of at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% of SEQ ID NOs: 219-229.

[0329] 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). 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 with a sequence identity of at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% of SEQ ID NOs: 208-218. In some embodiments, the second recombinase recognition site comprises an AttP sequence with a sequence identity of at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% of SEQ ID NOs: 219-229.

[0330] 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 circular plasmid 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 tRNA gene into the human genome at the target site (e.g., ROSA26, CCR5, and AAVS1). In some embodiments, installation of the suppressor tRNA gene at the target site (e.g., safe harbor locus site or general expression site) results in the indefinite expression of the suppressor tRNAs gene.

[0331] Other aspects relate to methods for treating a disease caused by premature termination codons, the method comprising mutating an endogenous tRNA gene into a suppressor tRNA gene using prime editing, the method comprising administering to a subject (i) a prime editor and (ii) a pegRNA, wherein the suppressor tRNA gene encodes a suppressor tRNA molecule comprising an anticodon sequence comprising ochre stop codon, an opal stop codon, or an amber stop codon.

[0332] Additional aspects of the disclosure relate to methods for treating a disease caused by premature termination codons, the method comprising installing a suppressor tRNA 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) a pegRNA, wherein the suppressor tRNA gene encodes a suppressor tRNA molecule comprising an anticodon sequence comprising ochre stop codon, an opal stop codon, or an amber stop codon,92 / 346Bl 195.70209 WOOO#14646633v2

[0333] Non-limiting examples of diseases caused by premature termination codons (e.g., nonsense mutations) include cystic fibrosis, beta thalassemia, Hurler syndrome, Dravet syndrome, Duchenne muscular dystrophy, Usher syndrome, and hemophilia.

[0334] Some aspects of the disclosure relate to a complex capable of editing an endogenous tRNA into a suppressor tRNA. In some embodiments, the complex comprises a prime editor and any one of the pegRNAs disclosed herein. In some embodiments, the complex comprises a prime editor and multiple pegRNA’s. In some embodiments, the complex comprises a pair of pegRNA’s (e.g., two pegRNA’s). In some embodiments, the complex comprises two pairs of pegRNA’s (e.g., four pegRNA’s). Non-limiting embodiments of pairs of pegRNAs designed, for example, to replace an endogenous tRNA sequence with a suppressor tRNA sequence via prime editing, may be found in Table 5 of PCT / US2024 / 011892. In some embodiments, the prime editor is selected from the group consisting of PE6a, PE6b, PE6c, PE6d, PE6d+MLHldn, PE6e, PE6f, PE6g, PEmax, PEmax+MLHldn, and PEmaxA. In some embodiments, the prime editor comprises PE6c. Methods for selecting suppressor tRNA genes

[0335] In some aspects, the current disclosure relates to one or more methods of selecting a suppressor tRNA gene. In some embodiments, the method comprises creating a reporter cell line comprising a reporter construct comprising a constitutively expressed fusion protein comprising a first biomarker protein, a premature termination codon (PTC) sequence, a ribosomal skipping element, and a second biomarker protein different than the first biomarker protein. In some embodiments, the methods comprise creating a gene library encoding the sequences of all human tRNA sequences. In some cases, the tRNA sequences comprise the same three-base pair anticodon that is complimentary to the PTC in the reporter construct. In some embodiments, the methods comprise introducing the library into the reporter cell line. In some embodiments, the methods comprise sorting cells that express the second biomarker protein and determining which tRNA sequences are enriched in the sorted population.

[0336] In some embodiments, the PTC is TGA. In other embodiments, the PTC is TAG. In other cases, the PTC is TAA, according to some embodiments. In some cases, the biomarker (e.g., first or second biomarker) is a fluorescent protein. In some instances, the first biomarker is a mCherry fluorescent protein. In some embodiments, the second biomarker is a green fluorescent protein (GFP).

[0337] In some embodiments, each cell contains a single copy of the reporter construct. Techniques to ensure that each cell only contains a single copy of the reporter construct are93 / 346Bl 195.70209 WOOO#14646633v2known in the art, and the skill artisan may use any of said techniques in any of the methods disclosed herein. In some embodiments, the PTC in the reporter construct can be replaced with any amino acid variant without altering expression levels of the second biomarker protein.

[0338] In some embodiments, the gene library is cloned into a lentiviral backbone, although other backbones may be used. Accordingly, in some embodiments, any suitable lentiviral backbone known in the art may be used as the lentiviral backbone in any of the methods disclosed herein.

[0339] In some embodiments, the lentiviral backbone comprises an exogenous promoter. Any suitable exogenous promoter known in the art by the skilled artisan may be used in any of the methods disclosed herein. In some embodiments, the promoter comprises a human U6. In other embodiments, the promoter comprises a minimal U6 promoters. Alternatively, in some embodiments, the lentiviral backbone does not comprise an exogenous promoter. For example, in some cases, the encoded tRNA sequence comprises an endogenous tRNA protomer capable of driving expression of the tRNA gene sequence.

[0340] In some embodiments, the genes within the gene library further comprise a leader sequence. Any suitable leader sequence known to the skilled artisan may be used in any of the methods disclosed herein, such as those disclosed in Table 3 of PCT / US2024 / 011892. In some embodiments, the leader sequence is positioned to precede the mature tRNA sequence. However, other arrangements are also possible in other embodiments.

[0341] In some embodiments, the genes within the gene library further comprise a termination sequence. Any suitable termination sequence known to the skilled artisan may be used in any of the methods as described herein, such as those disclosed in Table 4 of PCT / US2024 / 011892. For instance, in some embodiments, the termination sequence comprises a poly T tail. In some embodiments, the termination sequence comprises four, five, or seven thymidine tracks. In some embodiments, the termination sequence is within 100 bp of the mature tRNA sequence.

[0342] In some embodiments, the selected suppressor tRNA gene may encode for any known tRNA gene of any species known to the skilled artisan. For example, in some embodiments, the encoded tRNA gene is a human tRNA gene. In some embodiments, the selected suppressor tRNA gene encodes for Leu-TAA-1-1, Leu-TAA-2-1, Leu-TAA-3-1, or Leu-TAA-4-1.

[0343] In some embodiments, the methods relate to a method of selecting a pegRNA to edit an endogenous tRNA gene. In some embodiments, the methods comprise creating a 94 / 346Bl 195.70209 WOOO#14646633v2reporter cell line comprising a reporter construct comprising a constitutively expressed fusion protein comprising a first biomarker protein, a premature termination codon (PTC) sequence, a ribosomal skipping element, and a second biomarker protein different than the first fluorescent protein. In some embodiments, the methods further comprise creating a gene library encoding pegRNAs that target every tRNA sequence in the genome and which convert the natural tRNA anticodon of said tRNA sequence to a PTC. In some embodiments, the methods further comprise introducing the gene library and a prime editor into the cell line and sorting cells that express the second biomarker protein and determining which pegRNA sequences are enriched in the sorted population.

[0344] In some embodiments, the methods relate to a method for replacing an endogenous tRNA gene at its endogenous locus in a genome for treating Hurler Syndrome, the method comprising contacting a DNA sequence encoding an endogenous tRNA gene (e.g., see Table 7) with a prime editor and any one of the pegRNAs disclosed herein. In some embodiments, the prime editor replaces the endogenous tRNA gene with the suppressor tRNA gene. In some embodiments, the pegRNA comprises a DNA synthesis template encoding one or more mutations to be inserted into an anticodon loop of the endogenous tRNA.

[0345] Additional methods contemplated herein relate to treating Hurler Syndrome caused by a premature termination codon. In some embodiments, the methods comprise installing a suppressor tRNA 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) any one of the pegRNAs disclosed herein. In some embodiments, the installed suppressor tRNA gene encodes a suppressor tRNA comprising an one or more mutations in an anticodon loop, relative to an endogenous tRNA, and an anticodon sequence configured to bind to an ochre stop codon, an opal stop codon, or an amber stop codon. In some embodiments, the one or more mutations in the anticodon loop of the endogenous tRNA comprises a TA> CG mutation at hairpin position (hp) 12, a GOCG mutation at hp 13, a GOTA mutation at hp 13, a GOAT mutation at hp 13, a GOTA mutation at hp 14, and / or A> T mutation at amino acid position 38. In some embodiments, the one or more mutations in the anticodon loop of the endogenous tRNA is a hpl2 TA> CG, hpl3 GOCG, hpl3 GOTA, hpl3 GOAT, hpl4 GOTA, and / or mut38 A> T.

[0346] Additional methods relate to methods for replacing an endogenous tRNA gene with a suppressor tRNA gene in a genome for reading through a TAG or TGA premature termination codon. In some embodiments, the methods comprise contracting a DNA 95 / 346Bl 195.70209 WOOO#14646633v2sequence encoding the endogenous tRNA gene with a prime editor and any one of the pegRNAs disclosed herein. In some embodiments, the prime editor replaces at least a part of the endogenous tRNA gene with the suppressor tRNA gene.

[0347] In some embodiments, the methods disclosed herein are used to convert an endogenous tRNA into a sup-tRNA with minimal or no disruption to the endogenous tRNA transcriptome (e.g., wherein “minimal or no disruption,” as measured by a known assay, means in some embodiments between 0% change to the tRNA transcriptome or up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or up to 10% change to the tRNA transcriptome). For example, in some embodiments, the methods disclosed herein do not cause a stress response in cells where an endogenous tRNA has been converted into a sup-tRNA using said methods.

[0348] In some embodiments, the methods disclosed herein are used to convert an endogenous tRNA into a sup-tRNA with minimal or no disruption to tRNA homeostasis. For example, in some embodiments, the methods disclosed herein do not significantly alter gene expression in endogenous tRNAs, relative to tRNAs in wild type cells. As used herein, “do not significantly alter gene expression” means the conversion of an endogenous tRNA to a sup-tRNA does not statistically or biologically meaningfully change, as measured by recognized global assays, relative to a suitable control, within defined quantitative thresholds. In some embodiments, ‘do not significantly alter gene expression’ refers to a condition in which the expression level of endogenous genes, including endogenous tRNAs, in cells comprising a sup-tRNA differs from that of corresponding wild-type cells by no more than ±l-fold, ±1.2-fold, ±1.5-fold, or ±2-fold, ±4-fold, or ±5-fold as measured by RNA sequencing, tRNA-sequencing, microarray analysis, or a comparable transcriptomic assay. In some embodiments, fewer than 1%, 2%, 5%, or 10% of detected transcripts exhibit statistically significant differential expression (e.g., adjusted p-value < 0.05) relative to wildtype cells as a result of the conversion of an endogenous tRNA to a sup-tRNA.

[0349] In some embodiments, the sup-tRNAs installed using the prime editing methods disclosed herein do not significantly perturb the proteome and / or induce detectable NTC readthrough. In some embodiments, ‘do not significantly perturb the proteome’ refers to a condition in which the abundance of cellular proteins in cells comprising a sup-tRNA differs from that of wild-type cells by no more than ±l-fold, ±1.2-fold, ±1.5-fold, or ±2-fold, id-fold, or ±5-fold for the majority of detected proteins, as measured by quantitative mass spectrometry or a comparable proteomic assay. In some embodiments, the installed sup-tRNAs do not cause detectable or broad non-target codon (NTC) readthrough, wherein NTC readthrough is assessed using ribosome profiling, reporter assays, or proteome-wide peptide 96 / 346Bl 195.70209 WOOO#14646633v2mapping, and wherein readthrough at non-target stop codons occurs at a frequency of less than 0.1%, 0.5%, 1%, or 2% relative to baselineUses of pegRN As

[0350] Aspects of the disclosure relate to one or more uses of a pegRNA. In some embodiments, the use relates to using the pegRNA to edit a target DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor tRNA. In some embodiments, the pegRNA comprises a DNA synthesis template, the DNA synthesis template encoding a nonsense suppressor anticodon sequence to be inserted into the target DNA sequence encoding an anticodon sequence of the endogenous tRNA. In some embodiments, the DNA synthesis template further encodes one or more mutations to be inserted into an anticodon loop of the endogenous tRNA.

[0351] In some embodiments, the use relates to using a pegRNA to edit an endogenous tRNA-Leu-TAA gene, by prime editing, to produce a suppressor tRNA-Leu gene. In some embodiments, the endogenous tRNA-Leu-TAA gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to any one of SEQ ID NOs: 17-20 (tRNA-Leu-TAA). In some embodiments, the pegRNA comprises a DNA synthesis template, the DNA synthesis template encoding a 5'-CUA-3' or 5'-UCA-3' nonsense suppressor anticodon sequence to be inserted into the endogenous tRNA-Leu gene to produce the suppressor tRNA-Leu gene.

[0352] In some embodiments, the use relates to using a pegRNA to replace an endogenous tRNA gene with a suppressor tRNA gene in a genome for treating Hurler Syndrome. In some embodiments, the use comprises contacting a DNA sequence encoding the endogenous tRNA gene with a prime editor and a pegRNA. In some embodiments, the prime editor replaces the endogenous tRNA gene with the suppressor tRNA gene. In some embodiments, the pegRNA comprises a DNA synthesis template, the DNA synthesis template encoding a nonsense suppressor anticodon sequence to be inserted into the target DNA sequence encoding an anticodon sequence of the endogenous tRNA. In some embodiments, the DNA synthesis template further encodes one or more mutations to be inserted into an anticodon loop of the endogenous tRNA.

[0353] In some embodiments, the use relates to using a pegRNA to treat Hurler Syndrome caused by a premature termination codon. In some embodiments, the use comprises installing a suppressor tRNA gene into a target site in a human genome using prime editing, and further comprising administering to a subject (i) a prime editor and (ii) any one of the pegRNA disclosed herein. In some embodiments, the suppressor tRNA gene 97 / 346Bl 195.70209 WOOO#14646633v2encodes a suppressor tRNA comprising an one or more mutations in anticodon loop, relative to an endogenous tRNA, and an anticodon sequence comprising ochre stop codon, an opal stop codon, or an amber stop codon.Prime editors

[0354] 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 N-[napDNAbp]-[ polymerase] -C; or N-[polymerase]-[napDNAbp]-C, 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 N-[napDNAbp]-[RT]-C; or N-[RT]- [napDNAbp] -C, wherein each instance of “]-[” indicates the presence of an optional linker sequence.

[0355] 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. Nonlimiting examples of prime editors contemplated herein may be found in U. S. Provisional Application No. 62 / 820,813 (filed March 19, 2019), U. S. Provisional Application No.62 / 858,958 (filed June 7, 2019), U. S. Provisional Application No. 62 / 889,996 (filed August 21, 2019), U. S. Provisional Application No. 62 / 922,654 (filed August 21, 2019), U. S.Provisional Application No. 62 / 913,553 (filed October 10, 2019), U. S. Provisional Application No. 62 / 973,558 (filed October 10, 2019), U. S. Provisional Application No.62 / 931,195 (filed November 5, 2019), U. S. Provisional Application No. 62 / 944,231 (filed December 5, 2019), U. S. Provisional Application No. 62 / 974,537 (filed December 5, 2019), U. S. Provisional Application No. 62 / 991,069 (filed March 17, 2020), U. S. Provisional Application No. 63 / 100,548 (March 17, 2020), U. S. Provisional Application No. 63 / 022,397 (filed May 8, 2020), U. S. Provisional Application No. 63 / 116,785 (filed November 20, 2020),98 / 346Bl 195.70209 WOOO#14646633v2International PCT Application No. PCT / US2020 / 023721 (filed March 19, 2020), International PCT Application No. PCT / US2020 / 023553 (filed March 19, 2020), International PCT Application No. PCT / US2020 / 023583 (filed March 19, 2020), International PCT Application No. PCT / US2020 / 023730 (filed March 19, 2020), International PCT Application No. PCT / US2020 / 023713 (filed March 19, 2020), International PCT Application No. PCT / US2020 / 023712 (filed March 19, 2020), International PCT Application No. PCT / US2020 / 023727 (filed March 19, 2020), International PCT Application No. PCT / US2020 / 023724 (filed March 19, 2020), International PCT Application No. PCT / US2020 / 023725 (filed March 19, 2020), International PCT Application No. PCT / US2020 / 023728 (filed March 19, 2020), International PCT Application No. PCT / US2020 / 023732 (filed March 19, 2020), International PCT Application No. PCT / US2020 / 023723 (filed March 19, 2020), International PCT Application No. PCT / US2021 / 031439 (filed May 7, 2021), and U. S. Patent No. 11,440,770, issued on Sept. 20, 2022, U. S. Patent No. 12,435,330, issued on October 7, 2025, U. S. Patent No. 11,912,985, issued on February 27, 2024, U. S. Patent No. 12,031,126, issued on July 9, 2024, U. S. Patent No. 11,643,652, issued on May 9, 2023, U. S. Patent No.12,281,303, issued on April 22, 2025, U. S. Patent No. 11,447,770, issued on September 20, 2022, and U. S. Patent No. 11,795,452, issued on October 24, 2023, each of which is incorporated herein by reference in its entirety.

[0356] 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.

[0357] 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 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 99 / 346Bl 195.70209 WOOO#14646633v2indicates 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 “]-[1” indicates the presence of an optional linker sequence.

[0358] In various embodiments, the prime editor fusion protein may have the following structure (referred to herein as “PEI”), 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 PEI fusion protein has the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(wt)].

[0359] 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.

[0360] In one set of embodiments, a prime editor of the present disclosure may be a PEI, PE2, PE3, PE4, PE5, PEmax, PE6 series, 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).

[0361] In some embodiments, a PE3 prime editor comprises PE2 machinery and an additional sgRNA.

[0362] 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 doublestranded 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 100 / 346Bl 195.70209 WOOO#14646633v2programmable 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.

[0363] In other embodiments, e.g., installing a suppressor tRNA gene into a safe harbor site in the genome, may involve a prime editing system referred to herein and in the literature as PASSIGE (an acronym for prime-editing-assisted site- specific integrase gene editing) and PASSIGE variants evoPASSIGE and eePASSIGE, see Pandey, S., Gao, X. D., Krasnow, N. A. et al, “Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing,” Nat. Biomed. Eng. (June 10, 2024), https: / / doi.org / 10.1038 / s41551-024-01227-1, the entire contents of which are incorporated herein by reference.napDNAbp domain

[0364] 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, which is incorporated herein by reference in its entirety. For example, in various embodiments, the napDNAbp may be any Class 2 CRISPR-Cas system, including any type II, type V, or type VI CRISPR-Cas enzyme. 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.101 / 346Bl 195.70209 WOOO#14646633v2

[0365] 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).

[0366] In various embodiments described herein, the prime editors comprise a napDNAbp, such as a Cas9 protein. These proteins 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 sequence. However, in certain embodiments 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. Patent Applications, 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 102 / 346Bl 195.70209 WOOO#14646633v2Biotechnol. 2013, Vol.31: 397-405, each of which are incorporated herein by reference in their entireties.

[0367] Any suitable napDNAbp may be used in the prime editors described herein. In various embodiments, the napDNAbp may be any Class 2 CRISPR-Cas system, including any type II, type V, or type VI CRISPR-Cas enzyme. 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. The skilled person will be able to identify the specific CRISPR-Cas enzyme being referenced in this Application based on the nomenclature that is used, whether it is old (i.e., “legacy”) or new nomenclature. CRISPR-Cas nomenclature is extensively discussed in Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?,” The CRISPR Journal, Vol. 1. No. 5, 2018, the entire contents of which are incorporated herein by reference. The particular CRISPR-Cas nomenclature used in any given instance in this Application is not limiting in any way and the skilled person will be able to identify which CRISPR-Cas enzyme is being referenced.

[0368] For example, the following type II, type V, and type VI Class 2 CRISPR-Cas enzymes have the following art-recognized old (i.e., legacy) and new names. Each of these enzymes, and / or variants thereof, may be used with the prime editors described herein: Legacy nomenclature Current nomenclature*type II CRISPR-Cas enzymesCas9 sametype V CRISPR-Cas enzymesCpfl Casl2aCasX Casl2eC2cl Casl2blCasl2b2 sameC2c3 Casl2cCasY Casl2dC2c4 sameC2c8 sameC2c5 sameC2cl0 same103 / 346Bl 195.70209 WO00#14646633v2C2c9 sametype VI CRISPR-Cas enzymesC2c2 Casl3aCasl3d sameC2c7 Casl3cC2c6 Casl3b* See Makarova et al., The CR1SPR Journal, Vol. 1, No. 5, 2018.

[0369] The below description of various napDNAbps which can be used in connection with the presently disclose prime editors is not meant to be limiting in any way. The prime editors may comprise 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 which can 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 of 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).

[0370] The prime editors described herein may also comprise Cas9 equivalents, including Casl2a (Cpfl) and Casl2bl proteins which are the result of convergent evolution. The napDNAbps used herein (e.g., SpCas9, Cas9 variant, or Cas9 equivalents) may also contain various modifications that alter / enhance their PAM specificities. Lastly, the application contemplates any Cas9, Cas9 variant, or Cas9 equivalent which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% sequence identity to a reference Cas9 sequence, such as a references SpCas9 canonical sequence or a reference Cas9 equivalent (e.g., Casl2a (Cpfl)).

[0371] The napDNAbp can be a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. As outlined above, 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 CRISPR104 / 346Bl 195.70209 WO00#14646633v2systems 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 linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3 '-5' exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M. et al., Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference.

[0372] In some embodiments, the napDNAbp directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the napDNAbp directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In some embodiments, a vector encodes a napDNAbp that is mutated to with respect to a corresponding wild- type enzyme such that the mutated napDNAbp lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A in reference to the canonical SpCas9 sequence, or to equivalent amino acid positions in other Cas9 variants or Cas9 equivalents.

[0373] As used herein, the term “Cas protein” refers to a full-length Cas protein obtained from nature, a recombinant Cas protein having a sequences that differs from a naturally occurring Cas protein, or any fragment of a Cas protein that nevertheless retains all or a significant amount of the requisite basic functions needed for the disclosed methods, i.e., (i) possession of nucleic-acid programmable binding of the Cas protein to a target DNA, and (ii) ability to nick the target DNA sequence on one strand. The Cas proteins contemplated herein embrace CRISPR Cas 9 proteins, as well as Cas9 equivalents, variants (e.g., Cas9 nickase (nCas9) or nuclease inactive Cas9 (dCas9)) homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and may include a Cas9 equivalent from any Class 2 CRISPR system (e.g., type II, V, VI), including Casl2a (Cpfl), Casl2e (CasX), Casl2bl (C2cl), Casl2b2, Casl2c (C2c3), C2c4,105 / 346Bl 195.70209 WOOO#14646633v2C2c8, C2c5, C2cl0, C2c9 Casl3a (C2c2), Casl3d, Casl3c (C2c7), Casl3b (C2c6), and Casl3b. Further Cas-equivalents are described in Makarova et al., “C2c2 is a singlecomponent programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299) and Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?,” The CRISPR Journal, Vol. 1. No. 5, 2018, the contents of which are incorporated herein by reference.

[0374] The terms “Cas9” or “Cas9 nuclease” or “Cas9 moiety” or “Cas9 domain” embrace any naturally occurring Cas9 from any organism, any naturally-occurring Cas9 equivalent or functional fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a Cas9, naturally- occurring or engineered. The term Cas9 is not meant to be particularly limiting and may be referred to as a “Cas9 or equivalent.” Exemplary Cas9 proteins are further described herein and / or are described in the art and are incorporated herein by reference. The present disclosure is unlimited with regard to the particular Cas9 that is employed in the prime editor (PE) of the invention.

[0375] As noted herein, Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U. S. A. 98:4658-4663(2001); “CRISPR RNA maturation by transencoded 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).

[0376] Examples of Cas9 and Cas9 equivalents are provided as follows; however, these specific examples are not meant to be limiting. The primer editor of the present disclosure may use any suitable napDNAbp, including any suitable Cas9 or Cas9 equivalent.Wild type canonical SpCas9

[0377] In one embodiment, the primer editor constructs described herein may comprise the “canonical SpCas9” nuclease from S. pyogenes, which has been widely used as a tool for genome engineering and is categorized as the type II subgroup of enzymes of the Class 2 CRISPR-Cas systems. This Cas9 protein is a large, multi-domain protein containing two 106 / 346Bl 195.70209 WOOO#14646633v2distinct nuclease domains. Point mutations can be introduced into Cas9 to abolish one or both nuclease activities, resulting in a nickase Cas9 (nCas9) or dead Cas9 (dCas9), respectively, that still retains its ability to bind DNA in a sgRNA-programmed manner. In principle, when fused to another protein or domain, Cas9 or variant thereof (e.g., nCas9) can target that protein to virtually any DNA sequence simply by co-expression with an appropriate sgRNA.

[0378] The prime editors described herein may include canonical SpCas9, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with a wild type Cas9 sequence provided above.

[0379] Other wild type SpCas9 sequences that may be used in the present disclosure, include: SpCas9 (Streptococcus pyogenes MGAS1882 wild type, NC_017053.1), SpCas9 (Streptococcus pyogenes MGAS1882 wild type, NC_017053.1), SpCas9 (Streptococcus pyogenes wild type, SWBC2D7W014), SpCas9 Streptococcus pyogenes wild type (Encoded product of SWBC2D7W014), SpCas9 (Streptococcus pyogenes M1GAS wild type, NC_002737.2), SpCas9 (Streptococcus pyogenes M1GAS wild type, Encoded product of NC_002737.2 (100% identical to the canonical Q99ZW2 wild type),

[0380] The prime editors described herein may include any of the above SpCas9 sequences, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.Wild type Cas9 orthologs

[0381] In other 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.

[0382] LfCas9 (Lactobacillus fermentum wild type, GenBank: SNX31424.1 1), 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), EfCas9107 / 346Bl 195.70209 WOOO#14646633v2(Enterococcus faecalis, NCBI Reference Sequence: WP_016631044.1, Wild type), Staphylococcus aureus Cas9, Geobacillus thermodenitrificans Cas9, ScCas9 (S. canis, 1375 AA, 159.2 kDa),

[0383] The prime editors described herein may include any of the above Cas9 ortholog sequences, or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0384] The napDNAbp may include any suitable homologs and / or orthologs or naturally occurring enzymes, such as, Cas9. Cas9 homologs and / or orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Preferably, the Cas moiety is configured (e.g., mutagenized, recombinantly engineered, or otherwise obtained from nature) as a nickase 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 has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 protein as provided by any one of the Cas9 orthologs in the above tables.Dead Cas9 variant

[0385] In certain embodiments, the prime editors described herein may include a dead Cas9, e.g., dead SpCas9, which has no nuclease activity due to one or more mutations 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). The nuclease inactivation may be due to one or mutations that result in one or more substitutions and / or deletions in the amino acid sequence of the encoded protein, or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0386] As used herein, the term “dCas9” refers to a nuclease-inactive Cas9 or nuclease-dead Cas9, or a functional fragment thereof, and embraces any naturally occurring dCas9 from any organism, any naturally-occurring dCas9 equivalent or functional fragment thereof, any dCas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a dCas9, naturally-occurring or engineered. The term dCas9 is not meant to be particularly limiting and may be referred to as a “dCas9 or equivalent.” Exemplary dCas9 proteins and 108 / 346Bl 195.70209 WOOO#14646633v2method for making dCas9 proteins are further described herein and / or are described in the art and are incorporated herein by reference.

[0387] In other embodiments, dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. In other embodiments, Cas9 variants having mutations other than D10A and H840A are provided which may result in the full or partial inactivate of the endogenous Cas9 nuclease activity (e.g., nCas9 or dCas9, respectively). Such mutations, by way of example, include other amino acid substitutions at DIO and H820, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC 1 subdomain) with reference to a wild type sequence such as Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1). In some embodiments, variants or homologues of Cas9 (e.g., variants of Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1) are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% 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 NCBI Reference Sequence: NC_017053.1. In some embodiments, variants of dCas9 (e.g., variants of NCBI Reference Sequence: NC_017053.1) are provided having amino acid sequences which are shorter, or longer than NC_017053.1 by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more.

[0388] In one embodiment, the dead Cas9 may be based on the canonical SpCas9 sequence of Q99ZW2 and comprise D10A and an H810A substitutions.Cas9 nickase variant

[0389] In one embodiment, 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. The wild type Cas9 (e.g., the canonical SpCas9) comprises two separate nuclease domains, namely, the RuvC domain (which cleaves the non-protospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand). In one embodiment, the Cas9 nickase comprises a mutation in the RuvC domain which inactivates the RuvC nuclease activity. For example, mutations in aspartate (D) 10, histidine (H) 983, aspartate (D) 986, or glutamate (E) 762, have been reported as loss-of-function mutations of the RuvC nuclease domain and the 109 / 346Bl 195.70209 WOOO#14646633v2creation of a functional Cas9 nickase (e.g., Nishimasu et al., “Crystal structure of Cas9 in complex with guide RNA and target DNA,” Cell 156(5), 935-949, which is incorporated herein by reference). Thus, nickase mutations in the RuvC domain could include DI OX, H983X, D986X, or E762X, wherein X is any amino acid other than the wild type amino acid. In certain embodiments, the nickase could be D10A, of H983A, or D986A, or E762A, or a combination thereof.

[0390] In various embodiments, the Cas9 nickase can having a mutation in the RuvC nuclease domain. Exemplary embodiments include: Cas9 nickase, Cas9 nickase (Streptococcus pyogenes Q99ZW2 Cas9 with D10X, wherein X is any alternate amino acid), Cas9 nickase (Streptococcus pyogenes Q99ZW2 Cas9 with E762X, wherein X is any alternate amino acid), Cas9 nickase (Streptococcus pyogenes Q99ZW2 Cas9 with H983X, wherein X is any alternate amino acid), Cas9 nickase (Streptococcus pyogenes Q99ZW2 Cas9 with D986X, wherein X is any alternate amino acid), Cas9 nickase (Streptococcus pyogenes Q99ZW2 Cas9 with D10A), Cas9 nickase (Streptococcus pyogenes Q99ZW2 Cas9 with E762A), Cas9 nickase (Streptococcus pyogenes Q99ZW2 Cas9 with H983A), Cas9 nickase (Streptococcus pyogenes Q99ZW2 Cas9 with D986A).

[0391] In another embodiment, the Cas9 nickase comprises a mutation in the HNH domain which inactivates the HNH nuclease activity. For example, mutations in histidine (H) 840 or asparagine (R) 863 have been reported as loss-of-function mutations of the HNH nuclease domain and the creation of a functional Cas9 nickase (e.g., Nishimasu et al., “Crystal structure of Cas9 in complex with guide RNA and target DNA,” Cell 156(5), 935-949, which is incorporated herein by reference). Thus, nickase mutations in the HNH domain could include H840X and R863X, wherein X is any amino acid other than the wild type amino acid. In certain embodiments, the nickase could be H840A or R863A or a combination thereof.

[0392] In various embodiments, the Cas9 nickase can have a mutation in the HNH nuclease domain. Exemplary embodiments, include but are not limited to, Cas9 nickase (Streptococcus pyogenes Q99ZW2 Cas9 with H840X, wherein X is any alternate amino acid), Cas9 nickase (Streptococcus pyogenes Q99ZW2 Cas9 with H840A), Cas9 nickase (Streptococcus pyogenes Q99ZW2 Cas9 with R863X, wherein X is any alternate amino acid), Cas9 nickase (Streptococcus pyogenes Q99ZW2 Cas9 with R863A). Any amino acid sequence or variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any of the exemplary embodiments disclosed herein are also contemplated.110 / 346Bl 195.70209 WOOO#14646633v2

[0393] In some embodiments, the N-terminal methionine is removed from a Cas9 nickase, or from any Cas9 variant, ortholog, or equivalent disclosed or contemplated herein. For example, methionine-minus Cas9 nickases include Cas9 nickase ((Met minus) Streptococcus pyogenes Q99ZW2 Cas9 with H840X, wherein X is any alternate amino acid), Cas9 nickase ((Met minus) Streptococcus pyogenes Q99ZW2 Cas9 with H840A), Cas9 nickase ((Met minus) Streptococcus pyogenes Q99ZW2 Cas9 with R863X, wherein X is any alternate amino acid), Cas9 nickase ((Met minus) Streptococcus pyogenes Q99ZW2 Cas9 with R863A). Any amino acid sequence or variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any of the exemplary embodiments disclosed herein are also contemplated.Other Cas9 variants

[0394] Besides dead Cas9 and Cas9 nickase variants, the Cas9 proteins used herein may also include other “Cas9 variants” having 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 any reference Cas9 protein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art. In some embodiments, a 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 a reference Cas9. In some embodiments, the Cas9 variant comprises a fragment of a reference Cas9 (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

[0395] In some embodiments, the disclosure also may utilize Cas9 fragments which retain their functionality, and which are fragments of any herein disclosed Cas9 protein. In some embodiments, the Cas9 fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or 1300 amino acids in length.111 / 346Bl 195.70209 WOOO#14646633v2

[0396] In various embodiments, the prime editors disclosed herein may comprise one of the Cas9 variants described as follows, or a Cas9 variant thereof having 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 any reference Cas9 variants.Small-sized Cas9 variants

[0397] In some embodiments, the prime editors contemplated herein can include a Cas9 protein that is of smaller molecular weight than the canonical SpCas9 sequence. In some embodiments, the smaller-sized Cas9 variants may facilitate delivery to cells, e.g., by an expression vector, nanoparticle, or other means of delivery. In certain embodiments, the smaller-sized Cas9 variants can include enzymes categorized as type II enzymes of the Class 2 CRISPR-Cas systems. In some embodiments, the smaller-sized Cas9 variants can include enzymes categorized as type V enzymes of the Class 2 CRISPR-Cas systems. In other embodiments, the smaller-sized Cas9 variants can include enzymes categorized as type VI enzymes of the Class 2 CRISPR-Cas systems.

[0398] The canonical SpCas9 protein is 1368 amino acids in length and has a predicted molecular weight of 158 kilodaltons. The term “small-sized Cas9 variant”, as used herein, refers to any Cas9 variant — naturally occurring, engineered, or otherwise — that is less than at least 1300 amino acids, or at least less than 1290 amino acids, or than less than 1280 amino acids, or less than 1270 amino acid, or less than 1260 amino acid, or less than 1250 amino acids, or less than 1240 amino acids, or less than 1230 amino acids, or less than 1220 amino acids, or less than 1210 amino acids, or less than 1200 amino acids, or less than 1190 amino acids, or less than 1180 amino acids, or less than 1170 amino acids, or less than 1160 amino acids, or less than 1150 amino acids, or less than 1140 amino acids, or less than 1130 amino acids, or less than 1120 amino acids, or less than 1110 amino acids, or less than 1100 amino acids, or less than 1050 amino acids, or less than 1000 amino acids, or less than 950 amino acids, or less than 900 amino acids, or less than 850 amino acids, or less than 800 amino acids, or less than 750 amino acids, or less than 700 amino acids, or less than 650 amino acids, or less than 600 amino acids, or less than 550 amino acids, or less than 500 amino acids, but at least larger than about 400 amino acids and retaining the required functions of the Cas9 protein. The Cas9 variants can include those categorized as type II, type V, or type VI enzymes of the Class 2 CRISPR-Cas system.112 / 346Bl 195.70209 WOOO#14646633v2

[0399] In various embodiments, the prime editors disclosed herein may comprise any small-sized Cas9 variants known in the art, or a Cas9 variant thereof. Exemplary embodiments include: SaCas9 (Staphylococcus aureus, 1053 AA, 123 kDa), NmeCas9 (N. meningitidis, 1083 AA, 124.5 kDa), CjCas9 (C. jejuni. 984 AA, 114.9 kDa), GeoCas9 (G. stearothermophilus, 1087 AA, 127 kDa), LbaCasl2a (L. bacterium, 1228 AA, 143.9 kDa), BhCasl2b (B. hisashii, 1108 AA, 130.4 kDa). Any amino acid sequence known in the art having 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 any reference small-sized Cas9 protein are herein contemplated. Cas9 equivalents

[0400] In some embodiments, the prime editors described herein can include any Cas9 equivalent. As used herein, the term “Cas9 equivalent” is a broad term that encompasses any napDNAbp protein that serves the same function as Cas9 in the present prime editors despite that its amino acid primary sequence and / or its three-dimensional structure may be different and / or unrelated from an evolutionary standpoint. Thus, while Cas9 equivalents include any Cas9 ortholog, homolog, mutant, or variant described or embraced herein that are evolutionarily related, the Cas9 equivalents also embrace proteins that may have evolved through convergent evolution processes to have the same or similar function as Cas9, but which do not necessarily have any similarity with regard to amino acid sequence and / or three dimensional structure. The prime editors described here embrace any Cas9 equivalent that would provide the same or similar function as Cas9 despite that the Cas9 equivalent may be based on a protein that arose through convergent evolution. For instance, if Cas9 refers to a type II enzyme of the CRISPR-Cas system, a Cas9 equivalent can refer to a type V or type VI enzyme of the CRISPR-Cas system.

[0401] For example, Casl2e (CasX) is a Cas9 equivalent that reportedly has the same function as Cas9 but which evolved through convergent evolution. Thus, the Casl2e (CasX) protein described in Eiu et al., “CasX enzymes comprises a distinct family of RNA-guided genome editors,” Nature, 2019, Vol.566: 218-223, is contemplated to be used with the prime editors described herein. In addition, any variant or modification of Casl2e (CasX) is conceivable and within the scope of the present disclosure.

[0402] Cas9 is a bacterial enzyme that evolved in a wide variety of species. However, the Cas9 equivalents contemplated herein may also be obtained from archaea, which113 / 346Bl 195.70209 WOOO#14646633v2constitute a domain and kingdom of single-celled prokaryotic microbes different from bacteria.

[0403] In some embodiments, Cas9 equivalents may refer to Casl2e (CasX) or Casl2d (CasY), which have been described in, for example, Burstein et al., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21, the entire contents of which is hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR- Casl2e and CRISPR- Casl2d, which are among the most compact systems yet discovered. In some embodiments, Cas9 refers to Casl2e, or a variant of Casl2e. In some embodiments, Cas9 refers to a Casl2d, or a variant of Casl2d. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (napDNAbp), and are within the scope of this disclosure. Also see Liu et al., “CasX enzymes comprises a distinct family of RNA-guided genome editors,” Nature, 2019, Vol.566: 218-223. Any of these Cas9 equivalents are contemplated.

[0404] In some embodiments, the Cas9 equivalent comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally- occurring Casl2e (CasX) or Casl2d (CasY) protein. In some embodiments, the napDNAbp is a naturally- occurring Casl2e (CasX) or Casl2d (CasY) protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a wild-type Cas moiety or any Cas moiety provided herein.

[0405] In various embodiments, the nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), Casl2e (CasX), Casl2d (CasY), Casl2a (Cpfl), Casl2bl (C2cl), Casl3a (C2c2), Casl2c (C2c3), Argonaute,, and Casl2bl. One example of a nucleic acid programmable DNA-binding protein that has different PAM specificity than Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (i.e, Cas 12a (Cpfl)). Similar to Cas9, Cas 12a (Cpfl) is also a Class 2 CRISPR effector, but it is a member of type V subgroup of enzymes, rather than the type II subgroup. It has been shown that Cas 12a (Cpfl) mediates robust DNA interference 114 / 346Bl 195.70209 WOOO#14646633v2with features distinct from Cas9. Casl2a (Cpfl) is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich proto spacer- adjacent motif (TTN, TTTN, or YTN). Moreover, Cpfl cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpfl-family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. Cpfl proteins are known in the art and have been described previously, for example Yamano et al., “Crystal structure of Cpfl in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962; the entire contents of which is hereby incorporated by reference.

[0406] In still other embodiments, the Cas protein may include any CRISPR associated protein, including but not limited to, Casl2a, Casl2bl, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, or homologs thereof, or modified versions thereof, and preferably comprising a nickase mutation.

[0407] In various other embodiments, the napDNAbp can be any of the following proteins: a Cas9, a Casl2a (Cpfl), a Casl2e (CasX), a Casl2d (CasY), a Casl2bl (C2cl), a Casl3a (C2c2), a Casl2c (C2c3), a GeoCas9, a CjCas9, a Casl2g, a Casl2h, a Casl2i, a Casl3b, a Casl3c, a Casl3d, a Casl4, a Csn2, an xCas9, an SpCas9-NG, a circularly permuted Cas9, or an Argonaute (Ago) domain, or a variant thereof.

[0408] Exemplary Cas9 equivalents can include the following: AsCasl2a (previously known as Cpfl, Acidaminococcus sp. (strain BV3L6) UniProtKB U2UMQ6), AsCasl2a nickase (e.g., R1226A), LbCasl2a (previously known as Cpfl, Lachnospiraceae bacterium GAM79, Ref Seq. WP_119623382.1), PcCasl2a (previously known at Cpfl, Prevotella copri, Ref Seq. WP_119227726.1), ErCasl2a ( previously known at Cpfl, Eubacterium rectale, Ref Seq. WP_119223642.1), CsCasl2a (previously known at Cpfl Clostridium sp. AF34-10BH, Ref Seq. WP_118538418.1), BhCasl2b (Bacillus hisashii, Ref Seq.WP_095142515.1), ThCasl2b (Thermomonas hydrothermalis, Ref Seq. WP_072754838), LsCasl2b (Laceyella sacchari, WP_132221894), DtCasl2b (Dsulfonatronum thiodismutans, WP_031386437).

[0409] The prime editors described herein may also comprise Cas 12a (Cpfl) (dCpfl) variants that may be used as a guide nucleotide sequence-programmable DNA-binding protein domain. The Cas 12a (Cpfl) protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but does not have a HNH endonuclease domain, and the 115 / 346Bl 195.70209 WOOO#14646633v2N-terminal of Casl2a (Cpfl) does not have the alfa-helical recognition lobe of Cas9. It was shown in Zetsche et al., Cell, 163, 759-771, 2015 (which is incorporated herein by reference) that, the RuvC-like domain of Casl2a (Cpfl) is responsible for cleaving both DNA strands and inactivation of the RuvC-like domain inactivates Casl2a (Cpfl) nuclease activity.

[0410] In some embodiments, the napDNAbp is a single effector of a microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, without limitation, Cas9, Casl2a (Cpfl), Casl2bl (C2cl), Casl3a (C2c2), and Casl2c (C2c3). Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multi-subunit effector complexes, while Class 2 systems have a single protein effector. For example, Cas9 and Casl2a (Cpfl) are Class 2 effectors. In addition to Cas9 and Casl2a (Cpfl), three distinct Class 2 CRISPR-Cas systems (Casl2bl, Casl3a, and Casl2c) have been described by Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems”, Mol. Cell, 2015 Nov 5; 60(3): 385-397, the entire contents of which are hereby incorporated by reference.

[0411] Effectors of two of the systems, Casl2bl and Casl2c, contain RuvC-like endonuclease domains related to Cas 12a. A third system, Cas 13a contains an effector with two predicated HEPN RNase domains. Production of mature CRISPR RNA is tracrRNA-independent, unlike production of CRISPR RNA by Casl2bl. Casl2bl depends on both CRISPR RNA and tracrRNA for DNA cleavage. Bacterial Cas 13a has been shown to possess a unique RNase activity for CRISPR RNA maturation distinct from its RNA-activated singlestranded RNA degradation activity. These RNase functions are different from each other and from the CRISPR RNA-processing behavior of Cas 12a. See, e.g., East-Seletsky, et al., “Two distinct RNase activities of CRISPR-Cas 13a enable guide-RNA processing and RNA detection”, Nature, 2016 Oct 13;538(7624):270-273, the entire contents of which are hereby incorporated by reference. In vitro biochemical analysis of Cas 13a in Leptotrichia shahii has shown that Cas 13a is guided by a single CRISPR RNA and can be programed to cleave ssRNA targets carrying complementary protospacers. Catalytic residues in the two conserved HEPN domains mediate cleavage. Mutations in the catalytic residues generate catalytically inactive RNA-binding proteins. See e.g., Abudayyeh et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector”, Science, 2016 Aug 5;353(6299), the entire contents of which are hereby incorporated by reference.

[0412] The crystal structure of Alicyclobaccillus acidoterrastris Casl2bl (AacC2cl) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See e.g., Liu et al., “C2cl-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage 116 / 346Bl 195.70209 WOOO#14646633v2Mechanism”, Mol. Cell, 2017 Jan 19;65(2):310-322, the entire contents of which are hereby incorporated by reference. The crystal structure has also been reported in Alicyclobacillus acidoterrestris C2cl bound to target DNAs as ternary complexes. See e.g., Yang et al., “P AM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease”, Cell, 2016 Dec 15;167(7): 1814-1828, the entire contents of which are hereby incorporated by reference. Catalytically competent conformations of AacC2cl, both with target and non-target DNA strands, have been captured independently positioned within a single RuvC catalytic pocket, with C2cl -mediated cleavage resulting in a staggered seven-nucleotide break of target DNA. Structural comparisons between C2cl ternary complexes and previously identified Cas9 and Cpfl counterparts demonstrate the diversity of mechanisms used by CRISPR-Cas9 systems.

[0413] In some embodiments, the napDNAbp may be a C2cl, a C2c2, or a C2c3 protein. In some embodiments, the napDNAbp is a C2cl protein. In some embodiments, the napDNAbp is a Casl3a protein. In some embodiments, the napDNAbp is a Casl2c protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring Casl2bl (C2cl), Casl3a (C2c2), or Casl2c (C2c3) protein. In some embodiments, the napDNAbp is a naturally occurring Casl2bl (C2cl), Casl3a (C2c2), or Casl2c (C2c3) protein.Cas9 circular permutants

[0414] In various embodiments, the prime editors disclosed herein ma...

Claims

CLAIMSWhat is claimed is:

1. A prime editing guide RNA (pegRNA) for editing a target DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor tRNA, wherein the pegRNA comprises a DNA synthesis template,(i) wherein the DNA synthesis template encodes a nonsense suppressor anticodon sequence to be inserted into the anticodon sequence of the endogenous tRNA, and(ii) wherein the DNA synthesis template further encodes one or more additional mutations to be inserted into an anticodon arm domain of the endogenous tRNA.

2. The pegRNA of claim 1, wherein the pegRNA comprises a spacer sequence, a gRNA core, and an extension arm, wherein the extension arm comprises the DNA synthesis template and a primer binding site (PBS).

3. The pegRNA of claims 1 or 2, wherein the spacer sequence is configured to bind to a DNA sequence encoding an endogenous tRNA.

4. The pegRNA of any one of claims 1-3, further comprising a 3-tevopreQi motif.

5. The pegRNA of claim 2 or 3, wherein the spacer sequence and extension arms are any sequence listed in Table 2 of PCT / US2024 / 011892.

6. The pegRNA of any one of claims 2-5, wherein the spacer sequence binds the target DNA sequence adjacent a PAM sequence.

7. The pegRNA of claim 6, wherein the PAM sequence is 5'-NGG-3', wherein “N” is any nucleobase.

8. The pegRNA of any one of claims 1-7, wherein the one or more additional mutations to be inserted into the anticodon arm domain of the endogenous tRNA are a TA> CG mutation at hairpin(hp) position 12, GOCG mutation at hp position 13, GOTA328 / 346Bl 195.70209 WOOO#14646633v2mutation at hp position 13, GOAT mutation at hp position 13, GOTA mutation at hp position 14, or A> T mutation at nucleobase position 38.

9. The pegRNA of claim 8, wherein the one or more additional mutations to be inserted into an anticodon loop of the endogenous tRNA comprises hpl2 TA> CG, hpl3 GOCG, hpl3 GOTA, hpl3 GOAT, hpl4 GOTA, and / or aa A> T mutation at nucleobase position 38.

10. The pegRNA of any one of claims 1-9, wherein the nonsense suppressor anticodon sequence is 5'-UCA-3' or 5'-CUA-3'.

11. The pegRNA of any one of claims 1-10, wherein the DNA synthesis template further encodes for one or more PAM-disrupting mutations, MMR-evading mutations, or combinations thereof.

12. The pegRNA of any one of claims 1-11, wherein the suppressor tRNA enables readthrough of a L21 IX mutation13. The pegRNA as in claim 12, wherein the L21 IX mutation is in a TPP1 gene.

14. The pegRNA of any one of claims 1-11, wherein the suppressor tRNA enables readthrough of a L273X or L274X mutation in a HEXA gene.

15. The pegRNA of any one of claims 1-11, wherein the suppressor tRNA enables readthrough of a Q421X or Y423X mutation in a NPC1 gene.

16. The pegRNA of any one of claims 1-11, wherein the suppressor tRNA enables readthrough of a W402X mutation in a Idua gene.

17. The pegRNA of any one of claims 1-16, wherein the target DNA sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to anyone of SEQ ID NOs: 1-5 (Arg-CCT).329 / 346Bl 195.70209 WOOO#14646633v218. The pegRNA of any one of claims 1-16, wherein the target DNA sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to anyone of SEQ ID NOs: 6-9 (Leu-AAG).

19. The pegRNA of any one of claims 1-16, wherein the target DNA sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to anyone of SEQ ID NOs: 10-16 (Leu-CAA).

20. The pegRNA of any one of claims 1-16, wherein the target DNA sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to anyone of SEQ ID NOs: 17-20 (Leu-TAA).

21. The pegRNA of any one of claims 1-16, wherein the target DNA sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to anyone of SEQ ID NOs: 21-23 (Leu-TAG).

22. The pegRNA of any one of claims 1-16, wherein the target DNA sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to anyone of SEQ ID NOs: 24-27 (Ser- AGA).

23. The pegRNA of any one of claims 1-9, wherein DNA synthesis templates corresponding to SEQ ID NOs: 30, 36, or 54-60 encode the 5'-UCA-3' nonsense suppressor anticodon sequence.

24. The pegRNA of any one of claims 1-9, wherein DNA synthesis templates corresponding to SEQ ID NOs: 29, 31-35, 37-53 encode the 5'-CUA-3' nonsense suppressor anticodon sequence.

25. A prime editing guide RNA (pegRNA) for editing an endogenous tRNA-Leu-TAA gene by prime editing to produce a suppressor tRNA-Leu gene, wherein:(i) the endogenous tRNA-Leu-TAA gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 17-20 (tRNA-Leu-TAA- 1-1 to 4-1), and330 / 346Bl 195.70209 WOOO#14646633v2(ii) the pegRNA comprises a DNA synthesis template, the DNA synthesis template encoding a 5'-CUA-3' or 5'-UCA-3' nonsense suppressor anticodon sequence to be inserted into the endogenous tRNA-Leu gene to produce the suppressor tRNA-Leu gene.

26. The pegRNA of claim 25, wherein the endogenous tRNA-Leu gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity SEQ ID NO: 17 (tRNA-Leu-TAA-1-1)27. The pegRNA of claims 25 or 26, wherein the DNA synthesis template further encodes one or more additional mutations to be inserted into an anticodon loop of the endogenous tRNA-Leu gene.

28. The pegRNA of claim 27, wherein the one or more additional mutations to be inserted into an anticodon loop of the endogenous tRNA are hpl2 TA> CG, hpl3 GOCG, hpl3 GOTA, hpl3 GOAT, hpl4 GOTA, or mut38 A> T.

29. The pegRNA of any one of claims 25-28, wherein the DNA synthesis template further encodes one or more single nucleotide deletions in a D-loop, an anticodon-loop, a variable loop, or a T-loop of the endogenous tRNA-Leu gene, relative to any one of SEQ ID NOs: 17-20.

30. The pegRNA of any one of claims 25-29, wherein the DNA synthesis template further encodes for a variable loop having a nucleic acid sequence that is different than the variable loop sequence of the endogenous tRNA-Leu gene.

31. The pegRNA of claim 30, wherein the DNA synthesis template encodes for a variable loop of tRNA-Leu-TAA-3-1 (SEQ ID NO: 19) to be inserted into a variable loop domain of an endogenous tRNA-Leu-TAA-1-1 gene (SEQ ID NO: 17).

32. The pegRNA of any one of claims 25-31, wherein the pegRNA guides a prime editor to a tRNA-Leu-TAA-1-1 tRNA locus.331 / 346Bl 195.70209 WOOO#14646633v233. The pegRNA of claim 32, wherein the prime editor is selected from the group consisting of PE6a, PE6b, PE6c, PE6d, PE6d+MLHldn, PE6e, PE6f, PE6g, PEmax, PEmax+MLHldn, and PEmaxA.

34. The pegRNA of claim 33, wherein the prime editor comprises PE6c.

35. The pegRNA of any one of claims 25-32, wherein the pegRNA comprises an extension arm, and the extension arm comprises the DNA synthesis template (RTT) and a primer binding site (PBS).

36. The pegRNA of claim 35, wherein the PBS is between 8 and 16 nucleotides37. The pegRNA of any one of claims 25-36, wherein the DNA synthesis template is between 25 and 35 nucleotides.

38. The pegRNA of claim 37, wherein the DNA synthesis template is between 33 and 35 nucleotides.

39. The pegRNA of any one of claim 25-38, wherein the pegRNA further comprises a spacer sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 108-127.

40. The pegRNA of any one of claims 25-39, wherein the pegRNA further comprises a pegRNA scaffold having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28.

41. The pegRNA of any one of claims 35-40, wherein the PBS has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 61-86.

42. The pegRNA of any one of claims 25-41, wherein the DNA synthesis template has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 29-60.332 / 346Bl 195.70209 WOOO#14646633v243. A pegRNA for editing an endogenous tRNA-Arg-CCT-3-1 gene (SEQ ID NO: 3) or a tRNA-Arg-CCT-4-1 gene (SEQ ID NO: 4) to produce a suppressor tRNA capable of reading through a TAG premature termination codon comprising: a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 116, and a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 39.

44. The pegRNA of claim 43, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

45. The pegRNA of claim 43 or 44, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 71.

46. A pegRNA for editing an endogenous tRNA-Leu-TAA-4-1 gene (SEQ ID NO: 20) to produce a suppressor tRNA capable of reading through a TAG premature termination codon comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 40, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 117.

47. The pegRNA of claim 46, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 72.

48. The pegRNA of claim 46 or 47, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

49. A pegRNA for editing an endogenous tRNA-Arg-CCT-5-1 gene (SEQ ID NO: 5) to produce a suppressor tRNA capable of reading through a TAG premature termination codon comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 41, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 116.

50. The pegRNA of claim 49, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 73.333 / 346Bl 195.70209 WOOO#14646633v251. The pegRNA of claim 46 or 50, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

52. A pegRNA for editing an endogenous tRNA-Arg-CCT-5-1 gene (SEQ ID NO: 5), tRNA-Arg-CCT-1-1 gene (SEQ ID NO: 1), tRNA-Arg-CCT-3-1 gene (SEQ ID NO: 3), tRNA-Arg-CCT-4-1 gene (SEQ ID NO: 4), or a tRNA-Arg-CCT-2- 1 gene (SEQ ID NO: 2) to produce a suppressor tRNA capable of reading through a TAG premature termination codon comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 42, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 116.

53. The pegRNA of claim 52, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 71.

54. The pegRNA of claim 52 or 53, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

55. A pegRNA for editing an endogenous tRNA-Leu-AAG-2-1 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-3 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-2 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-4 gene (SEQ ID NO: 7), tRNA-Leu-AAG-3-1 gene (SEQ ID NO: 8), tRNA-Leu-AAG-1-2 gene (SEQ ID NO: 6), tRNA-Leu-AAG-1-3 gene (SEQ ID NO: 6), or a tRNA-Leu-AAG-1-1 gene (SEQ ID NO: 6) to produce a suppressor tRNA capable of reading through a TAG premature termination codon comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 43, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 118.

56. The pegRNA of claim 55, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 74.

57. The pegRNA of claim 55 or 56, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

58. A pegRNA for editing an endogenous tRNA-Leu-TAA-1-1 gene (SEQ ID NO: 17) to produce a suppressor tRNA capable of reading through a TAG premature termination 334 / 346Bl 195.70209 WOOO#14646633v2codon comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 44, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 110.

59. The pegRNA of claim 58, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 75.

60. The pegRNA of claim 58 or 59, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

61. A pegRNA for editing an endogenous tRNA-Ser- AGA-4-1 gene (SEQ ID NO: 27) to produce a suppressor tRNA capable of reading through a TAG premature termination codon comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 45, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 119.

62. The pegRNA of claim 61, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 76.

63. The pegRNA of claim 61 or 62, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

64. A pegRNA for editing an endogenous tRNA-Leu-AAG-2-1 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-3 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-2 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-4 gene (SEQ ID NO: 7), tRNA-Leu-AAG-3-1 gene (SEQ ID NO: 8), tRNA-Leu-AAG-1-2 gene (SEQ ID NO: 6), tRNA-Leu-AAG-1-3 gene (SEQ ID NO: 6), or a tRNA-Leu-AAG-1-1 gene (SEQ ID NO: 6) to produce a suppressor tRNA capable of reading through a TAG premature termination codon comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 46, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 118.

65. The pegRNA of claim 64, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 77.335 / 346Bl 195.70209 WOOO#14646633v266. The pegRNA of claim 64 or 65, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

67. A pegRNA for editing an endogenous tRNA-Tyr-GTA-1-1 gene (SEQ ID NO: 134) to produce a suppressor tRNA capable of reading through a TAG premature termination codon comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 47, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 120.

68. The pegRNA of claim 67, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 78.

69. The pegRNA of claim 67 or 68, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

70. A pegRNA for editing an endogenous tRNA-Leu-TAA-3-1 gene (SEQ ID NO: 19) to produce a suppressor tRNA capable of reading through a TAG premature termination codon comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 48, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 121.

71. The pegRNA of claim 70, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 79.

72. The pegRNA of claim 70 or 71, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

73. A pegRNA for editing an endogenous tRNA-Tyr-GTA-2-1 gene (SEQ ID NO: 135) to produce a suppressor tRNA capable of reading through a TAG premature termination codon comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 49, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 122.336 / 346Bl 195.70209 WOOO#14646633v274. The pegRNA of claim 73, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 80.

75. The pegRNA of claim 73 or 74, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

76. A pegRNA for editing an endogenous tRNA-Leu-TAG-1-1 gene (SEQ ID NO: 21), tRNA-Leu-AAG-2-1 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-3 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-2 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-4 gene (SEQ ID NO: 7), tRNA-Leu-TAG-2-1 gene (SEQ ID NO: 22), tRNA-Leu-AAG-3-1 gene (SEQ ID NO: 8), tRNA-Leu-AAG-1-2 gene (SEQ ID NO: 6), tRNA-Leu-AAG-1-3 gene (SEQ ID NO: 6), or a tRNA-Leu-AAG-1-1 gene (SEQ ID NO: 6) to produce a suppressor tRNA capable of reading through a TAG premature termination codon comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 50, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 123.

77. The pegRNA of claim 76, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 81.

78. The pegRNA of claim 76 or 77, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

79. A pegRNA for editing an endogenous tRNA-Tyr-GTA-5-5 gene (SEQ ID NO: 142) to produce a suppressor tRNA capable of reading through a TAG premature termination codon comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 51, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 124.

80. The pegRNA of claim 79, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 82.

81. The pegRNA of claim 79 or 80, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.337 / 346Bl 195.70209 WOOO#14646633v282. A pegRNA for editing an endogenous tRNA-Leu-TAG-1-1 gene (SEQ ID NO: 21), tRNA-Leu-AAG-2-1 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-3 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-2 gene (SEQ ID NO: 7), tRNA-Leu-AAG-2-4 gene (SEQ ID NO: 7), tRNA-Leu-TAG-2-1 gene (SEQ ID NO: 22), tRNA-Leu-AAG-3-1 gene (SEQ ID NO: 8), tRNA-Leu-AAG-1-2 gene (SEQ ID NO: 6), tRNA-Leu-AAG-1-3 gene (SEQ ID NO: 6), or a tRNA-Leu-AAG-1-1 gene (SEQ ID NO: 6) to produce a suppressor tRNA capable of reading through a TAG premature termination codon comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 50, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 123.

83. The pegRNA of claim 82, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 83.

84. The pegRNA of claim 82 or 83, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

85. A pegRNA for editing an endogenous tRNA-Arg-TCG-6-1 gene (SEQ ID NO: 133) to produce a suppressor tRNA capable of reading through a TGA premature termination codon comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 54, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 126.

86. The pegRNA of claim 85, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 85.

87. The pegRNA of claim 85 or 86, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

88. A pegRNA for editing an endogenous tRNA-Arg-TCG-1-1 gene (SEQ ID NO: 128), tRNA-Arg-TCG-2-1 gene (SEQ ID NO: 129), tRNA-Arg-TCG-3-1 gene (SEQ ID NO: 130), tRNA-Arg-TCG-4-1 gene (SEQ ID NO: 131), or a tRNA-Arg-TCG-5-1 gene (SEQ ID NO: 132), to produce a suppressor tRNA capable of reading through a TGA premature termination codon comprising: a DNA synthesis template sequence having a nucleic acid338 / 346Bl 195.70209 WOOO#14646633v2sequence identical to SEQ ID NO: 55, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 126.

89. The pegRNA of claim 88, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 85.

90. The pegRNA of claim 88 or 89, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

91. A pegRNA for editing an endogenous tRNA-Arg-CCT-1-1 gene (SEQ ID NO: 1) or a tRNA-Arg-CCT-2-1 gene (SEQ ID NO: 2) to produce a suppressor tRNA capable of reading through a TGA premature termination codon comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 56, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 116.

92. The pegRNA of claim 91, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 73.

93. The pegRNA of claim 91 or 92, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

94. A pegRNA for editing an endogenous tRNA-Arg-TCG-6-1 gene (SEQ ID NO: 133) to produce a suppressor tRNA capable of reading through a TGA premature termination codon, comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 54, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 126.

95. The pegRNA of claim 94, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 85.

96. The pegRNA of claim 94 or 95, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.339 / 346Bl 195.70209 WOOO#14646633v297. A pegRNA for editing an endogenous tRNA-Arg-TCG-1-1 gene (SEQ ID NO: 128), tRNA-Arg-TCG-2-1 gene (SEQ ID NO: 129), tRNA-Arg-TCG-3-1 gene (SEQ ID NO: 130), tRNA-Arg-TCG-4-1 gene (SEQ ID NO: 131), or a tRNA-Arg-TCG-5-1 gene (SEQ ID NO: 132), to produce a suppressor tRNA capable of reading through a TGA premature termination codon, comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 57, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 126.

98. The pegRNA of claim 97, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 85.

99. The pegRNA of claim 97 or 98, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

100. A pegRNA for editing an endogenous tRNA-Leu-TAA-1-1 gene (SEQ ID NO: 17) to produce a suppressor tRNA capable of reading through a TGA premature termination codon, comprising: a DNA synthesis template sequence having a nucleic acid sequence identical to SEQ ID NO: 58, and a pegRNA spacer sequence having a nucleic acid sequence identical to SEQ ID NO: 110.

101. The pegRNA of claim 100, further comprising a PBS having a nucleic acid sequence identical to SEQ ID NO: 75.

102. The pegRNA of claim 100 or 101, further comprising a pegRNA scaffold sequence having a nucleic acid sequence identical to SEQ ID NO: 28.

103. The pegRNA of any one of claims 43-100, wherein the DNA synthesis template further encodes one or more mutations to be inserted into the anticodon loop of the endogenous tRNA gene.

104. The pegRNA of claim 103, wherein the one or more mutations to be inserted into the anticodon loop of the endogenous tRNA is hpl2 TA> CG, hpl3 GOCG, hpl3 GOTA, hpl3 GOAT, hpl4 GOTA, or mut38 A> T.340 / 346Bl 195.70209 WOOO#14646633v2105. A complex comprising a prime editor and a pegRNA of any one of claims 1-104.

106. A polynucleotide comprising a first nucleic acid sequence encoding a prime editor and a second nucleic acid sequence encoding a pegRNA of any one of claims 1-104.

107. The polynucleotide of claim 106, wherein the pegRNA is configured to bind to a DNA sequence encoding an endogenous tRNA.

108. A cell comprising the pegRNAs of any one of claims 1-104, the complex of claim 64, or the polynucleotide of claims 65 or 66.

109. A composition comprising the pegRNA of any one of claims 1-104, the complex of claim 64, the polynucleotide of claims 65 or 66, or the cell of claim 67.

110. The composition of claim 109 further comprising a nicking guide RNA (ngRNA).

111. The composition of claim 110, wherein the nicking guide RNA (ngRNA) 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%, or 100% identical to any one of SEQ ID NOs: 91-100.

112. A composition comprising:(i) a first nucleotide sequence encoding a N-terminal portion of a prime editor fused at its C-terminus to an intein-N; and(ii) a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the prime editor,wherein the second nucleotide sequence further encodes for a pegRNA of any one of claims 1-104, the pegRNA operably linked to a promoter.

113. The composition of claim 112, wherein the N-terminal portion of the prime editor comprises a portion of any one of SEQ ID NOs: 147-207 that corresponds to amino acids 1-844 or 1-1024 of SEQ ID NO: 147.341 / 346Bl 195.70209 WOOO#14646633v2114. The composition of claim 112 or 113, wherein the C-terminal portion of the prime editor comprises a portion of any one of SEQ ID NOs: 147-207 that correspond to amino acids 845-1368 or 1025-1368 of SEQ ID NO: 147.

115. The composition of any one of claims 112-114, wherein the second nucleotide sequence further encodes for a nicking guide RNA (ngRNA) sequence operably linked to a promoter.

116. The composition of claim 115, wherein the nicking guide RNA (ngRNA) 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%, or 100% identical to any one of SEQ ID NOs: 91-100.

117. The composition of any one of claims 109-116, wherein three terminal amino acids at the N-terminus of the C-terminal portion are SEQ, SFQ, SFN, SEN, or CFN.

118. The composition of any one of claims 109-117, wherein the pegRNA comprises an evopreQl motif at its 3' end.

119. The composition of any one of claims 109-118, wherein the second nucleotide sequence further comprises a nucleotide encoding a MMLV RT ARNaseH codon optimized for expression in a mammalian cell.

120. The composition of any one of claims 109-119, wherein the one or more promoters is selected from the group consisting of EFS, Cbh, sCAG, hCMV, mPGK, hSYN, and U6.

121. The composition of any one of claims 112-120, wherein the first nucleotide sequence and / or second nucleotide sequence further encode for a SV40 late polyadenylation signal (SV40 late polyA).

122. A method for replacing an endogenous tRNA gene with a suppressor tRNA gene in a genome for treating Hurler Syndrome, the method comprising contacting a DNA sequence encoding the endogenous tRNA gene with a prime editor and a pegRNA of any one of claims 1-104, wherein the prime editor replaces the endogenous tRNA gene with the 342 / 346Bl 195.70209 WOOO#14646633v2suppressor tRNA gene, wherein the DNA synthesis template encodes one or more mutations to be inserted into an anticodon loop of the endogenous tRNA.

123. A method for treating Hurler syndrome caused by premature termination codons, the method comprising installing a suppressor tRNA 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) a pegRNA of any one of claims 1-104, wherein the suppressor tRNA gene encodes a suppressor tRNA comprising an one or more mutations in the anticodon loop, relative to an endogenous tRNA.

124. The method of claim 122 or 123, wherein the edited suppressor tRNA is configured to bind to an ochre stop codon, an opal stop codon, or an amber stop codon125. The method of claim 122 or 123, wherein the one or more mutations in the anticodon loop of the endogenous tRNA are a TA> CG mutation at hairpin position (hp) 12, GOCG mutation at hp 13, GOTA mutation at hp 13, GOAT mutation at hp 13, GOTA mutation at hp 14, or A> T mutation at amino acid position 38.

126. The method of claim 125, wherein one or more mutations in the anticodon loop of the endogenous tRNA are a hpl2 TA> CG, hpl3 GOCG, hpl3 GOTA, hpl3 GOAT, hpl4 GOTA, or mut38 A> T.

127. The method of any one of claims 123-126, wherein the target site in the human genome comprises a ROSA26 gene, an AAVS1 gene, a CCR5 gene, an endogenous tRNA loci, and / or a variable number tandem repeat loci.

128. A method for replacing an endogenous tRNA gene with a suppressor tRNA gene in a genome for reading through a TAG or TGA premature termination codon, the method comprising: contacting a DNA sequence encoding the endogenous tRNA gene with a prime editor and a pegRNA of any one of claims 1-104, wherein the prime editor replaces an anticodon sequence of the endogenous tRNA gene with a suppressor anticodon sequence configured to bind to an ochre stop codon, an opal stop codon, or an amber stop codon.343 / 346Bl 195.70209 WOOO#14646633v2129. Use of the pegRNA of any one of claims 1-104 for editing a target DNA sequence encoding an endogenous tRNA by prime editing to produce a DNA sequence encoding a suppressor tRNA, wherein(i) the pegRNA comprises a DNA synthesis template, wherein the DNA synthesis template encodes a nonsense suppressor anticodon sequence to be inserted into the anticodon sequence of the endogenous tRNA, and(ii) wherein the DNA synthesis template further encodes one or more additional mutations to be inserted into the anticodon loop of the endogenous tRNA.

130. Use of a pegRNA of any one of claims 25-42 for editing an endogenous tRNA-Leu-TAA gene, by prime editing to produce a suppressor tRNA-Leu gene, wherein:(i) the endogenous tRNA-Leu-TAA gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 17-20 (tRNA-Leu-TAA), and(ii) the pegRNA comprises a DNA synthesis template, wherein the DNA synthesis template encoding a 5'-CUA-3' or 5'-UCA-3' nonsense suppressor anticodon sequence is inserted into the endogenous tRNA-Leu gene to produce the suppressor tRNA-Leu gene.

131. Use of a prime editor and the pegRNA of any one of claims 1-22, 24, or 43-82 for replacing an endogenous tRNA gene with a suppressor tRNA gene in a genome for treating Hurler syndrome.

132. Use of the pegRNA of any one of claims 1-22, 24, or 43-82 for installing one or more mutations in an anticodon loop of an endogenous tRNA gene to produce a suppressor tRNA gene, wherein the suppressor tRNA gene encodes for a suppressor tRNA configured to bind to ochre stop codon, an opal stop codon, or an amber stop codon.

133. The method of any one of claims 122-128, wherein the method has minimal or no disruption to a transcriptome of an endogenous tRNA.

134. The method of any one of claims 122-128 or 133, wherein the method does not cause a stress response in a cell where an endogenous tRNA has been converted into a suppressor tRNA.344 / 346Bl 195.70209 WOOO#14646633v2135. The method of any one of claims 122-128 or 133-134, wherein the method causes minimal or no disruption to endogenous tRNA homeostasis.

136. The method of any one of claims 122-128 or 133-135, wherein the method does not significantly alter gene expression in endogenous tRNA relative to tRNAs in wild type cells.

137. The method of any one of claims 122-128 or 133-135, wherein the method does not broadly perturb the proteome and / or induce detected NTC readthrough.345 / 346Bl 195.70209 WOOO#14646633v2

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