Engineered prime editing guide rnas and methods of making and using same

ENGINEERED PRIME EDITING GUIDE RNAS AND METHODS OF MAKING AND USING SAME

RELATED APPLICATIONS

0003[0001] This application claims priority under 35 U. S. C. § 119(e) to U. S. Provisional Application, U. S. S. N. 63/719,236, filed November 12, 2024, which is incorporated herein by reference.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

0005[0002] The contents of the electronic sequence listing (B119570212WO00-SEQ-TNG.xml; Size: 107,741 bytes; and Date of Creation: November 6, 2025) is herein incorporated by reference in their entirety.

BACKGROUND

0007[0003] Prime editing is a versatile genome editing technology that enables precise installation of targeted DNA modifications without requiring double-strand breaks or donor DNA templates. While some of the protein and RNA components of the prime editing system have been engineered for enhanced performance, the structured RNA motifs sometimes appended to the 3 ' end of prime editing guide RNAs (pegRNAs) (as described in International PCT Application Publication No. WO 2022/067130, published March 31, 2022, and Nelson, J. W. et al., Nat. Biotechnol. 2022, 40, 402-410, each of which is incorporated herein by reference) remain largely unexplored. The discovery of new structured motifs for use in pegRNAs could therefore advance the field of prime editing.

SUMMARY

0009[0004] The present disclosure describes the use of high-throughput pooled screening to identify and optimize structured 3 ' RNA motifs that improve prime editing outcomes in human cells. 2,858 RNA motifs were evaluated across four libraries, including natural and engineered pseudoknots, G-quadruplexes, and reverse transcriptase recruitment elements. The screen nominated naturally occurring pseudoknots from viral and cellular RNAs with activity comparable to or exceeding that of the widely used tevopreQi motif. Structure-guided mutagenesis and combinatorial variant screening were applied to refine these hits, culminating in several optimized motifs, including the engineered and evolved pseudoknot variants “tevo2.0,” “eHAV,” and “eSBRMVl-A,” whose nucleotide sequences are provided 1/115

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0011#14568046vl herein. In a pooled correction screen of 847 pathogenic ClinVar variants, the top-performing motifs showed improved prime editing over tevopreQi for over 90% of edits. These optimized motifs increased prime editing efficiencies in clinically relevant contexts, including disease-associated mutations in primary human cells and in vivo via lipid nanoparticles (LNPs) and engineered virus-like particles (eVLPs). These motifs can be incorporated into pegRNA designs to increase prime editing performance.

0012[0005] Thus, in one aspect, the present disclosure provides prime editing guide RNAs (pegRNAs) comprising a guide RNA, an extension arm, and a nucleic acid moiety at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to, or comprising one, two, three, four, or five nucleic acid substitutions relative to, a sequence selected from the group consisting of:

0013GCCGGGCGCGGTGGCGCGTGCCTGTAGTCCCAGCTACTCGGGAGGCT (SEQ ID NO: 1), ATCTTCCGAAGCCGAACGGGAAAACCGGCATC (SEQ ID NO: 2), CGGGTGCAACCCCCCCTCCCCCCGTAGGTTAACGGGACCA (SEQ ID NO: 3), TGGGTGCAACCCCCCCGTCCATCTCGAACGTCATCGAGACCA (SEQ ID NO: 4), AGGGCGGGTACTGCCGTAAGTGCCA (SEQ ID NO: 5), CGTCGGTTCTACCTAGTTACGACGTTAAACCAACTAGGA (SEQ ID NO: 6), AGGCCATGGTGAGGGGCCTGATACCTCACCG (SEQ ID NO: 7), CCGCCTTGTCCCGGGCGTTAAAGGGAA (SEQ ID NO: 8), CCCAGACCTTGAGGGTGGTCTGGTCAATAAAAACCCTCC (SEQ ID NO: 9), CCTTCGCGTCGGAAGTAAAACGACC (SEQ ID NO: 10), ACGGTCGTGCAGTCATCGGTAAGACTGCACA (SEQ ID NO: 11), CGAGGGTGCGGTTGGCCTCGTAAAAAGCCGCA (SEQ ID NO: 12), CCGCCATGTCCCTGGCGTTAAAGGGAA (SEQ ID NO: 13), AGCGGTTCTATCTACTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 14), TTTGCGTCCAAGTCAGAGTCAACCTATAACCTTAAGA (SEQ ID NO: 15), AAGTCCATGGTGAGGGGACTTGATACCTCACCG (SEQ ID NO: 16), CCCAGCCTTGGGGTGGCTGGTCAATAAAAACCCCC (SEQ ID NO: 17), CCTTCGCTCGGAAGTAAAACGAC (SEQ ID NO: 18), ACGTCGTGCAGTACGGTAAACTGCACA (SEQ ID NO: 19), CCGCCTGTCCCGGCGTTAAAGGGAA (SEQ ID NO: 20), CGAGGGGCGGTTGGCCTCGTAAAAAGCCGC (SEQ ID NO: 21), and ACGTCGTGCCGTACGGTAAACGGCACA (SEQ ID NO: 22).

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0016#14568046vl [0006] In some embodiments, the pegRNA comprises a nucleic acid moiety at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to, or comprising one, two, three, four, or five nucleic acid substitutions relative to, the sequence CGTCGGTTCTACCTAGTTACGACGTTAAACCAACTAGGA (SEQ ID NO: 6). In certain embodiments, the pegRNA comprises the nucleic acid moiety CGTCGGTTCTACCTAGTTACGACGTTAAACCAACTAGGA (SEQ ID NO: 6). In some embodiments, the pegRNA comprises a nucleic acid moiety at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to, or comprising one, two, three, four, or five nucleic acid substitutions relative to, the sequence AGGCCATGGTGAGGGGCCTGATACCTCACCG (SEQ ID NO: 7). In certain embodiments, the pegRNA comprises the nucleic acid moiety AGGCCATGGTGAGGGGCCTGATACCTCACCG (SEQ ID NO: 7). In some embodiments, the pegRNA comprises a nucleic acid moiety at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to, or comprising one, two, three, four, or five nucleic acid substitutions relative to, the sequence AAGTCCATGGTGAGGGGACTTGATACCTCACCG (SEQ ID NO: 16). In certain embodiments, the pegRNA comprises the nucleic acid moiety AAGTCCATGGTGAGGGGACTTGATACCTCACCG (SEQ ID NO: 16). In some embodiments, the pegRNA comprises a nucleic acid moiety at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to, or comprising one, two, three, four, or five nucleic acid substitutions relative to, the sequence ACGTCGTGCCGTACGGTAAACGGCACA (SEQ ID NO: 22). In certain embodiments, the pegRNA comprises the nucleic acid moiety ACGTCGTGCCGTACGGTAAACGGCACA (SEQ ID NO: 22).

0017[0007] In some embodiments, the nucleic acid moiety is attached at the 3' end of the pegRNA. In certain embodiments, the pegRNA comprises the structure: 5'-[guide RNA]-[extension arm]-[nucleic acid moiety]-3' or 5'-[guide RNA]- [extension arm] -[linker] -[nucleic acid moiety] -3'.

0018[0008] In another aspect, the present disclosure provides systems for prime editing comprising a prime editor and any of the pegRNAs provided herein.

0019[0009] In another aspect, the present disclosure provides complexes comprising a prime editor and any of the pegRNAs provided herein.

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0022#14568046vl [0010] In another aspect, the present disclosure provides polynucleotides encoding any of the pegRNAs provided herein, or one or more polynucleotides encoding the pegRNA and the prime editor of any of the systems provided herein.

0023[0011] In another aspect, the present disclosure provides vectors comprising any of the polynucleotides provided herein.

0024[0012] In another aspect, the present disclosure provides cells comprising any of the pegRNAs, systems, complexes, polynucleotides, and/or vectors provided herein.

0025[0013] In another aspect, the present disclosure provides compositions comprising any of the pegRNAs, systems, complexes, polynucleotides, and/or vectors provided herein.

0026[0014] In another aspect, the present disclosure provides kits comprising any of the pegRNAs, systems, complexes, polynucleotides, vectors, cells, and/or compositions provided herein.

0027[0015] In another aspect, the present disclosure provides methods of prime editing comprising contacting a target nucleic acid with a prime editor and any of the pegRNAs provided herein.

0028[0016] In another aspect, any of the pegRNAs, systems, complexes, polynucleotides, vectors, cells, and/or compositions provided herein may be used in medicine.

0029[0017] In another aspect, any of the pegRNAs, systems, complexes, polynucleotides, vectors, cells, and/or compositions provided herein may be used in the manufacture of a medicament for treating a disease or disorder.

0030[0018] The foregoing concepts, and additional concepts discussed below, may be arranged in any suitable combination, as the present disclosure is not limited in this respect. Further, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

0032[0019] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

0033[0020] FIGs. 1A-1H show a self-targeting PE pooled reporter for identification of structured motifs. FIG. 1A shows that prime editors combine programmable nickases with reverse transcriptases and a prime editing guide RNA (pegRNA) that specifies the target site and the 4/115

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0035#14568046vl desired edit. Engineered pegRNAs (epegRNAs) with structured 3' motifs can improve pegRNA stability but remain underexplored. FIG. 1B shows systematic evaluation and optimization of structurally diverse motifs for prime editing to explore broader RNA sequence space. FIG. 1C shows phylogenetic classification of organisms with pseudoknot sequences used in this study, from PseudoBase⁵³. FIG. 1D provides an overview of the PE-PRISM screen. Each motif sequence was incorporated into an epegRNA construct paired with an adjacent matched target site, and the resulting library was packaged into lenti viruses. HEK293T cells were transduced with the epegRNA library, transfected with a prime editor expression plasmid, and prime editing outcomes were measured by HTS of the integrated cassette, which linked outcomes with input motifs. FIG. 1E shows the design of v1 PE-PRISM library. In FIG. IF, each dot represents the aggregate performance of a motif sequence in the vl PE-PRISM screen, averaging z-scores (x-axis) and counting element representation across editors, edits, and biological replicates after filtering (y-axis). A subset of library controls (left) and wild-type pseudoknot hits (right) are highlighted. FIG. 1G shows that PE-PRISM identified motifs with comparable activity to tevopreQi. Each dot represents the z-score of editing by a motif for a particular editor, edit, and replicate. Black bars represent mean ± standard deviation. FIG. 1H shows endogenous editing by arrayed plasmid transfection of top wild-type hit HSRP versus controls in HEK293T cells (n=3).

0036[0021] FIGs. 2A-2J show parallel directed evolution of pseudoknot motifs by iterative pooled screens. FIG. 2A shows targeted mutagenesis to preserve secondary structure of wild type pseudoknots. FIG. 2B (left) shows rank-order of motif variants by average z-score in v2 PE-PRISM screen in HEK293T cells. TevopreQi and wild type motifs outperformed negative controls. FIG. 2B (right) shows that the top-scoring variant of each pseudoknot family outperformed tevopreQi. Each dot represents the aggregate motif performance. FIG. 2C shows a comparison of parental motifs and the top variant per family. The dashed line shows the average z-score of tevopreQi. FIG. 2D shows a reversion analysis (left) of the top variant of FMDVA-2 to assess effects of trimming unpaired trailing nucleotides and alternative paired insertions, illustrated in the secondary structure diagram (right). In FIGs. 2C and 2D, each dot represents motif performance for a particular prime editor, edit, and replicate. FIG.

00372E shows a global performance penalty of mutagenesis measured by the decrease in average performance across all v2 variants, binned by change in nucleotide composition. FIG. 2F shows combinations of single variants from the v2 screen that were tested in the v3 PE-PRISM library. FIG. 2G shows rank-order of v3 PE-PRISM motifs by average z-score. For each motif family, top scoring combinations of variants showed moderate improvements over 5/115

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0039#14568046vl single variants alone. FIG. 2H shows a comparison of the average performance of the top two single variants and the top two combinations of variants for each family with >5 motifs. Connecting lines are drawn between motifs derived from the same parental origin. FIGs. 21 and 2J show arrayed validation of top motifs from the v3 library in HEK293T cells (n=3) for editing at (FIG. 21) synthetic sites with self-targeting lentiviral constructs and (FIG. 2J) endogenous genomic loci with plasmid transfection. The rank order of motifs was determined for each combination of editor, edit, and replicate. Bars represent the frequency of motif ranks, and segments are shaded by rank. Entries for tevopreQi are shown in bold for clarity. The edit for KCNE2 was a correction of a mutation absent in wild type HEK293T cells, and is therefore not in FIG. 21. For FIGs. 2C and 2H, P- values were calculated by Welch’s one-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

0040[0022] FIGs. 3A-3F show that engineered pseudoknots improved PE across cell types and delivery modalities. FIG. 3A shows a comparison of motif z- scores between cell types and target edits for self-targeting lentiviral panel. Each dot represents the outcome of editing in HEK293T, HeLa, K562, N2a, or U2OS cells, and horizontal lines represent the grand mean.

0041FIG. 3B shows a frequency distribution of motif ranks for the self-targeting lentiviral panel.

0042FIGs. 3C and 3D show prime editing efficiency in HEK293T (HEK3, FANCF) or N2a cells Dnmtl, Coll2al) with PEmax at the indicated targets using (FIG. 3C) the v3b PE-eVLP architecture or (FIG. 3D) low-dose plasmid transfection. Data represent the mean+s.d. for n=3 biological replicates. The horizontal dashed lines show the performance of tevopreQi in FIG. 3D. FIG. 3E shows the prime editing at the HBB locus in healthy donor-derived CD34+ HSPCs by RNA electroporation of PEmax mRNA, ngRNA, and epegRNAs programmed to correct the mutation that causes sickle-cell disease (n=5). FIG. 3F shows prime editing to correct the CFTR F508del mutation in immortalized bronchial epithelial 16HBEge cells (left) and in HEK293T cells (right) by RNA electroporation (n=3) or RNP lipofection (n=4), respectively. Vertical lines show the performance of tevopreQi.

0043[0023] FIGs. 4A-4G show that pegRNA pseudoknots improved editing of therapeutically relevant mutations. FIG. 4A shows that the design of the v4 library included 19 motifs in combination with 1,000 pegRNAs designed to revert pathogenic mutations in the ClinVar database⁵¹. FIG. 4B shows the distribution of prime edits in the v4 screen, including all possible transitions, transversions, insertions, and deletions. FIG. 4C shows motif performance in the v4 PE-PRISM screen in HEK293T cells using PEmax among data meeting minimum depth and signal thresholds (n=847). The vertical dashed line shows average z-score of tevopreQi. FIG. 4D shows a summary of motif rankings in the v4 library,

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0046#14568046vl showing the frequency of each motif yielding top-three performance tallied across all edits with PEmax. FIG. 4E shows mutations to parental pseudoknot sequences indicated on the secondary structure model, showing positions of paired double substitutions (hp mut), paired double deletions (hp del) and paired double insertions (hp ins) in hairpin regions of the top three scoring motifs: eHAV, HAV, and tevo2.0. eHAV is derived from HAV, and tevo(2)preQi (abbreviated tevo2.0) is derived from tevopreQi. FIG. 4F is a venn diagram of edits for which eHAV, HAV, or tevo2.0 were among the top 3 motifs when using PEmax. FIG. 4G shows a comparison of frequencies of each motif ranking among the top 3 when using PEmax, PE6c, and PE7.

0047[0024] FIGs. 5A-5C show that motifs improved in vivo prime editing with eVLP and LNP delivery modalities. FIG. 5A shows a summary of in vivo prime editing experiments with engineered pseudoknot motifs delivered by transient RNA and RNP-based delivery modalities, including lipid nanoparticles (LNPs) and engineered virus-like particles (eVLPs).

0048FIG. 5B shows that the prime editor v3b PEmax-eVLPs (5 x 10¹⁰) and eGFP: KASH lentivirus was injected into C57BL/6J mice at P0 by ICV administration, and editing was measured in bulk cortical nuclei at 21 days post- injection for tevopreQi and tevo2.0 structured motifs targeting Dnmtl +2GCTG> CAAC, previously optimized in vitro in N2a cells (see FIG. 3C). FIG. 5C shows PE-LNPs targeting Dnmtl (left) and Pcsk9 (right) using admixed formulations of prime editor mRNA, epegRNA, and ngRNA (1.0:0.9:0.1 ratio) were delivered as a single administration into adult C57BL/6J mice by retro-orbital injection and liver editing was assessed 1-week post-injection. Usage of PEmax for Dnmtl and PE6c for Pcsk9 was based on in vitro editing in Hepal-6 cells (see FIG. 13D). Horizontal bars show the average of n=3 independent biological replicates, and each dot represents data from one mouse. Vertical dashed lines show PE efficiency with tevopreQi.

0049[0025] FIGs. 6A-6J show the motif vl PE-PRISM library design and editing outcomes. FIG.

00506A is a schematic of secondary structures that facilitated steps of reverse transcription (primer generation, priming, initiation) by the Ec48 retron, the MMLV reverse transcriptase, and the Tfl retrotransposon. FIG. 6B shows that sequencing reads included the full 3' epegRNA extension, linkers, and barcodes, and synthetic target sites. Reads were aligned to the library reference with Bowtie2, separated into fastq files, trimmed to exclude the pegRNA scaffold, filtered for minimum read thresholds, and processed with CRISPResso2 to quantify editing and indels. FIG. 6C shows correlation between biological replicates. Each dot represents editing with a motif and a particular motif and prime editor. FIG. 6D shows correlation between prime editors. FIGs. 6E and 6F show PE outcomes by editor (FIG. 6E)

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0053#14568046vl and edit (FIG. 6F) after filtering. FIG. 6G shows motif performance by overall category. Each dot represents the average performance in all datasets. FIGs. 6H-6J show that individual motif classes including G-quadruplexes (FIG. 6H), RT-specific motifs (FIG. 61), and tevopreQi substitution variants (FIG. 6J) do not show substantial benefit for prime editing compared to wild type pseudoknots. For FIGs. 6G-6I, each dot represents the motif performance for programmed edit, prime editor, and biological replicate.

0054[0026] FIGs. 7A-7G show arrayed validation of vl PE- PRISM screen. FIG. 7A shows correlation of editing at a synthetic site in self-targeting lentiviral pool and an endogenous site in plasmid transfection in HEK293T cells. Each data point represents the average of biological replicates (n=2 for lentiviral pool, n=2-3 for arrayed plasmid transfection).

0055Different shapes and shades were used for different programmed edits. FIGs. 7B and 7C show the distribution of editing outcomes by programmed edit and prime editor in arrayed plasmid transfection. FIGs. 7D-7F provide raw editing values for each motif and edit tested for transfection with PEmax (FIG. 7D), PE6c (FIG. 7E), and PE7 (FIG. 7F) editor expression plasmids in HEK293T cells. FIG. 7G shows a direct comparison of editing with structured HSRP and tevopreQi versus unstructured dead motifs with PEmax and PE7.

0056[0027] FIGs. 8A-8F provide further analysis and validation of v2 PE-PRISM results. FIG.

00578A shows correlation between replicate editing values with PEmax, PE6c, and PE7. Each dot represents the performance of a single library element. FIG. 8B shows editing outcomes by programmed edit and prime editor in the v2 screen. Editing with tevopreQi is highlighted as a red dot. FIG. 8C shows a heatmap of editing fold-change vs. tevopreQi for each top scoring single variant of a given parental motif origin (x-axis) at four target edits (y-axis) with PEmax. FIG. 8D shows arrayed validation experiments in HEK293T cells (n=2-3) for arrayed lentiviral correlation with pooled screen (n=2). Each dot represents the average editing for a single library element. Pearson correlation for all editors with P &lt; 0.0001. FIG.

00588E shows representative performance of a top-scoring variant of FMDVA-2 across the pooled v2 screen (left) and arrayed lentiviral transduction (right). FIG. 8F shows the global performance penalty of trimming unpaired nucleotides. Each dot represents the change in average z-score upon trimming the specified number of nucleotides (x-axis) from a wild-type parental sequence from either N- or C-terminal positions.

0059[0028] FIGs. 9A-9F show combinations of mutations and analysis of v3 PE-PRISM. FIG.

00609A shows a summary of editing in the v3 PE-PRISM screen by prime editor and programmed edit. Each dot represents the average editing of a library element across n=4 biological replicates. FIG. 9B shows a reversion analysis of the top tevopreQi combination where the 8/115

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0062#14568046vl combination of two single variants (double insertion variant ins hp 2UA and double substitution variant hplO UA> CG) outperformed the single variants in isolation. Each dot represents the average z- score (n=4 biological replicates) of the indicated motif for a combination of prime editor and programmed edit. Horizontal lines show the grand mean in FIGs. 9A and 9B. FIGs. 9C and 9D provide raw editing values in HEK293T cells at (FIG.

00639C) synthetic sites with self-targeting and lentiviral constructs and (FIG. 9D) endogenous genomic loci with epegRNA plasmid transfection. FIG. 9E provides a correlation matrix between replicates in the pooled v3 library (Pool Iv, n=4), arrayed transfection (Array tfx, n=2-3), and arrayed transduction (Array Iv, n=2-3). FIG. 9F shows fold-change improvement for endogenous editing with PEmax in HEK293T cells (n=2-3).

0064[0029] FIGs. 10A-10C show motif efficiencies across diverse cell types. FIG. 10A shows differential expression of ribonucleases, including components of the exosome and cellular RNases. Raw expression data (nTPM) from Human Protein Atlas^79,80 were re-normalized to the average of the cell types shown and plotted as the z-score. FIG. 10B shows a correlation matrix of z-scores between cell types and prime editors for elements in the self-targeting lentiviral panel. FIG. 10C shows editing efficiency across cell types with PEmax for the pooled self-targeting lentiviral panel.

0065[0030] FIGs. 11A-11F provide improvements with DNA, RNA, and RNP-based delivery. FIGs. 11A and 11B show prime editing efficiency in HEK293T and N2a cells for the indicated programmed edits using (FIG. 11 A) the v3b PE-eVLP architecture and (FIG. 11B) plasmid transfection with motifs shown. Values depict the mean for n=3 biological replicates. Comparisons between motifs were made with eVLPs produced, concentrated, and transduced to minimize variability between preparations (see Methods). FIG. 11C provides a comparison of editing efficiencies between plasmid transfection and eVLP transduction in vitro in HEK293T and N2a cells using data from FIGs. 11A and 11B. FIG. 11D shows twin prime editing to install an attP site at the AAVS1 locus in HEK293T cells by plasmid transfection. FIGs. HE and HF show prime editing by electroporation of PEmax mRNA and chemically synthesized epegRNAs programmed to target (FIG. HE) the HBB locus in primary CD34+ HSPCs to install a +5 G> A substitution used in the corrective strategy for sickle cell disease, or (FIG. HF) the Atpla3 locus in mouse primary fibroblasts to correct the p. E815K C.2443A mutation associated with alternating hemiplegia of childhood. Bars represent the mean of n=3 biological replicates, which are shown as individual dots. The horizontal dashed line in FIG. HF represents the heterozygous baseline genotype.

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0068#14568046vl [0031] FIGs. 12A-12F show editing outcomes and arrayed validation of v4 PE-PRISM. FIG.

006912A shows a histogram of editing outcomes across programmed edits after applying minimum read and editing thresholds, binned by average editing values excluding negative controls. The vertical dashed lines show mean editing across all edits. FIG. 12B shows correlation matrices between biological replicates (n=4) for each editor. FIG. 12C shows motif performance with PE6c (left) and PE7 (right). FIG. 12D shows a summary of motif rankings in the v4 library, showing frequency of each motif yielding top-three performance tallied across all edits with PE6c (left) and PE7 (right). FIG. 12E shows correspondence between the pooled v4 screen (n=3-4) and arrayed lentiviral validation (n=2-3) in HEK293T cells, where each dot reflects mean editing values of a single element. FIG. 12F shows arrayed lentiviral correction of the indicated mutation in HEK293T cells at synthetic target sites with PEmax (n=2-3). Vertical dashed line shows efficiency of tevopreQi.

0070[0032] FIGs. 13A-13E show in vivo performance with eVLP and LNP delivery. FIG. 13A shows quantification of PEmax-eVLPs produced following ultracentrifugation and sucrose gradient concentration by MMLV-p30 and Cas9 ELISA to normalize particle count. FIG. 13B shows editing efficiency in N2a cells to measure per-particle potency with dose-normalized eVLPs. Values depict mean+s.d. for n=3 biological replicates. Best fit lines show nonlinear regression to three-parameter logistic curves. FIG. 13C shows editing with v3b PE-eVLPs (5 x 10¹⁰) injected into C57BL/6J mice at P0 by ICV administration was measured in highly transduced GFP+ cortical nuclei at 21 days post-injection for tevopreQi and tevo2.0 structured motifs targeting Dnmtl +2GCTG> CAAC. Data show mean+s.d. of six mice per group and each dot represents data from one mouse. FIG. 13D shows PE-LNPs targeting Dnmtl and Pcsk9 using admixed formulations of prime editor mRNA, epegRNA, and ngRNA (1.0:0.9:0.1 ratio) were delivered into Hepal-6 cells. Data show mean for n=3 biological replicates. FIG. 13E shows correlation of in vitro and in vivo LNP editing with structured motifs for data from FIG. 13D normalized to the efficiency of tevopreQi for both Dnmtl and Pcsk9 edits.

DEFINITIONS

0072[0033] 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);

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0075#14568046vl The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale &amp; Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

0076Cas9

0077[0034] 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 fully or partly inactive cleavage domain of Cas9 and/or the gRNA binding domain of Cas9. A “Cas9 protein” is a full length Cas9 protein. A Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, correct processing of pre-crRNA requires a 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 strand in the target DNA not complementary to crRNA is first cut endonucleolytically, then trimmed 3 '-5' exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the contents of which are incorporated herein 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.

007898:4658-4663(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 11/115

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0080#14568046vl 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, S. aureus, and S. thermophilus. In some embodiments, a Cas9 protein is an S. pyogenes Cas9 (SpCas9) protein, or a variant thereof.

0081DNA synthesis template

0082[0035] 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. When the polymerase of a prime editor is a reverse transcriptase, the DNA synthesis template of the pegRNA may also be referred to as a “reverse transcription template” or “RT template.” The extension arm, including the DNA synthesis template, may be comprised of DNA or RNA. In the case of RNA, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (e.g., a reverse transcriptase). In the case of DNA, the polymerase of the prime editor can be a DNA-dependent DNA polymerase. In various embodiments, the DNA synthesis template comprises an the “edit template” and a “homology arm.”

0083[0036] In some embodiments, the DNA synthesis template is a single- stranded portion of the PEgRNA that is 5' of the PBS and comprises a region of complementarity to the PAM strand (i.e., the non-target strand or the edit strand) and comprises one or more nucleotide edits compared to the endogenous sequence of the double stranded target DNA. In some embodiments, the DNA synthesis template is complementary or substantially complementary to a sequence on the non-target strand that is downstream of a nick site, except for one or more non-complementary nucleotides at the intended nucleotide edit positions. In some embodiments, the DNA synthesis template is complementary or substantially complementary to a sequence on the non-target strand that is immediately downstream (i.e., directly downstream) of a nick site, except for one or more non-complementary nucleotides at the intended nucleotide edit positions. In some embodiments, one or more of the non-complementary nucleotides at the intended nucleotide edit positions are immediately downstream of a nick site. In some embodiments, the DNA synthesis template comprises one or more nucleotide edits relative to the double- stranded target DNA sequence. In some 12/115

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0085#14568046vl embodiments, the DNA synthesis template comprises one or more nucleotide edits relative to the non-target strand of the double- stranded target DNA sequence. In some embodiments, the DNA synthesis template comprises more than one nucleotide edit relative to the doublestranded target DNA sequence.

0086Edit template

0087[0037] 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 or an RNA-dependent DNA polymerase e.g., a reverse transcriptase).

0088Extension arm

0089[0038] The term “extension arm” refers to a nucleotide sequence component of a pegRNA that comprises a primer binding site and a DNA synthesis template (e.g., an edit template and a homology arm) for a polymerase (e.g., a 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 comprises a DNA synthesis template and a primer binding site. In some embodiments, the extension arm comprises the following components in a 5' to 3' direction: the DNA synthesis template, and the primer binding site. 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.

0090Fusion protein

0091[0039] The term “fusion protein” as used herein refers to a hybrid polypeptide that 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, an Cas9 protein fused to a polymerase such as a reverse transcriptase (i.e., a prime editor). Any of the fusion proteins provided herein may be produced by any method known in the art. For example, the fusion proteins provided herein may be produced via recombinant protein 13/115

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0093#14568046vl 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 (4^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2012)), the entire contents of which is incorporated herein by reference.

0094Guide RNA (“gRNA”)

0095[0040] As used herein, the term “guide RNA” is a particular type of guide nucleic acid which is commonly associated with a Cas9 protein, directing the Cas9 protein to a specific sequence in a DNA molecule that includes complementarity to the protospacer sequence of the guide RNA. For example, a gRNA may direct a Cas9 protein (e.g., as part of a prime editor) to a target site in a gene. Functionally, guide RNAs associate with a Cas9 protein, directing (or programming) the Cas9 protein to a specific sequence in a DNA molecule that includes a sequence complementary to the protospacer sequence for the guide RNA. A gRNA is a component of the CRISPR/Cas system. The sequence specificity of a Cas9 DNA-binding protein is determined by gRNAs, which have nucleotide base-pairing complementarity to target DNA sequences. The native gRNA comprises a 20 nucleotide (nt) spacer, which specifies the DNA sequence to be targeted, and is immediately followed by an 80 nt scaffold sequence, which associates the gRNA with the Cas9 protein. In some embodiments, a spacer of the present disclosure has a length of 15 to 100 nucleotides, or more. For example, a spacer may have a length of 15 to 90, 15 to 85, 15 to 80, 15 to 75, 15 to 70, 15 to 65, 15 to 60, 15 to 55, 15 to 50, 15 to 45, 15 to 40, 15 to 35, 15 to 30, or 15 to 20 nucleotides. In some embodiments, the spacer is 20 nucleotides long. For example, the spacer may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. At least a portion of the target DNA sequence is complementary to the spacer of the gRNA. For a Cas9 protein to successfully bind to the DNA target sequence, a region of the target sequence is complementary to the spacer of the gRNA sequence and is immediately followed by the correct protospacer adjacent motif (PAM) sequence. In some embodiments, a spacer is 100% complementary to its target sequence. In some embodiments, the spacer sequence is less than 100% complementary to its target sequence and is, thus, considered to be partially complementary to its target sequence. For example, a targeting sequence may be 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% complementary to its target sequence. In some embodiments, the protospacer of template DNA or target DNA may differ from a complementary spacer region of a gRNA by 1, 2, 3, 4, or 5 nucleotides.

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0098#14568046vl [0041] In some embodiments, the guide RNA is about 15-120 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the guide RNA is 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, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 nucleotides long. In some embodiments, the guide RNA comprises a sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides that is complementary to a target sequence. Sequence complementarity refers to distinct interactions between adenine and thymine (DNA) or uracil (RNA), and between guanine and cytosine. In any of the guide RNA, pegRNA, or epegRNA sequences provided herein, thymines and uracils may be used interchangeably, and a person of ordinary skill in the art will appreciate that the sequences comprise uracils because they are RNA.

0099[0042] As described further herein, in some embodiments, a guide RNA may be attached to an extension arm to form a prime editing guide RNA (pegRNA). In some embodiments, a pegRNA may be further attached a structured nucleic acid moiety as described herein.

0100Linker

0101[0043] 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 components of a fusion protein. For example, a Cas9 protein can be fused to a polymerase (e.g., 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 (e.g., in a pegRNA). In other embodiments, the linker is a non-peptide linker. In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-200 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.

0102napDNAbp

0103[0044] As used herein, the term “nucleic acid programmable DNA binding protein” or “napDNAbp,” of which Cas proteins such as Cas9 and variants thereof are examples, refers to a protein that uses 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.,

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0106#14568046vl guide RNA), which localizes the napDNAbp to a DNA sequence that comprises a DNA strand (z.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 a variant thereof) to localize and bind to a complementary sequence.

0107[0045] 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 comprise a nuclease activity that cuts the non-target strand at a first location, and/or cuts the target strand at a second location. Depending on the nuclease activity, the target DNA can be cut to form a “double- stranded break” whereby both strands are cut. In other embodiments, the target DNA can be cut at only a single site, i.e., the DNA is “nicked” on one strand.

0108Nickase

0109[0046] As used herein, a “nickase” refers to a napDNAbp, such as a Cas9 protein, that is capable of cleaving only one of the two complementary strands of a double- stranded target DNA sequence, thereby generating a nick in that strand. In some embodiments, the nickase cleaves a non-target strand of a double stranded target DNA sequence. In some embodiments, the nickase comprises an amino acid sequence with one or more mutations in a catalytic domain of a Cas9 protein, wherein the one or more mutations reduces or abolishes nuclease activity of the catalytic domain. In some embodiments, the nickase is a Cas9 that comprises one or more mutations in a RuvC-like domain relative to a wild type Cas9 sequence or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 that comprises one or more mutations in an HNH-like domain relative to a wild type Cas9 sequence or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 that comprises a D10A mutation in the RuvCl catalytic domain of Cas9 relative to a canonical SpCas9 sequence. In some embodiments, the nickase is a Cas9 that comprises an H840A mutation relative to a canonical SpCas9 sequence. In some embodiments, the term “Cas9

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0112#14568046vl nickase” refers to a Cas9 with one of the two nuclease domains inactivated. This enzyme is capable of cleaving only one strand of a target DNA.

0113Nuclear localization sequence (NLS)

0114[0047] 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. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., international PCT application, PCT/EP2000/011690, filed November 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference for its disclosure of exemplary nuclear localization sequences. In some embodiments, a base editor or prime editor comprises one or more NLS as described herein.

0115Nucleic acid molecule

0116[0048] The term “nucleic acid,” as used herein, (also referred to as a “polynucleotide” or “oligonucleotide”) refers to a polymer of nucleotides. The polymer may include natural nucleosides (z.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyl uridine, C5 propynyl cytidine, C5 methylcytidine, 7 deazaadenosine, 7 deazaguanosine, 8 oxoadenosine, 8 oxoguanosine, 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., 2 ’-fluororibose, ribose, 2'-deoxyribose, 2'-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5’ N phosphoramidite linkages).

0117pegRNA

0118[0049] As used herein, the terms “prime editing guide RNA,” “PEgRNA,” “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 prime editing as described herein. As described herein, the prime editing guide RNAs comprise one or more “extended regions,” also referred to herein as “extension arms,” of the nucleic acid sequence. The extended regions may comprise, but are not limited to, single- stranded RNA or DNA.

0119Further, 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 17/115

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0121#14568046vl 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” or “reverse transcriptase template” that encodes (by the polymerase/reverse transcriptase of the prime editor) a single- stranded DNA which, in turn, has been designed to be (a) homologous with the endogenous target DNA to be edited, and (b) which comprises at least one desired nucleotide change (e.g., a transition, a transversion, a deletion, or an insertion) to be introduced or integrated into the endogenous target DNA. The extended region may also comprise other functional sequence elements, such as, but not limited to, a “primer binding site” and a “linker” sequence, or other structural elements, such as, but not limited to, aptamers, stem loops, hairpins, toe-loops (e.g., a 3' toeloop), or an RNA-protein recruitment domain (e.g., MS2 hairpin). 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.

0122[0050] In certain embodiments, the pegRNAs have a 3' extension arm, a spacer, and a gRNA core. The 3' extension arm further comprises in the 5' to 3' direction a DNA synthesis template, a primer binding site, and a linker. The DNA synthesis 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.

0123[0051] 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 DNA synthesis template, a primer binding site, and a linker. The DNA synthesis 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.

0124[0052] In still other embodiments, the pegRNAs have in the 5' to 3' direction a spacer, a gRNA core, and an extension arm. The extension arm is at the 3' end of the pegRNA. The extension arm further comprises, in the 5' to 3' direction, a homology arm, an edit template, and a primer binding site. The extension arm 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.

0125[0053] In still other embodiments, the pegRNAs have in the 5' to 3' direction an extension arm, a spacer, and a gRNA core. The extension arm is at the 5' end of the pegRNA. The extension arm further comprises in the 3' to 5' direction a primer binding site, an edit 18/115

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0127#14568046vl template, and a homology arm. The extension arm 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.

0128[0054] In some embodiments, the spacer sequence of the pegRNA is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides in length. In certain embodiments, the spacer sequence of the pegRNA is about 20 nucleotides in length. In some embodiments, the primer binding site is about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, or about 17 nucleotides in length. In certain embodiments, the primer binding site is about 9, about 10, about 11, about 12, about 13, about 14, or about 15 nucleotides in length. In some embodiments, the homology arm of the pegRNA is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length. In some embodiments, the DNA synthesis template is from about 14 to about 21 nucleotides in length.

0129[0055] In some embodiments, a pegRNA is an “engineered pegRNA” (“epegRNA”).

0130Relative to a pegRNA, an epegRNA comprises an additional structured motif, for example, attached to its 3' end. Such additional structured motifs may stabilize the pegRNA or otherwise prevent it from being degraded. Suitable structured motifs include, but are not limited to, toe-loops, hairpins, stem-loops, pseudoknots, aptamers, G-quadruplexes, tRNAs, riboswitches, and ribozymes. In some embodiments, a pegRNA comprises one of the nucleic acid moieties described herein, including:

0131GCCGGGCGCGGTGGCGCGTGCCTGTAGTCCCAGCTACTCGGGAGGCT (SEQ ID NO: 1), ATCTTCCGAAGCCGAACGGGAAAACCGGCATC (SEQ ID NO: 2), CGGGTGCAACCCCCCCTCCCCCCGTAGGTTAACGGGACCA (SEQ ID NO: 3), TGGGTGCAACCCCCCCGTCCATCTCGAACGTCATCGAGACCA (SEQ ID NO: 4), AGGGCGGGTACTGCCGTAAGTGCCA (SEQ ID NO: 5), CGTCGGTTCTACCTAGTTACGACGTTAAACCAACTAGGA (SEQ ID NO: 6), AGGCCATGGTGAGGGGCCTGATACCTCACCG (SEQ ID NO: 7), CCGCCTTGTCCCGGGCGTTAAAGGGAA (SEQ ID NO: 8), CCCAGACCTTGAGGGTGGTCTGGTCAATAAAAACCCTCC (SEQ ID NO: 9), CCTTCGCGTCGGAAGTAAAACGACC (SEQ ID NO: 10), ACGGTCGTGCAGTCATCGGTAAGACTGCACA (SEQ ID NO: 11),

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0134#14568046vl CGAGGGTGCGGTTGGCCTCGTAAAAAGCCGCA (SEQ ID NO: 12), CCGCCATGTCCCTGGCGTTAAAGGGAA (SEQ ID NO: 13), AGCGGTTCTATCTACTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 14), TTTGCGTCCAAGTCAGAGTCAACCTATAACCTTAAGA (SEQ ID NO: 15), AAGTCCATGGTGAGGGGACTTGATACCTCACCG (SEQ ID NO: 16), CCCAGCCTTGGGGTGGCTGGTCAATAAAAACCCCC (SEQ ID NO: 17), CCTTCGCTCGGAAGTAAAACGAC (SEQ ID NO: 18), ACGTCGTGCAGTACGGTAAACTGCACA (SEQ ID NO: 19), CCGCCTGTCCCGGCGTTAAAGGGAA (SEQ ID NO: 20), CGAGGGGCGGTTGGCCTCGTAAAAAGCCGC (SEQ ID NO: 21), and ACGTCGTGCCGTACGGTAAACGGCACA (SEQ ID NO: 22).

0135[0056] PegRNAs are further described, e.g., in International Patent Application No.

0136PCT/US2020/023721, filed March 19, 2020, which published as WO 2020/191239;

0137International Patent Application No. PCT/US2021/031439, filed May 7, 2021, which published as WO 2021/226558; International Patent Application No. PCT/2021/052097, filed September 24, 2021, which published as WO 2022/067130; International Patent Application No. PCT/US2022/012054, filed January 11, 2022, which published as WO 2022/150790; International Patent Application No. PCT/US2022/078655, filed October 25, 2022, which published as WO 2023/076898; and International Patent Application No.

0138PCT/US2022/074628, filed August 5, 2022, which published as WO 2023/015309; each of which is incorporated by reference herein.

0139[0057] In the pegRNAs described herein, and any other RNA sequences provided herein, T’ s and U’s may be used interchangeably in the sequences. A person of ordinary skill in the art will understand that T’s in any provided pegRNA or other RNA sequence should be construed as U’s.

0140Prime editing

0141[0058] As used herein, the term “prime editing” refers to an approach for gene editing using napDNAbps e.g., a Cas9 protein), 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. Prime editing is described in 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.

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0144#14568046vl [0059] Prime editing represents a platform for genome editing that is a versatile and precise method to directly write new genetic information into a specified DNA site using a nucleic acid programmable DNA binding protein (“napDNAbp”) working in association with a polymerase (z.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 sequence as the endogenous strand (or is homologous to it) 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 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 that is installed in place of the corresponding target site endogenous DNA strand.

0145Prime editor

0146[0060] The term “prime editor” refers to the polypeptide or polypeptide components involved in prime editing as described herein. In some embodiments, a prime editor comprises a fusion construct comprising a napDNAbp (e.g., a Cas9 protein) and a reverse transcriptase (e.g., any of the reverse transcriptases provided herein). In some embodiments, a prime editor is capable of carrying out prime editing on a target nucleotide sequence in the presence of a pegRNA (or “extended guide RNA”). In some embodiments, a prime editor comprises a napDNAbp (e.g., a Cas9 protein) and a reverse transcriptase provided in trans, i.e., the napDNAbp and the reverse transcriptase are not fused. The in trans napDNAbp and the reverse transcriptase may be tethered via a non-peptide linkage, e.g., an MS2 RNA-protein binding RNA sequence and a MS2 coat protein fused to either the napDNAbp or the reverse transcriptase, or may be unlinked to each other and simply recruited by the pegRNA. In some embodiments, a prime editor composition, system, or complex provided herein comprises a fusion protein or a fusion protein complexed with a pegRNA, and/or further complexed with a second-strand nicking sgRNA. In some embodiments, the prime editor system may also refer to the complex comprising a fusion protein (reverse transcriptase fused to a

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0149#14568046vl napDNAbp), a pegRNA, and a regular guide RNA capable of directing the second-site nicking step of the non-edited strand as described herein.

0150Primer binding site

0151[0061] The term “primer binding site” or “the PBS” refers to the portion of nucleotide sequence located on a pegRNA as component of the extension arm (typically for example, at the 3' end of the extension arm). The term “primer binding site” refers to a single- stranded portion of the PEgRNA as a component of the extension arm that comprises a region of complementarity to a sequence on the non-target strand. In some embodiments, the primer binding site is complementary to a region upstream of a nick site in a non-target strand. In some embodiments, the primer binding site is complementary to a region immediately upstream of a nick site in the non-target strand. In some embodiments, the primer binding site is capable of binding to the primer sequence that is formed after nicking of the target sequence by the prime editor. When the prime editor nicks one strand of the target DNA sequence (e.g., by a Cas nickase component of the prime editor), 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. In some embodiments, the PBS is complementary to or substantially complementary to, and can anneal to a free 3' end on the non-target strand of the double stranded target DNA at the nick site. In some embodiments, the PBS annealed to the free 3' end on the non-target strand can initiate target-primed DNA synthesis.

0152Protein, peptide, and polypeptide

0153[0062] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein, or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a famesyl group, an isofamesyl 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 22/115

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0155#14568046vl 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 contents of which are incorporated herein by reference.

0156Protospacer

0157[0063] 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 a guide RNA (e.g., a pegRNA). 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). 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.

0158Reverse transcriptase

0159[0064] 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 &amp; 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 RNaseH activity has been presented by Berger et al., Biochemistry 22:2365-2372 (1983). Another reverse transcriptase that is used extensively in molecular biology is reverse transcriptase originating from Moloney murine 23/115

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0161#14568046vl leukemia virus (M-MLV or “MMLV”). 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. Any such reverse transcriptases, or variants or mutants thereof, may be used in the prime editors described herein.

0162Spacer sequence

0163[0065] As used herein, the term “spacer sequence” in connection with a guide RNA (e.g., a pegRNA) refers to the portion of the guide RNA (generally of about 20 nucleotides) that 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.

0164Subject

0165[0066] The term “subject,” as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex, and at any stage of development.

0166Target nucleic acid sequence

0167[0067] The term “target nucleic acid sequence” refers to a sequence within a nucleic acid molecule that is modified (e.g., edited) by a prime editor. The target nucleic acid sequence further refers to the sequence within a nucleic acid molecule to which a complex of, for example, a prime editor and a pegRNA, binds.

0168Variant

0169[0068] As used herein, the term “variant” means the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant Cas9 or reverse transcriptase comprising one or more changes in amino acid residues (i.e., substitutions) as compared to a wild type Cas9 or reverse transcriptase. The term “variant” encompasses homologous proteins having 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%, or at least 99% identity with a reference 24/115

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0171#14568046vl sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term “variant” also encompasses homologous proteins having one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more mutations relative to a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term further encompasses mutants, insertions, truncations, or domains of a reference sequence that display the same or substantially the same functional activity or activities as the reference sequence.

0172Vector

0173[0069] The term “vector,” as used herein, refers to a nucleic acid that encodes a gene of interest, and that is able to enter 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

0175[0070] The present disclosure reports the use of a high-throughput screening system to enable discovery of RNA motifs that enhance editing efficiency when incorporated into pegRNAs. Using this platform, 2858 RNA motif candidates derived from diverse natural and synthetic origins were assessed, and several candidates that supported robust prime editing were identified. Smaller motifs with improved functionality were generated by creating and testing variants of these candidates through structure-informed mutational scanning approaches. The broad applicability of these engineered motifs was demonstrated in the prime editing-mediated correction of 847 pathogenic mutations, and refined motifs that consistently outperformed the previous most commonly used motif (tevopreQi) were ultimately discovered. The applicability of these motifs was further demonstrated across cell types and delivery modalities, including engineered virus-like particles (eVLPs) and lipid nanoparticles (LNPs) in vivo.

0176[0071] Thus, the present disclosure provides engineered prime editing guide RNAs (pegRNAs) including the new 3' motifs useful for increasing prime editing efficiency. The present disclosure also provides systems and complexes for prime editing using the disclosed pegRNAs. Polynucleotides and vectors encoding the pegRNAs are also provided herein. The present disclosure also provides cells, compositions, and kits comprising the pegRNAs.

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0179#14568046vl Methods of prime editing using the pegRNAs are also provided herein. The present disclosure further provides for the use of the pegRNAs in medicine or in the manufacture of a medicament for treating a disease or disorder.

0180Prime Editing Guide RNAs (pegRNAs)

0181[0072] In various aspects, the present disclosure provides modified pegRNAs comprising nucleic acid moieties (e.g., attached at the 3' end of the pegRNA). In some embodiments, the nucleic acid moiety prevents or reduces degradation of the pegRNA in a cell relative to a pegRNA that does not comprise the nucleic acid moiety. Reduced degradation of the pegRNA has several benefits when the pegRNA is used in the context of prime editing, including increasing prime editing efficiency. As described further herein, the inventors have shown that the use of the newly disclosed nucleic acid moieties leads to increased prime editing efficiency relative to editing strategies that use other nucleic acid moieties known in the art. In some embodiments, the nucleic acid moieties are shorter than those previously used in the art, simplifying production (e.g., chemical synthesis) of the pegRNAs.

0182[0073] Thus, in one aspect, the present disclosure provides pegRNAs comprising a guide RNA, an extension arm, and a nucleic acid moiety at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to, or comprising one, two, three, four, or five nucleic acid substitutions relative to, a sequence selected from the group consisting of:

0183GCCGGGCGCGGTGGCGCGTGCCTGTAGTCCCAGCTACTCGGGAGGCT (SEQ ID NO: 1), ATCTTCCGAAGCCGAACGGGAAAACCGGCATC (SEQ ID NO: 2), CGGGTGCAACCCCCCCTCCCCCCGTAGGTTAACGGGACCA (SEQ ID NO: 3), TGGGTGCAACCCCCCCGTCCATCTCGAACGTCATCGAGACCA (SEQ ID NO: 4), AGGGCGGGTACTGCCGTAAGTGCCA (SEQ ID NO: 5), CGTCGGTTCTACCTAGTTACGACGTTAAACCAACTAGGA (SEQ ID NO: 6), AGGCCATGGTGAGGGGCCTGATACCTCACCG (SEQ ID NO: 7), CCGCCTTGTCCCGGGCGTTAAAGGGAA (SEQ ID NO: 8), CCCAGACCTTGAGGGTGGTCTGGTCAATAAAAACCCTCC (SEQ ID NO: 9), CCTTCGCGTCGGAAGTAAAACGACC (SEQ ID NO: 10), ACGGTCGTGCAGTCATCGGTAAGACTGCACA (SEQ ID NO: 11), CGAGGGTGCGGTTGGCCTCGTAAAAAGCCGCA (SEQ ID NO: 12), CCGCCATGTCCCTGGCGTTAAAGGGAA (SEQ ID NO: 13), AGCGGTTCTATCTACTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 14), TTTGCGTCCAAGTCAGAGTCAACCTATAACCTTAAGA (SEQ ID NO: 15),

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0186#14568046vl AAGTCCATGGTGAGGGGACTTGATACCTCACCG (SEQ ID NO: 16), CCCAGCCTTGGGGTGGCTGGTCAATAAAAACCCCC (SEQ ID NO: 17), CCTTCGCTCGGAAGTAAAACGAC (SEQ ID NO: 18), ACGTCGTGCAGTACGGTAAACTGCACA (SEQ ID NO: 19), CCGCCTGTCCCGGCGTTAAAGGGAA (SEQ ID NO: 20), CGAGGGGCGGTTGGCCTCGTAAAAAGCCGC (SEQ ID NO: 21), and ACGTCGTGCCGTACGGTAAACGGCACA (SEQ ID NO: 22). In some embodiments, the nucleic acid moiety comprises a sequence selected from the group consisting of:

0187GCCGGGCGCGGTGGCGCGTGCCTGTAGTCCCAGCTACTCGGGAGGCT (SEQ ID NO: 1), ATCTTCCGAAGCCGAACGGGAAAACCGGCATC (SEQ ID NO: 2), CGGGTGCAACCCCCCCTCCCCCCGTAGGTTAACGGGACCA (SEQ ID NO: 3), TGGGTGCAACCCCCCCGTCCATCTCGAACGTCATCGAGACCA (SEQ ID NO: 4), AGGGCGGGTACTGCCGTAAGTGCCA (SEQ ID NO: 5), CGTCGGTTCTACCTAGTTACGACGTTAAACCAACTAGGA (SEQ ID NO: 6), AGGCCATGGTGAGGGGCCTGATACCTCACCG (SEQ ID NO: 7), CCGCCTTGTCCCGGGCGTTAAAGGGAA (SEQ ID NO: 8), CCCAGACCTTGAGGGTGGTCTGGTCAATAAAAACCCTCC (SEQ ID NO: 9), CCTTCGCGTCGGAAGTAAAACGACC (SEQ ID NO: 10), ACGGTCGTGCAGTCATCGGTAAGACTGCACA (SEQ ID NO: 11), CGAGGGTGCGGTTGGCCTCGTAAAAAGCCGCA (SEQ ID NO: 12), CCGCCATGTCCCTGGCGTTAAAGGGAA (SEQ ID NO: 13), AGCGGTTCTATCTACTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 14), TTTGCGTCCAAGTCAGAGTCAACCTATAACCTTAAGA (SEQ ID NO: 15), AAGTCCATGGTGAGGGGACTTGATACCTCACCG (SEQ ID NO: 16), CCCAGCCTTGGGGTGGCTGGTCAATAAAAACCCCC (SEQ ID NO: 17), CCTTCGCTCGGAAGTAAAACGAC (SEQ ID NO: 18), ACGTCGTGCAGTACGGTAAACTGCACA (SEQ ID NO: 19), CCGCCTGTCCCGGCGTTAAAGGGAA (SEQ ID NO: 20), CGAGGGGCGGTTGGCCTCGTAAAAAGCCGC (SEQ ID NO: 21), and ACGTCGTGCCGTACGGTAAACGGCACA (SEQ ID NO: 22).

0188[0074] In some embodiments, the nucleic acid moiety is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to, or comprises one, two, three, four, or five nucleic acid substitutions relative to, the sequence CGTCGGTTCTACCTAGTTACGACGTTAAACCAACTAGGA (SEQ ID NO: 6). In 27/115

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0190#14568046vl certain embodiments, the nucleic acid moiety comprises the sequence CGTCGGTTCTACCTAGTTACGACGTTAAACCAACTAGGA (SEQ ID NO: 6) (also referred to herein as “tevo2.0”). In some embodiments, the nucleic acid moiety is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to, or comprises one, two, three, four, or five nucleic acid substitutions relative to, the sequence AGGCCATGGTGAGGGGCCTGATACCTCACCG (SEQ ID NO: 7). In certain embodiments, the nucleic acid moiety comprises the sequence AGGCCATGGTGAGGGGCCTGATACCTCACCG (SEQ ID NO: 7) (also referred to herein as “eHAV”). In some embodiments, the nucleic acid moiety is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to, or comprises one, two, three, four, or five nucleic acid substitutions relative to, the sequence AAGTCCATGGTGAGGGGACTTGATACCTCACCG (SEQ ID NO: 16). In certain embodiments, the nucleic acid moiety comprises the sequence AAGTCCATGGTGAGGGGACTTGATACCTCACCG (SEQ ID NO: 16) (also referred to herein as “HAV”). In some embodiments, the nucleic acid moiety is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to, or comprises one, two, three, four, or five nucleic acid substitutions relative to, the sequence ACGTCGTGCCGTACGGTAAACGGCACA (SEQ ID NO: 22). In certain embodiments, nucleic acid moiety comprises the sequence ACGTCGTGCCGTACGGTAAACGGCACA (SEQ ID NO: 22) (also referred to herein as “eSBRMVl-A”).

0191[0075] In various aspects, the pegRNAs provided herein comprise a guide RNA (gRNA), which itself comprises a spacer sequence complementary to a site in a target nucleic acid of interest and a backbone sequence capable of binding to an napDNAbp (e.g., a Cas9 protein). In some embodiments, the guide RNA comprises a backbone sequence that binds a nucleic acid-programmable DNA binding protein (napDNAbp). Several exemplary napDNAbps are provided herein, and any napDNAbp known in the art may be used in conjunction with the disclosed pegRNAs. In some embodiments, the napDNAbp is selected from the group consisting of Cas9, Casl2e, Casl2d, Casl2a, Casl2bl, Casl2b2, Casl2c, Casl2h, Casl2i, Casl2g, Casl2f (Casl4), Casl2fl, Casl2j (Cas ), and Argonaute. In some embodiments, the napDNAbp is a nickase. In certain embodiments, the guide RNA comprises a core that binds a Cas9 protein. In certain embodiments, the Cas9 protein is a Cas9 nickase.

0192[0076] The design of a spacer sequence for use in the pegRNAs provided herein will depend upon the nucleotide sequence of a target site of interest (i.e., the desired site to be edited, e.g., within the genome of a cell), among other factors, such as PAM sequence locations, percent 28/115

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0194#14568046vl G/C content in the target sequence, the degree of microhomology regions, and secondary structures. The sequences of suitable guide RNAs for use in the pegRNAs described herein to target specific genomic sites for prime editing will be apparent to those of skill in the art based on the instant disclosure. Such suitable guide RNA sequences typically comprise spacer sequences that are complementary to a nucleic sequence within about 50 nucleotides upstream or downstream of the target nucleotide(s) to be edited.

0195[0077] In general, a spacer sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of, e.g., a prime editor, to the target sequence. In some embodiments, the degree of complementarity between a spacer sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

0196[0078] In some embodiments, a spacer sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a spacer sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or fewer nucleotides in length. In some embodiments, a spacer sequence is 10-30 or 15-25 nucleotides in length. In certain embodiments, a spacer sequence is about 20 nucleotides in length. The ability of a spacer sequence to direct sequence-specific binding of a fusion protein to a target sequence may be assessed by any suitable assay. For example, the components of a prime editor and pegRNA, including the spacer sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of a prime editor and pegRNA disclosed herein, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a prime editor and pegRNA, including the spacer sequence to be tested and a control spacer sequence different from the test spacer sequence, and comparing binding or rate of cleavage at the target sequence between the test and control 29/115

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0198#14568046vl spacer sequence reactions. Other assays are possible, and will be apparent to those skilled in the art.

0199[0079] The pegRNAs provided herein also comprise an extension arm. In some embodiments, the nucleic acid moiety is attached to the extension arm of the pegRNA. In some embodiments, the extension arm is attached to the 3' end of the guide RNA. In some embodiments, the nucleic acid moiety is attached to the 3' end of the extension arm. In certain embodiments, the pegRNA comprises the structure: 5'-[guide RNA] -[extension arm]-[nucleic acid moiety] -3'.

0200[0080] In some embodiments, the nucleic acid moiety is attached to the extension arm via a linker. In some embodiments, the linker is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, or at least 30 nucleotides in length. In some embodiments, the linker is no longer than 50 nucleotides in length. In certain embodiments, the linker is 4-30 nucleotides in length. In certain embodiments, the linker is about 8 nucleotides in length. In certain embodiments, the pegRNA comprises the structure 5'-[guide RNA] -[extension arm] -[linker] -[nucleic acid moiety] -3'.

0201[0081] The structures of pegRNA extension arms (and pegRNAs more generally) are well known in the art and are described further, for example, in International Patent Application Publication Nos. WO 2020/191248, published September 24, 2020, and WO 2022/067130, published March 31, 2022, each of which is incorporated herein by reference. A traditional guide RNA includes an approximately 20 nucleotide spacer sequence and a gRNA backbone region, which binds with the napDNAbp (e.g., Cas9). In some embodiments, the guide RNA includes the extension arm at the 5' end, i.e., a 5' extension arm. In some embodiments, the 5' extension arm includes a reverse transcription (RT) template sequence, a reverse transcription (RT) primer binding site, and an optional 5-20 nucleotide linker sequence. The RT primer binding site hybridizes to the free 3' end that is formed after a nick is formed in the non-target strand of the R-loop, thereby priming reverse transcriptase for DNA polymerization in the 5'-3' direction. In some embodiments, the guide RNA includes the extension arm at the 3' end, i.e., a 3' extension arm. In some embodiments, the 3' extension arm includes a reverse 30/115

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0203#14568046vl transcription template sequence, and a reverse transcription primer binding site. The RT primer binding site hybridizes to the free 3' end that is formed after a nick is formed in the non-target strand of the R-loop, thereby priming reverse transcriptase for DNA polymerization in the 5 '-3' direction. In some embodiments, the guide RNA includes the extension arm at an intermolecular position within the gRNA backbone sequence, i.e., an intramolecular extension arm. In some embodiments, the intramolecular extension arm includes a reverse transcription template sequence, and a reverse transcription primer binding site. The RT primer binding site hybridizes to the free 3' end that is formed after a nick is formed in the non-target strand of the R-loop, thereby priming reverse transcriptase for DNA polymerization in the 5 '-3' direction.

0204[0082] The length of the extension arm (which includes at least the RT template and primer binding site) can be any useful length. In various embodiments, the RNA extension is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

0205[0083] The RT template sequence within the extension arm can also be any suitable length. For example, the RT template sequence can be at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

0206[0084] In some embodiments, the reverse transcription primer binding site (PBS) sequence within the extension arm is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at 31/115

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0208#14568046vl least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

0209[0085] The RT template sequence, in certain embodiments, encodes a single- stranded DNA molecule that is homologous to the non-target strand (and thus, complementary to the corresponding site of the target strand) but includes one or more nucleotide changes. The one or more nucleotide changes may include one or more single-base nucleotide changes, one or more deletions, and/or one or more insertions.

0210[0086] The synthesized single- stranded DNA product of the RT template sequence is homologous to the non-target strand and contains one or more nucleotide changes. The single- stranded DNA product of the RT template sequence hybridizes in equilibrium with the complementary target strand sequence, thereby displacing the homologous endogenous target strand sequence. The displaced endogenous strand may be referred to in some embodiments as a 5' endogenous DNA flap species. This 5' endogenous DNA flap species can be removed by a 5' flap endonuclease (e.g., FEN1), and the single- stranded DNA product, now hybridized to the endogenous target strand, may be ligated, thereby creating a mismatch between the endogenous sequence and the newly synthesized strand. The mismatch may be resolved by the cell’s innate DNA repair and/or replication processes. In various embodiments, the nucleotide sequence of the RT template sequence corresponds to the nucleotide sequence of the non-target strand that becomes displaced as the 5' flap species and that overlaps with the site to be edited.

0211[0087] In various embodiments of the pegRNAs, the reverse transcription template sequence may encode a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to a nick site, wherein the single-strand DNA flap comprises a desired nucleotide change. The single- stranded DNA flap may displace an endogenous single-strand DNA at the nick site. The displaced endogenous single-strand DNA at the nick site can have a 5' end and form an endogenous flap, which can be excised by the cell. In various embodiments, excision of the 5' end endogenous flap can help drive product formation, since removing the 5' end endogenous flap encourages hybridization of the single- strand 3' DNA flap to the corresponding complementary DNA strand, and the incorporation or assimilation of the desired nucleotide change carried by the single- strand 3' DNA flap into the target 32/115

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0213#14568046vl DNA. In various embodiments of the pegRNAs, the cellular repair of the single- strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.

0214[0088] In still other embodiments, the desired nucleotide change is installed in an editing window that is between about -5 to +5 of the nick site, or between about -10 to +10 of the nick site, or between about -20 to +20 of the nick site, or between about -30 to +30 of the nick site, or between about -40 to + 40 of the nick site, or between about -50 to +50 of the nick site, or between about -60 to +60 of the nick site, or between about -70 to +70 of the nick site, or between about -80 to +80 of the nick site, or between about -90 to +90 of the nick site, or between about -100 to +100 of the nick site, or between about -200 to +200 of the nick site.

0215[0089] In other embodiments, the desired nucleotide change is installed in an editing window that is between about +1 to +2 from the nick site, or about +1 to +3, +1 to +4, +1 to +5, +1 to +6, +1 to +7, +1 to +8, +1 to +9, +1 to +10, +1 to +11, +1 to +12, +1 to +13, +1 to +14, +1 to +15, +1 to +16, +1 to +17, +1 to +18, +1 to +19, +1 to +20, +1 to +21, +1 to +22, +1 to +23, +1 to +24, +1 to +25, +1 to +26, +1 to +27, +1 to +28, +1 to +29, +1 to +30, +1 to +31, +1 to +32, +1 to +33, +1 to +34, +1 to +35, +1 to +36, +1 to +37, +1 to +38, +1 to +39, +1 to +40, +1 to +41, +1 to +42, +1 to +43, +1 to +44, +1 to +45, +1 to +46, +1 to +47, +1 to +48, +1 to +49, +1 to +50, +1 to +51, +1 to +52, +1 to +53, +1 to +54, +1 to +55, +1 to +56, +1 to +57, +1 to +58, +1 to +59, +1 to +60, +1 to +61, +1 to +62, +1 to +63, +1 to +64, +1 to +65, +1 to +66, +1 to +67, +1 to +68, +1 to +69, +1 to +70, +1 to +71, +1 to +72, +1 to +73, +1 to +74, +1 to +75, +1 to +76, +1 to +77, +1 to +78, +1 to +79, +1 to +80, +1 to +81, +1 to +82, +1 to +83, +1 to +84, +1 to +85, +1 to +86, +1 to +87, +1 to +88, +1 to +89, +1 to +90, +1 to +90, +1 to +91, +1 to +92, +1 to +93, +1 to +94, +1 to +95, +1 to +96, +1 to +97, +1 to +98, +1 to +99, +1 to +100, +1 to +101, +1 to +102, +1 to +103, +1 to +104, +1 to +105, +1 to +106, +1 to +107, +1 to +108, +1 to +109, +1 to +110, +1 to +111, +1 to +112, +1 to +113, +1 to +114, +1 to +115, +1 to +116, +1 to +117, +1 to +118, +1 to +119, +1 to +120, +1 to +121, +1 to +122, +1 to +123, +1 to +124, or +1 to +125 from the nick site.

0216[0090] In still other embodiments, the desired nucleotide change is installed in an editing window that is between about +1 to +2 from the nick site, or about +1 to +5, +1 to +10, +1 to +15, +1 to +20, +1 to +25, +1 to +30, +1 to +35, +1 to +40, +1 to +45, +1 to +50, +1 to +55, +1 to +100, +1 to +105, +1 to +110, +1 to +115, +1 to +120, +1 to +125, +1 to +130, +1 to +135, +1 to +140, +1 to +145, +1 to +150, +1 to +155, +1 to +160, +1 to +165, +1 to +170,

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0219#14568046vl +1 to +175, +1 to +180, +1 to +185, +1 to +190, +1 to +195, or +1 to +200, from the nick site.

0220[0091] Any of the pegRNAs described herein may be expressed from an encoding nucleic acid, or synthesized chemically. Methods are well known in the art for obtaining or otherwise synthesizing pegRNAs, and for determining the appropriate sequence of the pegRNA, including the spacer sequence that interacts and hybridizes with the target strand of a genomic target site of interest.

0221[0092] In another aspect, the present disclosure provides RNAs comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to, or comprising one, two, three, four, or five nucleic acid substitutions relative to, a sequence selected from the group consisting of:

0222GCCGGGCGCGGTGGCGCGTGCCTGTAGTCCCAGCTACTCGGGAGGCT (SEQ ID NO: 1), ATCTTCCGAAGCCGAACGGGAAAACCGGCATC (SEQ ID NO: 2), CGGGTGCAACCCCCCCTCCCCCCGTAGGTTAACGGGACCA (SEQ ID NO: 3), TGGGTGCAACCCCCCCGTCCATCTCGAACGTCATCGAGACCA (SEQ ID NO: 4), AGGGCGGGTACTGCCGTAAGTGCCA (SEQ ID NO: 5), CGTCGGTTCTACCTAGTTACGACGTTAAACCAACTAGGA (SEQ ID NO: 6), AGGCCATGGTGAGGGGCCTGATACCTCACCG (SEQ ID NO: 7), CCGCCTTGTCCCGGGCGTTAAAGGGAA (SEQ ID NO: 8), CCCAGACCTTGAGGGTGGTCTGGTCAATAAAAACCCTCC (SEQ ID NO: 9), CCTTCGCGTCGGAAGTAAAACGACC (SEQ ID NO: 10), ACGGTCGTGCAGTCATCGGTAAGACTGCACA (SEQ ID NO: 11), CGAGGGTGCGGTTGGCCTCGTAAAAAGCCGCA (SEQ ID NO: 12), CCGCCATGTCCCTGGCGTTAAAGGGAA (SEQ ID NO: 13), AGCGGTTCTATCTACTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 14), TTTGCGTCCAAGTCAGAGTCAACCTATAACCTTAAGA (SEQ ID NO: 15), AAGTCCATGGTGAGGGGACTTGATACCTCACCG (SEQ ID NO: 16), CCCAGCCTTGGGGTGGCTGGTCAATAAAAACCCCC (SEQ ID NO: 17), CCTTCGCTCGGAAGTAAAACGAC (SEQ ID NO: 18), ACGTCGTGCAGTACGGTAAACTGCACA (SEQ ID NO: 19), CCGCCTGTCCCGGCGTTAAAGGGAA (SEQ ID NO: 20), CGAGGGGCGGTTGGCCTCGTAAAAAGCCGC (SEQ ID NO: 21), and ACGTCGTGCCGTACGGTAAACGGCACA (SEQ ID NO: 22).

022334/115

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0225#14568046vl Nucleic Acid-Programmable DNA Binding Proteins (napDNAbp)

0226[0093] In various embodiments, the prime editors contemplated by the present disclosure comprise a nucleic acid programmable DNA binding protein (napDNAbp). In some embodiments, the napDNAbp is a Cas protein (e.g., a Cas9 protein, including a Cas9 nickase or a nuclease-inactivated (dCas9) protein). Suitable napDNAbp sequences that can be used in prime editors will be apparent to those of skill in the art based on this disclosure, and such proteins 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; which is incorporated herein by reference. Exemplary Cas variants and homologs include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), Cpfl, CasX, CasY, C2cl, C2c2, C2c3, GeoCas9, CjCas9, Casl2a, Casl2b, Casl2g, Casl2h, Casl2i, Casl3b, Casl3c, Casl3d, Casl4, Csn2, xCas9, SpCas9-NG, Nme2Cas9, circularly permuted Cas9, Argonaute (Ago), Cas9-KKH, SmacCas9, Spy-macCas9, SpCas9-VRQR, SpCas9-NRRH, SpaCas9-NRTH, SpCas9-NRCH, LbCasl2a, AsCasl2a, CeCasl2a, MbCasl2a, Cas3, Cas, and circularly permuted Cas9 domains such as CP1012, CP1028, CP 1041, CP 1249, and CP 1300, and variants and homologs thereof.

0227[0094] For example, a prime editor may include a napDNAbp domain having a wild type Cas9 sequence, including, for example, the canonical Streptococcus pyogenes Cas9 sequence, shown as follows:

0228napDNAbp Sequence SEQ ID NO

0229Wild type MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGN 23 Streptococcus TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRR pyogenes KNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH

0230Cas9 ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD

0231(SpCas9) LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQL VQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLI AQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAK LQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKAL VRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFI KPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVG PLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS FIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKV KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQ LKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG

0232<img file="WO2026106945A2_D0001.tif" />

KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS

023435/115

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0236#14568046vl GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGI KELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYV DQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILD SRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFV YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFF KTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKL KSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLII KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEII EQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAEN IIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDAT

0237<img file="WO2026106945A2_D0002.tif" />

LIHQSITGLYETRIDLSQLGGD

0239[0095] In some embodiments, a prime editor may include a napDNAbp domain having a modified Cas9 sequence, including, for example, nickase or nuclease-inactivated (dead) variants of Streptococcus pyogenes Cas9, shown as follows:

0240napDNAbp Sequence SEQ ID NO

0241Cas9 nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD 24 Streptococcus RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI pyogenes Cas9 CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF with H840A GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNRE KIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEE VVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE

0242<img file="WO2026106945A2_D0003.tif" />

VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA

024436/115

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0246#14568046vl VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENII HLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD

0247Cas9 nickase MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTD 25 Streptococcus RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI pyogenes Cas9 CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF with D10A GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNRE KIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEE VVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENII HLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD

0248Nuclease- MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTD 26 inactivated RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI Cas9 (dCas9) CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF Streptococcus GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL

0249<img file="WO2026106945A2_D0004.tif" />

0250pyogenes Cas9 AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE 37/115

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0252#14568046vl with D10A and NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF H840A GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNRE KIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEE VVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENII HLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD PE6e Cas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD 27 (derived from RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI Streptococcus CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF pyogenes GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL Cas9) AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNRE KIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP

0253<img file="WO2026106945A2_D0005.tif" />

ENIVIEMARENQTTQKGQRNSRERMKRIEEGIKELGSQIL 38/115

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0256#14568046vl KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEE VVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIARQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENII HLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD PE6fCas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD 28 (derived from RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI Streptococcus CYLQEIFSNEMAKVDDSFFRRLEESFLVEEDKKHERHPIF pyogenes GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL Cas9) AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNRE KIEKILTFRIPYYVGPLARGNSRFAWMTRKSEKTITPWNFE EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMVEERLKT YAHLFDNKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQ GDSLYEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHK PENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEE VVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIARQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENII HLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ

0257<img file="WO2026106945A2_D0006.tif" />

SITGLYETRIDLSQLGGD

025939/115

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0261#14568046vl PE6g Cas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD (derived from RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI Streptococcus CYLQEIFSNEMAKVDDSFFRRLEESFLVEEDKKHERHPIF pyogenes GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL

0262Cas9) AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNRE KIEKILTFRIPYYVGPLARGNSRFAWMTRKSEKTITPWNFE EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMVEERLKT YAHLFDNKVMKQLKRCRYTGWGRLSRKLINGIRDKQSG KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQ GDSLYEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHK PENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEE VVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENII HLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ

0263<img file="WO2026106945A2_D0007.tif" />

SITGLYETRIDLSQLGGD

0265[0096] In some embodiments, the Cas9 protein included in a prime editor can be a wild type Cas9 ortholog from another bacterial species different from the canonical Cas9 from. S'. pyogenes. For example, modified versions of Cas9 orthologs can be used in connection with the prime editors described in this specification by making mutations at positions corresponding to D10A and/or H840A or any other amino acids of interest in wild type SpCas9. In some embodiments, a prime editor may include a napDNAbp domain having a modified Cas9 sequence, including, for example, Cas9 proteins with alternative PAM specificities.

0266[0097] Additional suitable napDNAbp sequences that can be used in prime editors in the compositions, complexes, and methods described herein will be apparent to those of skill in 40/115

B1195.70212WO00

0268#14568046vl the art based on this disclosure. A person of ordinary skill in the art will also understand that a napDNAbp may be used in a prime editor with or without an N-terminal methionine if one is shown in any of the sequences above (z.e., if the napDNAbp is not located at the N-terminus of the gene editor, the N-terminal methionine normally present in the napDNAbp sequence (due to the start codon in the nucleotide sequence encoding the napDNAbp) does not need to be present in the gene editor).

0269Reverse Transcriptases

0270[0098] In various embodiments, the prime editors of the present disclosure comprise a reverse transcriptase, optionally joined to a napDNAbp (e.g., a Cas9) by a linker. The use of any suitable reverse transcriptase is contemplated, including any of those whose sequences are provided herein. Since reverse transcriptases are well-known in the art, and amino acid sequences of reverse transcriptases are readily available, this disclosure is not meant in any way to be limited to those specific reverse transcriptases identified herein.

0271[0099] In some embodiments, the reverse transcriptase domain is a wild type MMLV reverse transcriptase. In some embodiments, the reverse transcriptase domain is a variant of wild type MMLV reverse transcriptase. In some embodiments, a variant MMLV reverse transcriptase comprises the amino acid substitutions D200N, T306K, W313F, T330P, and L603W relative to the wild type MMLV RT of SEQ ID NO: 30.

0272[0100] The prime editor fusion proteins provided herein may also comprise other variant RTs. Exemplary reverse transcriptases that can be fused to Cas9, or provided as individual proteins in trans according to various embodiments of this disclosure, are provided below. Exemplary reverse transcriptases include the following sequences, and variants with at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the following sequences:

0273Reverse Sequence SEQ ID Transcriptase NO Moloney TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGG 30 murine MGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHI leukemia virus QRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLR reverse EVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDA transcriptase FFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFK

(M-MLV RT) NSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATS

0275wild type ELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYL GYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGT AGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKA

0276<img file="WO2026106945A2_D0008.tif" />

YQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL

027841/115

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0280#14568046vl Reverse Sequence SEQ ID Transcriptase NO TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAV LTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQH NCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQE GQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQA LKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSE GKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEAR GNRMADQAARKAAITETPDTSTLLIENSSP

0281M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGG 31 D200N T306K MGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHI W313F T330P QRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLR L603W EVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDA FFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFK NSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATS ELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYL GYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGK AGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAY QEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLT QKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNAR MTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN CLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQAL KMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSE GKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEAR GNRMADQAARKAAITETPDTSTLLIENSSP

0282Moloney TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGG 32 murine MGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHI leukemia virus QRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLR reverse EVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDA transcriptase FFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFK

0283(M-MLV RT) NSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATS comprising ELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYL RNaseH GYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGT domain AGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKA truncation YQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL (between D497 TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAV and 1498 of LTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA wildtype) RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQH NCLD

0284M-MLV RT TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGG 33 D200N T306K MGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHI W313F T330P QRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLR comprising EVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDA RNaseH FFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFK domain NSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATS truncation ELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYL

0285<img file="WO2026106945A2_D0009.tif" />

0286(between D497 GYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGK

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0289#14568046vl Reverse Sequence SEQ ID Transcriptase NO and 1498 of AGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAY wildtype) QEIKQAEETAPAEGEPDETKPFEEFVDEKQGYAKGVET QKEGPWRRPVAYESKKEDPVAAGWPPCERMVAAIAVE TKDAGKETMGQPEVIEAPHAVEAEVKQPPDRWESNAR MTHYQAEEEDTDRVQFGPVVAENPATEEPEPEEGEQHN CED

0290Schizosacchar ISSSKHTESQMNKVSNIVKEPEEPDIYKEFKDITADTNTE 34 omyces pombe KEPKPIKGLEFEVEETQENYREPIRNYPEPPGKMQAMND

0291Tfl reverse EINQGLKSGIIRESKAINACPVMFVPKKEGTERMVVDYK transcriptase PENKYVKPNIYPEPEIEQEEAKIQGSTIFTKEDEKSAYHEI RVRKGDEHKEAFRCPRGVFEYEVMPYGISTAPAHFQYF INTIEGEAKESHVVCYMDDIEIHSKSESEHVKHVKDVLQ KEKNANLIINQAKCEFHQSQVKFIGYHISEKGFTPCQENI DKVLQWKQPKNRKEERQFEGSVNYERKFIPKTSQLTHP ENKEEKKDVRWKWTPTQTQAIENIKQCLVSPPVLRHFD FSKKIEEETDASDVAVGAVLSQKHDDDKYYPVGYYSA KMSKAQENYSVSDKEMEAIIKSLKHWRHYEESTIEPFKI ETDHRNLIGRITNESEPENKREARWQLFEQDFNFEINYR PGSANHIADAESRIVDETEPIPKDSEDNSINFVNQISI PE6b reverse ISSSKHTESQMNKVSNIVKEPEEPDIYKEFKDITADTNTE 35 transcriptase KEPKPIKGLEFEVEETQENYREPIRNYPETPVKMQAMN (derived from DEINQGLKGGIIRESKAINACPVIFVPRKEGTERMVVDY Schizosacchar RPENKYVKPNVYPEPEIEQEEAKIQGSTIFTKEDEKSAY omyces pombe HQIRVRKGDEHKEAFRCPRGVFEYEVMPYGISTAPAHF

0292Tfl reverse QYFINTIEGEAKESHVVCYMDDIEIHSKSESEHVKHVKD transcriptase) VLQKEKNANLIINQAKCEFHQSQVKFIGYHISEKGLTPC QENIDKVLQWKQPKNRKEERQFEGSVNYERKFIPKTSQ ETHPENKEEKKDVRWKWTPTQTQAIENIKQCLVSPPVL RHFDFSKKIEEETDVSDVAVGAVLSQKHDDDKYYPVG YYSAKMSKAQENYSVSDKEMEAIIKSLEHWRHYEESTI EPFKIETDHRNLIGRITNESEPENKREARWQLFEQDFNFE INYRPGSANHIADAESRIVDETEPIPKDNEDNSINFVNQIS I PE6c reverse ISSSKHTESQMNKVSNIVKEPEEPDIYKEFKDITADTNTE 36 transcriptase KEPKPIKGLEFEVEETQENYREPIRNYPETPVKMQAMN (derived from DEINQGLKGGIIRESKAINACPVIFVPRKEGTERMVVDY Schizosacchar RPENKYVKPNVYPEPEIEQEEAKIQGSTIFTKEDEKSAY omyces pombe HQIRVRKGDEHKEAFRCPRGVFEYEVMPYGIKTAPAHF

0293Tfl reverse QYFINTIEGEAKESHVVCYMDDIEIHSKSESEHVKHVKD transcriptase) VLQKEKNANLIINQAKCEFHQSQVKFEGYHISEKGLTPC QENIDKVLQWKQPKNQKEERQFEGQVNYERKFIPKTSQ ETHPENKEEKKDVRWKWTPTQTQAIENIKQCLVSPPVL RHFDFSKKIEEETDVSDVAVGAVLSQKHDDDKYYPVG YYSAKMSKAQENYSVSDKEMEAIIKSLEHWRHYEESTI EPFKIETDHRNLIGRITNESEPENKREARWQLFEQDFNFE INYRPGSANHIADAESRIVDETEPIPKDNEDNSINFVNQIS

0294<img file="WO2026106945A2_D0010.tif" />

I

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0298#14568046vl Reverse Sequence SEQ ID Transcriptase NO PE6d reverse TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGG 37 transcriptase MGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHI (derived from QRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLR truncated M- EVNKRVEDIHPNVPNPYNLLSGLPPSHQWYTVLDLKDA MLV RT) FFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFK NSPTLFCEALHRDLADFRIQHPDLILLQYYDDLLLAATS ELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYL GYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGK AGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAY QEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLT QKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVL TKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNAR MTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN CLD

0299Escherichia GRPYVTLNLNGMFMDKFKPYSKSNAPITTLEKLSKALSI 38 coli Ec48 SVEELKAIAELSLDEKYTLKEIPKIDGSKRIVYSLHPKMR reverse LLQSRINKRIFKELVVFPSFLFGSVPSKNDVLNSNVKRD transcriptase YVSCAKAHCGAKTVLKVDISNFFDNIHRDLVRSVFEEIL HIKDEALEYLVDICTKDDFVVQGALTSSYIATLCLFAVE GDVVRRAQRKGLVYTRLVDDITVSSKISNYDFSQMQSH IERMLSEHDLPINKHKTKIFHCSSEPIKVHGLRVDYDSPR LPSDEVKRIRASIHNLKLLAAKNNTKTSVAYRKEFNRC MGRVNKLGRVGHEKYESFKKQLQAIKPMPSKRDVAVI DAAIKSLELSYSKGNQNKHWYKRKYDLTRYKMIILTRS ESFKEKLECFKSRLASLKPL PE6a reverse GRPYVTLNLNGMFMDKFKPYSKSNAPITTLEKLSKALSI 39 transcriptase SVEELKAIAELSLDEKYTLKKIPKIDGSKRIVYSLHPKMR (derived from LLQSRINERIFKELVVFPSFLFGSVPSKNDVLNSNVKRDY Escherichia VSCAKAHCGAKTVLKVDISNFFDNIHRDLVRSVFEEILH coli Ec48 IKDEALDYLVDICTKDDFVVQGALTSSYIATLCLFAVEG reverse DVVRRAQRKGLVYTRLVDDITVSSKISNYDFSQMQSHI transcriptase) ERMLSEHNLPINKHKTKIFHCSSEPIKVHGLIVDYDSPRL PSDKVKRIRASIHNLKLLAAKNNTKTSVAYRKEFNRCM GRVNELGRVGHEKYESFKKQLQAIKPMPSNRDVAVIDA AIKSLELSYSKGNQNKHWYKRKYDLTRYKMIILTRSESF

0300<img file="WO2026106945A2_D0011.tif" />

KEKLECFKSRLASLKPL

0302Additional Prime Editor Domains

0303[0101] In various embodiments, the prime editors provided in the present disclosure may comprise one or more nuclear localization sequences (NLS), which help promote translocation of the fusion protein into the cell nucleus. In some embodiments, the prime editors described herein may comprise one or more NLS. Such sequences are known in the art and can include the following examples:

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0306#14568046vl NLS Sequence SEQ ID NO PKKKRKV 40 MKRTADGSEFESPKKKRKV 41 MDSLLMNRRKFLYQFKNVRWAKGRRETYLC 42 AVKRPAATKKAGQAKKKKLD 43 MSRRRKANPTKLSENAKKLAKEVEN 44 PAAKRVKLD 45 KLKIKRPVK 46 VSRKRPRP 47 EGAPPAKRAR 48 PPQPKKKPLDGE 49 KRTADGSEFEPKKKRKV 50

0307<img file="WO2026106945A2_D0012.tif" />

KRTADGSEFESPKKKRKV 51

0309[0102] The NLS examples above are non-limiting. The prime editors provided herein may comprise any known NLS sequence, including any of those described in Cokol et al., “Finding nuclear localization signals,” EMBO Rep., 2000, 1(5): 411-415; and Freitas et al., “Mechanisms and Signals for the Nuclear Import of Proteins,” Current Genomics, 2009, 10(8): 550-7, each of which are incorporated herein by reference.

0310[0103] In various embodiments, the prime editors disclosed herein further comprise one or more, preferably at least two, nuclear localization sequences. In certain embodiments, the prime editors comprise at least two NLSs. In embodiments with at least two NLSs, the NLSs can be the same NLSs, or they can be different NLSs. In some embodiments, one or more of the NLSs are bipartite NLSs (“bpNLS”). In certain embodiments, the disclosed prime editors comprise two bipartite NLSs. In some embodiments, the disclosed prime editors comprise more than two bipartite NLSs. The location of the NLS fusion can be at the N-terminus, the C-terminus, or within a sequence of a prime editor.

0311[0104] In certain embodiments, a prime editor comprises an NLS of the amino acid sequence PKKKRKV (SEQ ID NO: 40). In certain embodiments, a prime editor comprises an NLS of the amino acid sequence MKRTADGSEFESPKKKRKV (SEQ ID NO: 41). In certain embodiments, a prime editor comprises an NLS of the amino acid sequence KRTADGSEFEPKKKRKV (SEQ ID NO: 50).

0312[0105] In some embodiments, a prime editor comprises one or more linkers. In some embodiments, a linker is a peptide linker. Exemplary peptide linkers for use in the prime editors contemplated by the present disclosure include, but are not limited to, (GGGGS)_n (SEQ ID NO: 52), (G)_n (SEQ ID NO: 78), (EAAAK)_n (SEQ ID NO: 53), (GGS)_n (SEQ ID NO: 79), (SGGS)n (SEQ ID NO: 54), (XP)_n (SEQ ID NO: 80), SGSETPGTSESATPES (SEQ ID NO: 55), SGSETPGTSESA (SEQ ID NO: 56), SGSETPGTSESATPEGGSGGS (SEQ ID 45/115

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0314#14568046vl NO: 57), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 58), SGGSGGSGGS (SEQ ID NO: 59), SGGS (SEQ ID NO: 60), SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSS GGS (SEQ ID NO: 61), GGSGGS (SEQ ID NO: 62), GGSGGSGGS (SEQ ID NO: 63), and SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 64), or any combination thereof, wherein each n is independently an integer between 1 and 30, and wherein X is any amino acid.

0315Prime Editors

0316[0106] The present disclosure contemplates the use of prime editors with the pegRNAs provided herein. A prime editor comprises at least a napDNAbp and a polymerase (e.g., DNA-dependent DNA polymerase or RNA-dependent DNA polymerase, such as a reverse transcriptase), optionally joined by a linker. The use of any suitable napDNAbp and polymerase (e.g., DNA-dependent DNA polymerase or RNA-dependent DNA polymerase, such as a reverse transcriptase) in a prime editor is contemplated. Examples of napDNAbps and reverse transcriptases are each provided herein.

0317[0107] In various embodiments, prime editors may comprise any suitable structural configuration. For example, the fusion protein may comprise from the N-terminus to the C-terminus direction, a napDNAbp (e.g., a Cas9 protein) fused to a polymerase (e.g., a reverse transcriptase). In other embodiments, the fusion protein may comprise from the N-terminus to the C-terminus direction, a reverse transcriptase fused to a napDNAbp. The fused domain may optionally be joined by a linker, e.g., an amino acid sequence. In other embodiments, the fusion proteins may comprise the structure NH2-[napDNAbp]-[polymerase]-COOH; or NH2-[polymerase]-[napDNAbp]-COOH, wherein each instance of “]-[” indicates the presence of an optional linker sequence. In embodiments wherein the polymerase is a reverse transcriptase (RT), the fusion proteins may comprise the structure NH2- [napDNAbp] -[RT]-COOH; or NH2-[RT]-[napDNAbp]-COOH, wherein each instance of “]-[” indicates the presence of an optional linker sequence. In some embodiments, a prime editor comprises one or more additional domains. In some embodiments, a prime editor comprises one or more NLS domains.

0318[0108] In some embodiments, prime editor contemplated for use in the present disclosure include prime editors comprising any of the following amino acid sequences, or comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at

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0321#14568046vl least 97%, at least 98%, or at least 99% identical to any of the following amino acid sequences:

0322Prime Description SEQ ID Editor NO PEI MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVIT 65

0323DEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLEN LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQK AQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDN VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAY LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI TGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSG GSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAW AETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGI KPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQD LREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDA FFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN SPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELD CQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLK EGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLW IPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLT

0324<img file="WO2026106945A2_D0013.tif" />

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0327#14568046vl AYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDR VQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDL TDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIW AKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYA FATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKR ESIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTST EEIENSSPSGGSKRTADGSEFEPKKKRKV _

0328PE2 MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVIT DEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLEN LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQK AQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDN VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAY LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI TGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSG GSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAW AETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGI KPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQD LREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDA FFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN SPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELD

0329<img file="WO2026106945A2_D0014.tif" />

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0332#14568046vl EGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFI PGFAEMAAPEYPETKPGTEFNWGPDQQKAYQEIKQAEET APAEGEPDETKPFEEFVDEKQGYAKGVETQKEGPWRRPV AYESKKEDPVAAGWPPCERMVAAIAVETKDAGKETMGQ PEVIEAPHAVEAEVKQPPDRWESNARMTHYQAEEEDTDR VQFGPVVAENPATEEPEPEEGEQHNCEDIEAEAHGTRPDE TDQPEPDADHTWYTDGSSEEQEGQRKAGAAVTTETEVIW AKAEPAGTSAQRAEEIAETQAEKMAEGKKENVYTDSRYA FATAHIHGEIYRRRGWETSEGKEIKNKDEIEAEEKAEFEPK RESIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTS TEEIENSSPSGGSKRTADGSEFEPKKKRKV PE3 PE2 prime editor sequence + second strand nicking gRNA

0333PE4 PE2 prime editor sequence + disruptor of DNA mismatch repair

0334pathway

0335PE5 PE2 prime editor sequence + disruptor of DNA mismatch repair

0336pathway + second strand nicking gRNA

0337PE6a MKRTADGSEFESPKKKRKV(SEQ ID NO: - see in 41)[CAS9]SGGSSGGSKRTADGSEFESPKKKRKVSGGSSGG sequence - SGRPYVTENENGMFMDKFKPYSKSNAPITTEEKESKAESIS VEEEKAIAEESEDEKYTEKKIPKIDGSKRIVYSEHPKMREE QSRINERIFKEEVVFPSFEFGSVPSKNDVENSNVKRDYVSC AKAHCGAKTVEKVDISNFFDNIHRDEVRSVFEEIEHIKDEA EDYEVDICTKDDFVVQGAETSSYIATECEFAVEGDVVRRA QRKGEVYTREVDDITVSSKISNYDFSQMQSHIERMESEHN EPINKHKTKIFHCSSEPIKVHGEIVDYDSPREPSDKVKRIRA SIHNEKEEAAKNNTKTSVAYRKEFNRCMGRVNEEGRVGH EKYESFKKQEQAIKPMPSNRDVAVIDAAIKSEEESYSKGN QNKHWYKRKYDETRYKMIIETRSESFKEKEECFKSREASE KPEKRTADGSEFESPKKKRKVPAAKRVKED(SEQ ID NO:

033867), wherein [CAS9] comprises any Cas9 protein

0339PE6b MKRTADGSEFESPKKKRKV(SEQ ID NO: - see in 41)[CAS9]SGGSSGGSKRTADGSEFESPKKKRKVSGGSSGG sequence - SISSSKHTESQMNKVSNIVKEPEEPDIYKEFKDITADTNTEK EPKPIKGEEFEVEETQENYREPIRNYPETPVKMQAMNDEIN QGEKGGIIRESKAINACPVIFVPRKEGTERMVVDYRPENKY VKPNVYPEPEIEQEEAKIQGSTIFTKEDEKSAYHQIRVRKG DEHKEAFRCPRGVFEYEVMPYGISTAPAHFQYFINTIEGEA KESHVVCYMDDIEIHSKSESEHVKHVKDVEQKEKNANEII NQAKCEFHQSQVKFIGYHISEKGETPCQENIDKVEQWKQP KNRKEERQFEGSVNYERKFIPKTSQETHPENKEEKKDVRW KWTPTQTQAIENIKQCEVSPPVERHFDFSKKIEEETDVSDV AVGAVESQKHDDDKYYPVGYYSAKMSKAQENYSVSDKE MEAIIKSEEHWRHYEESTIEPFKIETDHRNEIGRITNESEPEN KREARWQEFEQDFNFEINYRPGSANHIADAESRIVDETEPI PKDNEDNSINFVNQISIKRTADGSEFESPKKKRKVPAAKRV KED(SEQ ID NO: 68), wherein [CAS9] comprises any Cas9

0340protein

0341PE6c MKRTADGSEFESPKKKRKV(SEQ ID NO: - see in 41)[CAS9]SGGSSGGSKRTADGSEFESPKKKRKVSGGSSGG sequence -

0342<img file="WO2026106945A2_D0015.tif" />

SISSSKHTESQMNKVSNIVKEPEEPDIYKEFKDITADTNTEK

034449/115

B1195.70212WO00

0346#14568046vl LPKPIKGLEFEVELTQENYRLPIRNYPLTPVKMQAMNDEIN QGLKGGIIRESKAINACPVIFVPRKEGTLRMVVDYRPLNKY VKPNVYPLPLIEQLLAKIQGSTIFTKLDLKSAYHQIRVRKG DEHKLAFRCPRGVFEYLVMPYGIKTAPAHFQYFINTILGEA KESHVVCYMDDILIHSKSESEHVKHVKDVLQKLKNANLII NQAKCEFHQSQVKFLGYHISEKGLTPCQENIDKVLQWKQP KNQKELRQFLGQVNYLRKFIPKTSQLTHPLNKLLKKDVR WKWTPTQTQAIENIKQCLVSPPVLRHFDFSKKILLETDVSD VAVGAVLSQKHDDDKYYPVGYYSAKMSKAQLNYSVSDK EMLAIIKSLEHWRHYLESTIEPFKILTDHRNLIGRITNESEPE NKRLARWQLFLQDFNFEINYRPGSANHIADALSRIVDETEP IPKDNEDNSINFVNQISIKRTADGSEFESPKKKRKVPAAKR VKLD(SEQ ID NO: 69), wherein [CAS9] comprises any Cas9

0347protein

0348PE6d MKRTADGSEFESPKKKRKV(SEQ ID NO: - see in 41)[CAS9]SGGSSGGSKRTADGSEFESPKKKRKVSGGSSGG sequence - STLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGM GLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRL LDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKR VEDIHPNVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLH PTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFCEA LHRDLADFRIQHPDLILLQYYDDLLLAATSELDCQQGTRA LLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLT EARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMA APLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPD LTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHA VEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVA LNPATLLPLPEEGLQHNCLDKRTADGSEFESPKKKRKVPA AKRVKLD(SEQ ID NO: 70), wherein [CAS9] comprises any

0349Cas9 protein

0350PE6e MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVIT - see in DEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT sequence - RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLEN LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQK

0351<img file="WO2026106945A2_D0016.tif" />

AQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV

035350/115

B1195.70212WO00

0355#14568046vl MGRHKPENIVIEMARENQTTQKGQRNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDN VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIARQLVETRQITKHVAQILDSRMNTKYDENDK LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAY LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI TGLYETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKR KVSGGSSGGS(SEQ ID NO: 71)[REVERSE TRANSCRIPTASE]KRTADGSEFESPKKKRKVPAAKRVKL D(SEQ ID NO: 72), wherein [REVERSE TRANSCRIPTASE] comprises any reverse transcriptase

0356PE6f MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVIT - see in DEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT sequence - RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFRRLE ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLEN LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEKTITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MVEERLKTYAHLFDNKVMKQLKRRRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ KAQVSGQGDSLYEHIANLAGSPAIKKGILQTVKVVDELVK VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIK ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG GLSELDKAGFIARQLVETRQITKHVAQILDSRMNTKYDEN DKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNG ETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV

0357<img file="WO2026106945A2_D0017.tif" />

AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGY

035951/115

B1195.70212WO00

0361#14568046vl KEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYL DEIIEQISEFSKRVIEADANEDKVESAYNKHRDKPIREQAE NIIHEFTETNEGAPAAFKYFDTTIDRKRYTSTKEVEDATEIH QSITGEYETRIDESQEGGDSGGSSGGSKRTADGSEFESPKK KRKVSGGSSGGS(SEQ ID NO: 73)[REVERSE TRANSCRIPTASE]KRTADGSEFESPKKKRKVPAAKRVKE D(SEQ ID NO: 72), wherein [REVERSE TRANSCRIPTASE] comprises any reverse transcriptase

0362PE6g MKRTADGSEFESPKKKRKVDKKYSIGEDIGTNSVGWAVIT - see in DEYKVPSKKFKVEGNTDRHSIKKNEIGAEEFDSGETAEAT sequence - REKRTARRRYTRRKNRICYEQEIFSNEMAKVDDSFFRREE ESFEVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHERKKEV DSTDKADEREIYEAEAHMIKFRGHFEIEGDENPDNSDVDK EFIQEVQTYNQEFEENPINASGVDAKAIESARESKSRREEN EIAQEPGEKKNGEFGNEIAESEGETPNFKSNFDEAEDAKEQ ESKDTYDDDEDNEEAQIGDQYADEFEAAKNESDAIEESDI ERVNTEITKAPESASMIKRYDEHHQDETEEKAEVRQQEPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPIEEKMDGT EEEEVKENREDEERKQRTFDNGSIPHQIHEGEEHAIERRQE DFYPFEKDNREKIEKIETFRIPYYVGPEARGNSRFAWMTRK SEKTITPWNFEEVVDKGASAQSFIERMTNFDKNEPNEKVE PKHSEEYEYFTVYNEETKVKYVTEGMRKPAFESGEQKKAI VDEEFKTNRKVTVKQEKEDYFKKIECFDSVEISGVEDRFN ASEGTYHDEEKIIKDKDFEDNEENEDIEEDIVETETEFEDRE MVEEREKTYAHEFDNKVMKQEKRCRYTGWGRESRKEIN GIRDKQSGKTIEDFEKSDGFANRNFMQEIHDDSETFKEDIQ KAQVSGQGDSEYEHIANEAGSPAIKKGIEQTVKVVDEEVK VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIK EEGSQIEKEHPVENTQEQNEKEYEYYEQNGRDMYVDQEE DINRESDYDVDAIVPQSFEKDDSIDNKVETRSDKNRGKSD NVPSEEVVKKMKNYWRQEENAKEITQRKFDNETKAERG GESEEDKAGFIKRQEVETRQITKHVAQIEDSRMNTKYDEN DKEIREVKVITEKSKEVSDFRKDFQFYKVREINNYHHAHD AYENAVVGTAEIKKYPKEESEFVYGDYKVYDVRKMIAKS EQEIGKATAKYFFYSNIMNFFKTEITEANGEIRKRPEIETNG ETGEIVWDKGRDFATVRKVESMPQVNIVKKTEVQTGGFS KESIEPKRNSDKEIARKKDWDPKKYGGFDSPTVAYSVEVV AKVEKGKSKKEKSVKEEEGITIMERSSFEKNPIDFEEAKGY KEVKKDEIIKEPKYSEFEEENGRKRMEASAGEEQKGNEEA EPSKYVNFEYEASHYEKEKGSPEDNEQKQEFVEQHKHYE DEIIEQISEFSKRVIEADANEDKVESAYNKHRDKPIREQAE NIIHEFTETNEGAPAAFKYFDTTIDRKRYTSTKEVEDATEIH QSITGEYETRIDESQEGGDSGGSSGGSKRTADGSEFESPKK KRKVSGGSSGGS[REVERSE TRANSCRIPTASE]KRTADGSEFESPKKKRKVPAAKRVKE D(SEQ ID NO: 72), wherein [REVERSE TRANSCRIPTASE] comprises any reverse transcriptase

0363PEmax MKRTADGSEFESPKKKRKVDKKYSIGEDIGTNSVGWAVIT 75

0364<img file="WO2026106945A2_D0018.tif" />

DEYKVPSKKFKVEGNTDRHSIKKNEIGAEEFDSGETAEAT

036652/115

B1195.70212WO00

0368#14568046vl RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRKLEN LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQK AQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDN VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAY LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI TGLYETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKR KVSGGSSGGSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQ AWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEAR LGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPV QDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLK DAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGF KNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSE LDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYL LKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCR LFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQAL LTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRR PVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM GQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRP DLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEV IWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSR YAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFL

0369<img file="WO2026106945A2_D0019.tif" />

PKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPD 53/115

B1195.70212WO00

0372#14568046vl TSTLLIENSSPSGGSKRTADGSEFESPKKKRKVGSGPAAKR VKLD PE7 PEmax + the N-terminal domain RNA-binding domain of La: 76

0373MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVIT DEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRKLEN LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQK AQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDN VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAY LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI TGLYETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKR KVSGGSSGGSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQ AWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEAR LGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPV QDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLK DAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGF KNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSE LDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYL LKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCR LFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQAL LTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRR PVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM

0374<img file="WO2026106945A2_D0020.tif" />

GQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT 54/115

B1195.70212WO00

0377#14568046vl DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRP DLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEV IWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSR YAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFL PKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPD TSTLLIENSSPSGGSSGGSSGSETPGTSESATPESSGGSSGGS MAENGDNEKMAALEAKICHQIEYYFGDFNLPRDKFLKEQI KLDEGWVPLEIMIKFNRLNRLTTDFNVIVEALSKSKAELM EISEDKTKIRRSPSKPLPEVTDEYKNDVKNRSVYIKGFPTD ATLDDIKEWLEDKGQVLNIQMRRTLHKAFKGSIFVVFDSI ESAKKFVETPGQKYKETDLLILFKDDYFAKKNESGGSKRT

0378<img file="WO2026106945A2_D0021.tif" />

ADGSEFESPKKKRKVGSGPAAKRVKLD

0380[0109] In addition, any prime editor known in the art may be used with the pegRNAs of the present disclosure. Prime editors are described, for example, in: Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR. “Search-and-replace genome editing without double-strand breaks or donor DNA.” Nature. 2019 Dec;576(7785): 149-157. doi: 10.1038/s41586-019- 1711-4. Epub 2019 Oct 21. PMID: 31634902; PMCID: PMC6907074.; Anzalone AV, Koblan LW, Liu DR. “Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors.” Nat Biotechnol. 2020 Jul;38(7):824-844. doi: 10.1038/s41587-020-0561-9. Epub 2020 Jun 22. PMID: 32572269.; Doman JL, Sousa AA, Randolph PB, Chen PJ, Liu DR. “Designing and executing prime editing experiments in mammalian cells.” Nat Protoc. 2022

0381Nov; 17(11):2431-2468. doi: 10.1038/s41596-022-00724-4. Epub 2022 Aug 8. PMID:

038235941224; PMCID: PMC9799714.; Chen PJ, Liu DR. “Prime editing for precise and highly versatile genome manipulation.” Nat Rev Genet. 2023 Mar;24(3):161-177. doi:

038310.1038/S41576-022-00541-1. Epub 2022 Nov 7. PMID: 36344749; PMCID:

0384PMC10989687.; Miller SM, Wang T, Randolph PB, Arbab M, Shen MW, Huang TP, Matuszek Z, Newby GA, Rees HA, Liu DR. “Continuous evolution of SpCas9 variants compatible with non-G PAMs.” Nat Biotechnol. 2020 Apr;38(4):471-481. doi:

038510.1038/S41587-020-0412-8. Epub 2020 Feb 10. PMID: 32042170; PMCID: PMC7145744.; Hsu JY, Griinewald J, Szalay R, Shih J, Anzalone AV, Lam KC, Shen MW, Petri K, Liu DR, Joung JK, Pinello L. “PrimeDesign software for rapid and simplified design of prime editing guide RNAs.” Nat Commun. 2021 Feb 15;12(1): 1034. doi: 10.1038/s41467-021-21337-7. PMID: 33589617; PMCID: PMC7884779; and Chen PJ, Hussmann JA, Yan J, Knipping F, Ravisankar P, Chen PF, Chen C, Nelson JW, Newby GA, Sahin M, Osborn MJ, Weissman JS, Adamson B, Liu DR. “Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.” Cell. 2021 Oct 28;184(22):5635-5652.e29. doi:

038655/115

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0388#14568046vl 10.1016/j.cell.2021.09.018. Epub 2021 Oct 14. PMID: 34653350; PMCID: PMC8584034, each of which is incorporated by reference.

0389[0110] In addition, any of the prime editors and/or other prime editing components described in the following references may be used with the pegRNAs provided herein. The contents of the following references are incorporated by reference.

0390WO 2023/015309 A2 IMPROVED PRIME EDITORS AND METHODS OF USE WO 2025/064678 A2 PRIME EDITING-MEDIATED READTHROUGH OF FRAMESHIFT MUTATIONS (PERF)

0391WO 2024/155741 Al PRIME EDITING-MEDIATED READTHROUGH OF PREMATURE TERMINATION CODONS (PERT)

0392WO 2021/226558 Al METHODS AND COMPOSITIONS FOR SIMULTANEOUS EDITING OF BOTH STRANDS OF A TARGET DOUBLESTRANDED NUCLEOTIDE SEQUENCE WO 2023/076898 Al METHODS AND COMPOSITIONS FOR EDITING A GENOME WITH PRIME EDITING AND A RECOMBINASE WO 2025/096936 A2 USE OF PRIME EDITING IN CORRECTING MUTATIONS IN CDKL5

0393WO 2023/205687 Al IMPROVED PRIME EDITING METHODS AND COMPOSITIONS WO 2024/168147 A2 EVOLVED RECOMBINASES FOR EDITING A GENOME IN COMBINATION WITH PRIME EDITING WO 2022/150790 A2 PRIME EDITOR VARIANTS, CONSTRUCTS, AND METHODS FOR ENHANCING PRIME EDITING EFFICIENCY AND PRECISION WO 2024/243415 Al EVOLVED AND ENGINEERED PRIME EDITORS WITH IMPROVED EDITING EFFICIENCY WO 2020/191153 A2 METHODS AND COMPOSITIONS FOR EDITING NUCLEOTIDE SEQUENCES WO 2020/191171 Al METHODS AND COMPOSITIONS FOR EDITING NUCLEOTIDE SEQUENCES WO 2020/191242 Al METHODS AND COMPOSITIONS FOR EDITING NUCLEOTIDE SEQUENCES WO 2024/206125 Al USE OF PRIME EDITING FOR TREATING SICKLE CELL DISEASE WO 2024/108092 Al PRIME EDITOR DELIVERY BY AAV

0394WO 2020/191245 Al METHODS AND COMPOSITIONS FOR EDITING NUCLEOTIDE SEQUENCES WO 2021/072328 Al METHODS AND COMPOSITIONS FOR PRIME EDITING RNA WO 2023/102538 Al SELF-ASSEMBLING VIRUS-LIKE PARTICLES FOR DELIVERY OF PRIME EDITORS AND METHODS OF MAKING AND USING SAME WO 2024/077267 Al PRIME EDITING METHODS AND COMPOSITIONS FOR TREATING TRIPLET REPEAT DISORDERS WO 2023/102550 A2 COMPOSITIONS AND METHODS FOR EFFICIENT IN VIVO DELIVERY

039556/115

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0397#14568046vl WO 2020/191243 Al METHODS AND COMPOSITIONS FOR EDITING NUCLEOTIDE SEQUENCES WO 2020/191241 Al METHODS AND COMPOSITIONS FOR EDITING NUCLEOTIDE SEQUENCES WO 2020/191249 Al METHODS AND COMPOSITIONS FOR EDITING NUCLEOTIDE SEQUENCES WO 2020/191246 Al METHODS AND COMPOSITIONS FOR EDITING NUCLEOTIDE SEQUENCES WO 2020/191248 Al METHOD AND COMPOSITIONS FOR EDITING NUCLEOTIDE SEQUENCES WO 2020/191234 Al METHODS AND COMPOSITIONS FOR EDITING NUCLEOTIDE SEQUENCES WO 2020/191239 Al METHODS AND COMPOSITIONS FOR EDITING NUCLEOTIDE SEQUENCES WO 2022/212926 Al METHODS AND COMPOSITIONS FOR EDITING NUCLEOTIDE SEQUENCES WO 2022/256714 A2 GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF WILSON'S DISEASE WO 2023/288332 A2 GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF WILSON'S DISEASE WO 2023/004439 A2 GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF CHRONIC GRANULOMATOUS DISEASE WO 2023/015318 A2 GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF CYSTIC FIBROSIS WO 2023/015014 Al GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF MYOTONIC DYSTROPHY WO 2023/028180 A2 GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF RETINOPATHY WO 2023/070062 A2 GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF USHER SYNDROME TYPE 3

0398WO 2023/070110 A2 GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF RETINITIS PIGMENTOSA WO 2023/081787 A2 GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF FANCONI ANEMIA WO 2023/081426 Al GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF FRIEDREICH'S ATAXIA

0399WO 2023/086389 Al GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF AMYOTROPHIC LATERAL SCLEROSIS

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0402#14568046vl WO 2023/086842 Al GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF FUCHS ENDOTHELIAL CORNEAL DYSTROPHY WO 2023/086558 Al GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF FRAGILE X SYNDROME WO 2023/096847 A2 METHODS AND COMPOSITIONS FOR INHIBITING MISMATCH REPAIR WO 2023/192655 A2 METHODS AND COMPOSITIONS FOR EDITING NUCLEOTIDE SEQUENCES WO 2024/119098 Al LIPID NANOPARTICLE (LNP) DELIVERY SYSTEMS AND FORMULATIONS WO 2024/148313 A2 GENOME EDITING COMPOSITIONS AND METHODS OF USE WO 2024/163679 Al GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF CYSTIC FIBROSIS WO 2024/163680 A2 GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF CYSTIC FIBROSIS WO 2024/168116 Al GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF MYOTONIC DYSTROPHY WO 2024/178144 Al METHODS AND COMPOSITIONS FOR EDITING NUCLEOTIDE SEQUENCES WO 2024/220807 A2 LIPID NANOPARTICLE (LNP) DELIVERY SYSTEMS AND FORMULATIONS WO 2024/233655 Al GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF FRAGILE X SYNDROME WO 2024/259228 Al CYCLIC PEPTIDE LIPIDS FOR NANOPARTICLE FORMULATIONS WO 2025/090524 Al METHODS FOR DETECTING SINGLE

0403STRANDED BREAK SITES IN GENOMIC DNA INDUCED BY NICKING ENDONUCLEASES WO 2025/090637 A2 GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF RETINITIS PIGMENTOSA WO 2025/111430 Al CHEMICAL MODIFICATIONS IN mRNA

POLY(A) TAIL

0405WO 2025/111452 A2 CHEMICAL MODIFICATIONS IN PEgRNA and ngRNAs

0406Methods of Use

0407[0111] Some aspects of the present disclosure provide methods of using the pegRNAs provided herein. In one aspect, the present disclosure provides methods for prime editing comprising contacting a target nucleic acid molecule with a prime editor and any of the pegRNAs provided herein. In some embodiments, the methods described herein are 58/115

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0409#14568046vl performed in vitro. In some embodiments, the methods described herein are performed in vivo. In some embodiments, the methods described herein are performed ex vivo. In certain embodiments, the method is performed in a subject. A subject may have been diagnosed with a disease or disorder, or be at risk for having a disease or disorder.

0410[0112] In some embodiments, the target sequence comprises a sequence associated with a disease or disorder. In some embodiments, the target sequence comprises one or more mutations associated with a disease or disorder. In some embodiments, editing the target nucleic acid results in correction of the one or more mutations (e.g., by prime editing). In some embodiments, the target sequence encodes a protein, and the one or more mutations are in codons that result in a change in the amino acids encoded by the mutant codons as compared to the wild-type codons. In some embodiments, editing of the mutant codons results in a change in the amino acids encoded by the mutant codons. In some embodiments, editing of the mutant codons results in codons encoding the wild-type amino acids. In some embodiments, the prime editor and pegRNA are used to replace a sequence associated with a disease or disorder with a sequence that is not associated with a disease or disorder.

0411[0113] The present disclosure provides methods for the treatment of a subject diagnosed with a disease associated with or caused by a point mutation that can be corrected by a prime editor and pegRNA provided herein. The methods disclosed herein may be used to treat any disease by prime editing, including, for example, any disease as described in International PCT Application Publication No. WO 2020/191239, published September 24, 2020, which is incorporated herein by reference. For example, in some embodiments, a method is provided that comprises administering to a subject having such a disease an effective amount of a prime editor and a pegRNA that forms a complex with the prime editor, that corrects one or more mutations or introduces a deactivating mutation into a disease-associated gene. In some embodiments, a method is provided that comprises administering to a subject having such a disease, an effective amount of a prime editor-pegRNA complex that corrects one or more mutations or introduces a deactivating mutation into a disease-associated gene. Further provided herein are methods comprising administering to a subject one or more polynucleotides or vectors that contain a nucleotide sequence that expresses the prime editor and a pegRNA disclosed herein that forms a complex with the prime editor.

0412[0114] In some aspects, the present disclosure contemplates use of any of the pegRNAs, systems, polynucleotides, vectors, cells, compositions, and kits disclosed herein in the manufacture of a medicament for the treatment of a disease or disorder. In some aspects, any

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0415#14568046vl of the pegRNAs, systems, polynucleotides, vectors, cells, compositions, and kits disclosed herein may be used in medicine.

0416Delivery Methods

0417[0115] The present disclosure provides, in some aspects, methods comprising delivering any of the pegRNAs, systems, polynucleotides, vectors, and compositions described herein, e.g., to a cell. The pegRNAs, systems, polynucleotides, vectors, and compositions can be delivered in any form, e.g., each may independently be delivered in DNA, RNA, or (for the prime editor portion) protein form. Conventional viral and non- viral based gene transfer methods can be used to introduce nucleic acids to cells (e.g., mammalian cells) or target tissues. Such methods can be used to administer nucleic acids encoding components of a pegRNA and/or prime editor to cells in culture, or in a host organism. Non- viral vector delivery systems include ribonucleoprotein (RNP) complexes, DNA plasmids, RNA, naked nucleic acid, and nucleic acid complexed with, part of, or associated with a delivery vehicle, such as a liposome, lipid nanoparticle (LNP), or virus-like particle (VLP).

0418[0116] A VLP (or “engineered virus-like particle (eVLP),” which is used interchangeably with the term “VLP” herein) consists of a supra-molecular assembly comprising: (a) an envelope comprising (i) a lipid membrane (e.g., single-layer or bi-layer membrane), and a (ii) viral envelope glycoprotein; and (b) a multi-protein core region comprising (ii) a Gag protein, (ii) a first fusion protein comprising a Gag protein and Pro-Pol, and (iii) a second fusion protein comprising a Gag protein fused to a cargo via a protease-cleavable linker. In some embodiments, the gag protein comprises a nucleocapsid protein variant as described herein. In various embodiments, the cargo is pegRNA-prime editor ribonucleoprotein complex. In various embodiments, the VLPs are prepared in a producer cell that is transiently transformed with plasmid DNA that encodes the various protein and nucleic acid (pegRNA) components of the VLPs. The components self-assemble at the cell membrane and bud out in accordance with the naturally occurring mechanism of retroviral budding in order to release from the cell fully-matured VLPs. Once formed, the Pol-Pro cleaves the protease-sensitive linker joining the Gag-cargo linker (e.g., the linker joining a Gag to a prime editor complexed with a pegRNA) to release the cargo within the VLP. Once the VLP is administered to a recipient target cell and taken up by said target cell, the contents of the VLP are released, including cargo (e.g., a prime editor complexed with a pegRNA). Once in the cell, the cargo may translocate to the nuclease of the cell (in particular, where NLSs are associated with the cargo), where DNA editing may occur at target sites specified by the pegRNA.

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0421#14568046vl [0117] In some embodiments, a VLP comprises additional agents for targeting the VLP for delivery to particular cell types. For example, such additional targeting agents may be incorporated into the outer lipid membrane encapsulation layer of the VLP. In some embodiments, the additional targeting agent is a protein. In certain embodiments, the additional targeting agent is an antibody or fragment thereof. In certain embodiments, the additional targeting is a ligand (e.g., a receptor ligand). In certain embodiments, the additional targeting agent is a receptor or a fragment thereof. In certain embodiments, the additional targeting agent is an aptamer or a fragment thereof.

0422[0118] VLPs are described further, for example, in International PCT Publication Nos. WO 2023/102537, WO 2023/102538, WO 2023/102550, WO 2024/215652, and WO 2024/254346, each of which is incorporated herein by reference. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel &amp; Feigner, TIBTECH 11:211-217 (1993); Mitani &amp; Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer &amp; Perricaudet, British Medical Bulletin 51 (1 ):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bihm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

0423[0119] In some embodiments, a prime editor and a pegRNA are delivered or administered as a proteimRNA complex. In certain embodiments, the method of delivery comprises delivering an RNP complex. For example, RNP delivery of gene editors markedly increases the DNA specificity of gene editing and leads to fewer off-target effects. RNP delivery can also ablate off-target editing at non-repetitive sites while maintaining on-target editing comparable to plasmid delivery, and greatly reduce off-target editing. See Rees, H. A. et al., “Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery.” Nat. Commun. 8, 15790 (2017), which is incorporated herein by reference.

0424[0120] Additional methods of non- viral delivery include RNP complexes, lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in, e.g., U. S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are sold commercially (e.g., Lipofectamine®, Lipofectamine® 2000, Lipofectamine® 3000, TransfectamTM, and 61/115

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0426#14568046vl LipofectinTM). In certain embodiments of the disclosed methods of editing, a cationic lipid comprising Lipofectamine® 2000 is used for delivery of nucleic acids to cells. Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner (see International Patent Application Publication Nos. WO 1991/17424 and WO 1991/16024, each of which is incorporated herein by reference).

0427Delivery of, e.g., Cas9 proteins and gRNAs using cationic lipids and cationic polymers is also described in International Patent Application Publication Nos. WO 2015/035136, published March 12, 2015, and WO 2016/070129, published May 6, 2016, each of which is incorporated herein by reference. Delivery can be to cells e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).

0428[0121] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao e

0429<img file="WO2026106945A2_D0022.tif" />

0430t al., Gene Therapy (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U. S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,946,787, 9,526,784, and 9,737,604).

0431[0122] The use of RNA or DNA viral based systems for the delivery of nucleic acids (e.g., nucleic acids encoding a pegRNA and prime editor as described herein) take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo), or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated, and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

0432[0123] In some embodiments, a viral particle-based system is used for delivery of nucleic acid molecule(s) encoding a pegRNA and prime editor. In some embodiments, the viral particle-based system is an adeno-associated virus (AAV)-based system. Particularly in applications where transient expression is preferred, adenoviral-based systems may be used. Adenoviral-based vectors are capable of very high transduction efficiency in many different cell types and do not require cell division. With such vectors, high titer and levels of 62/115

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0434#14568046vl expression have been obtained. This vector can be produced in large quantities in a relatively simple system. AAV vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West etal., Virology 160:38-47 (1987); U. S. Pat. No.

04354,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors is described in a number of publications, including U. S. Pat. No. 5,173,414; Tratschin etal., Mol. Cell. Biol.

04365:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &amp; Muzyczka, PNAS 81:6466-6470 (1984); Samulski et al., J. Virol. 63:03822-3828 (1989); and International Patent Application No. PCT/US2023/066389, filed April 28, 2023.

0437[0124] Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and q/2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.

0438[0125] In various embodiments, the constructs for expressing a pegRNA and prime editor described herein may be engineered for delivery in one or more AAV vectors. An AAV as related to any of the methods and compositions provided herein may be of any serotype including any derivative or pseudotype e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 2/1, 2/5, 2/8, 2/9, 3/1, 3/5, 3/8, or 3/9).

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0441#14568046vl Compositions

0442[0126] Other aspects of the present disclosure relate to compositions comprising any of the pegRNAs, systems, polynucleotides, and/or vector described herein. In some embodiments, the composition is a pharmaceutical composition. The term “pharmaceutical composition,” as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g., for specific delivery, increasing half-life, or other therapeutic compounds).

0443[0127] As used here, the term “pharmaceutically-acceptable carrier” (or “pharmaceutically acceptable excipient”) 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.). Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, and antioxidants can also be 64/115

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0445#14568046vl present in the formulation. Terms such as “excipient,” “carrier,” “pharmaceutically acceptable carrier,” “pharmaceutically acceptable excipient,” or the like are used interchangeably herein.

0446[0128] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject for prime editing.

0447[0129] The pharmaceutical compositions described herein may be administered or packaged as a unit dose, for example. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

0448[0130] In some embodiments, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierce-able by a hypodermic injection needle. The active agent in the composition is a compound of the invention. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer’s solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

0449Polynucleotides, Vectors, Cells, and Kits

0450[0131] The present disclosure provides, in some aspects, polynucleotides and vectors encoding any of the pegRNAs, systems, or pegRNA-prime editor complexes described herein. In some embodiments, a polynucleotide encodes a pegRNA described herein. In some embodiments, a vector (e.g., a plasmid) comprises such a polynucleotide. In some embodiments, one or more polynucleotides encodes a pegRNA described herein and a prime editor. In some embodiments, one or more vectors comprise one or more such polynucleotides. In some embodiments, the polynucleotides and vectors provided herein 65/115

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0452#14568046vl comprise DNA (e.g., plasmid DNA or viral DNA). In some embodiments, the polynucleotides and vectors provided herein comprise RNA e.g., mRNA or viral RNA).

0453[0132] Cells that may contain any of the pegRNAs, systems, polynucleotides, and/or vectors described herein are also provided by the present disclosure. In the methods described herein, a pegRNA and prime editor (or one or more polynucleotides encoding the same) may be delivered into a eukaryotic cell (e.g., a mammalian cell, such as a human cell). In some embodiments, the cell is in vitro (e.g., a cultured cell). In some embodiments, the cell is in vivo (e.g., in a subject, such as a human subject). In some embodiments, the cell is ex vivo (e.g., isolated from a subject and may be administered back to the same or a different subject).

0454[0133] In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. In some embodiments, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with the components of a prime editing system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a prime editing complex, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence.

0455[0134] The pegRNAs, systems, polynucleotides, vectors, and/or cells described herein may also be assembled into kits. In some embodiments, the kit comprises polynucleotides for expression of the pegRNAs described herein, and optionally a prime editor. In some embodiments, the kit comprises appropriate pegRNAs as described herein, or nucleic acid vectors for the expression of such pegRNAs, to target a prime editor provided herein to a desired target sequence, e.g., a sequence comprising one or more mutations associated with a disease or disorder.

0456[0135] The kits described herein may include one or more containers housing components for performing the methods described herein, and optionally instructions for use. Any of the kits described herein may further comprise components needed for performing the methods described herein. Each component of the kits, where applicable, may be provided in liquid form (e.g., in solution) or in solid form, (e.g., a dry powder). In certain cases, some of the components may be reconstitutable or otherwise processible (e.g., to an active form), for 66/115

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0458#14568046vl example, by the addition of a suitable solvent or other species (for example, water), which may or may not be provided with the kit.

0459[0136] In some embodiments, the kits may optionally include instructions for use of the components provided. As used herein, “instructions” can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which can also reflect approval by the agency of manufacture, use, or sale for animal administration. Additionally, the kits may include other components depending on the specific application, as described herein.

0460[0137] The kits may contain any one or more of the components described herein in one or more containers. The components may be prepared sterilely, packaged in a syringe, and shipped refrigerated. Alternatively, they may be housed in a vial or other container for storage. A second container may have other components prepared sterilely. Alternatively, the kits may include the active agents premixed and shipped in a vial, tube, or other container.

0461[0138] The kits may have a variety of forms, such as a blister pouch, a shrink-wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box, or a bag. The kits may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kits may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, and/or a support for the agents prior to administration.

EXAMPLES

0463Example 1. Directed evolution of small RNA-stabilizing motifs for improved prime editing [0139] Prime editing is a powerful genome editing technology that enables the precise installation of targeted DNA changes including base substitutions, small insertions, and deletions without requiring double- stranded breaks or exogenous donor DNA¹. Prime editing 67/115

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0465#14568046vl uses a reverse transcriptase (RT) and a programmable nickase such as Cas9 nickase, guided by a prime editing guide RNA (pegRNA) that specifies both the target locus and the desired edit. Upon Cas9 binding and nicking, the pegRNA’s primer binding site (PBS) anneals to the nicked DNA strand, priming reverse transcription of an RT template (RTT) and incorporating the desired edit directly into the genome (FIG. 1A)^2-4. The versatility and precision of prime editing have led to its broad application for basic science^5-9, agriculture^10-14, and therapeutics^15-25. For example, prime editing to correct a 2-bp deletion in NCF1 resulted in successful restoration of NADPH oxidase activity in the hematopoietic system to treat patients with chronic granulomatous disease (CGD)²⁶. Most applications of prime editing benefit from systems with increased efficiency that could open new applications, reduce dosing for clinical use, and increase desired signals in basic science studies.

0466[0140] Improvements have been made to various components of the prime editing platform. These efforts include the use of protein engineering and continuous evolution campaigns to identify Cas9 variants and reverse transcriptase orthologs that support more efficient prime editing²⁷; genetic screens that have identified cellular determinants of prime editing outcomes, leading to strategies for enhancing prime editing efficiency through mismatch repair evasion^28-30 or other endogenous factors³¹; and machine learning-based tools that guide pegRNA design across many variables including PBS length, RTT length, and the strategic co-installation of silent mutations^32-34. The design of engineered pegRNAs (epegRNAs) that include a structured pseudoknot motif positioned 3' of the PBS to shield the pegRNA from exonucleolytic degradation³⁵ have also been reported, and similar protective designs have been described^36,37. However, while the protein and RNA components of the prime editing system have been systematically improved via evolution or large-scale screening efforts, the structured motifs within epegRNAs have remained underexplored by comparison, with the vast majority of current prime editing efforts using the motif first reported in 2021³⁵. Despite their demonstrated potential to substantially improve editing efficiency, only a modest number of structured RNA motifs have been functionally evaluated in prime editing studies to date^35-37.

0467[0141] The most common motif in epegRNAs for current prime editing workflows is a trimmed and engineered variant of the preQ1 riboswitch aptamer pseudoknot (tevopreQi)^35,38. Although its inclusion modestly increases pegRNA length by 37 nucleotides, this protective motif can substantially enhance prime editing efficiencies and is compatible with U6 promoter-driven expression systems such as those in plasmids and AAV vectors. Nevertheless, the limited commercial availability of long synthetic pegRNAs containing 68/115

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0469#14568046vl tevopreQi remains a factor due to current challenges in chemical synthesis^39,40. Alternative semi- synthetic approaches have been proposed to bypass these limitations⁴¹. In addition, fusion of the prime editor to the N-terminal domain of La, termed PE7, has been shown to stabilize pegRNAs lacking structural motifs by binding to the poly-U tail appended by Pol III during transcription²⁹. However, the increased size of this fusion protein renders it incompatible with current dual- AAV delivery architectures⁴² and the added protein component increases the complexity of prime editing systems. Mining a broader diversity of structured 3' motifs from naturally occurring RNAs could reveal elements capable of protecting epegRNA structure and supporting efficient prime editing without increasing the size of the prime editor protein or epegRNA constructs (FIG. IB).

0470[0142] A systematic evaluation and iterative optimization of structured 3 ' RNA motifs could broaden the capabilities of prime editing in several ways. First, because structured motifs can influence pegRNA secondary structure in an edit- dependent manner^35,43, a diverse set of high-performance motifs would facilitate screening designs that minimize adventitious unproductive RNA folding. Second, the identification of smaller, more compact motifs would improve the synthetic accessibility of epegRNAs, particularly for RNA- or RNP-based delivery platforms including lipid-nanoparticle (LNP) formulations, since shorter RNA oligomers are synthesized with higher average yield and purity⁴⁴. Third, the availability of sequence-orthogonal motifs would support twin- and multiplexed-prime editing applications by allowing simultaneous use of multiple epegRNAs while reducing risks of recombination or template switching in viral vectors^45-47. Finally, structured motifs — especially those derived from diverse natural RNAs — could confer additional benefits, such as enhanced transcript expression⁴⁸, improved Cas9-binding or target site engagement⁴⁹, and altered subcellular localization⁵⁰. Motivated by these opportunities, a platform for the discovery and optimization of epegRNA structured 3' motifs was developed from a broad search much larger than the process we used to identify tevopreQi³⁵.

0471[0143] The present disclosure reports the design of a pooled prime editing reporter system, PE-PRISM (prime editing pooled reporter for identification of structured motifs), which enabled high-throughput discovery of RNA motifs that enhanced editing efficiency. Using this platform, 2,858 total RNA motif candidates derived from diverse natural and synthetic origins were assessed and several candidates that supported robust prime editing were identified. To generate smaller motifs with improved functionality, variants of these candidates were created and tested through structure-informed mutational scanning approaches, testing motif variants with up to four mutations from wild-type sequences. The 69/115

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0473#14568046vl broad applicability of these engineered 3' motifs was demonstrated in the prime editing-mediated correction of 847 pathogenic mutations in the ClinVar⁵¹ database. The resulting data revealed three refined motifs that consistently outperformed tevopreQi. The applicability of these 3 ' motifs was further demonstrated across different cell types and delivery modalities, including engineered virus-like particles (eVLPs) and lipid nanoparticle (LNP) settings in vivo.

0474Results

0475Design of PE pooled reporter for identification of structured motifs (PE-PRISM)

0476[0144] A diverse library of structured RNA motif sequences was curated from both natural and synthetic origins. Pseudoknots, compact and highly structured RNA elements, play roles in cellular processes such as programmed ribosomal frameshifting, telomere maintenance, and metabolite sensing via riboswitches⁵². Given the prior success of the tevopreQi pseudoknot, pseudoknot sequences⁵³ derived from a variety of organisms and functional RNA contexts were selected, including viral 5' or 3' UTRs, frameshifting signals, and tRNA-like structures (FIG. 1C). In addition to pseudoknots, G-quadruplexes, a structurally privileged class of functional RNA motifs⁵⁴, were also included. The library comprised both naturally occurring RNA quadruplexes with variable architectures, as well as synthetic designs of the form (G≥N1)4, (G≥N2)4, or (G≥N3)4 in which three-guanine tracts were separated by loop sequences of 1-3 nucleotides^55-57.

0477[0145] Structural RNA features resembling native RT priming or initiation motifs may similarly improve prime editing outcomes in an RT-dependent fashion. To test this possibility, RNA substructures (hairpins, loops, and helices) derived from naturally occurring sequences associated with priming or initiation for the reverse transcriptases were selected from the Ec48 retron⁵⁸ (PE6a), M-MLV⁵⁹ (PEmax and PE6d), and Tfl retrotransposon⁶⁰ (PE6c; FIG. 6A). Finally, tevopreQi was diversified to generate variants with all possible single nucleotide substitutions.

0478[0146] Sequences were selected for naturally occurring RNA pseudoknots (N=189), G-quadruplexes (N=67), RT recruitment or initiation motifs (N=39), and variants of tevopreQi (N=143), as well as previously reported sequences as controls (N=22), for a total curated list of 460 structured sequences for assessment in prime editing.

0479[0147] To rapidly test the functional activities of the selected RNA motif sequences, a pooled lentiviral library of self-targeting elements was designed in which each construct contained an epegRNA bearing a unique 3' motif adjacent to its corresponding target site for prime 70/115

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0481#14568046vl editing (FIG. ID). This design was inspired by recent pooled pegRNA screens used to inform machine learning models for pegRNA optimization^9,33’³⁴. Although alternative strategies were considered — such as phage-assisted continuous evolution^27,61 or enrichment-based PE reporter selections^29,62-64 — a screening format that evaluated motif performance across diverse edit types and within the context of human cells was prioritized. Applicability across a broad range of prime edits and target sites was essential given the strong edit-dependence of prime editing outcomes and the likely influence of cellular processes such as RNA expression and degradation³⁵.

0482[0148] For the vl PE-PRISM library, 460 curated RNA motifs were incorporated into nine previously reported pegRNAs^27,28 targeting a representative set of edit types, including transitions, transversions, and a small deletion. The nine epegRNAs with the standard tevopreQi 3' motif spanned a wide range of reported editing efficiencies from 3% to 55%. Each epegRNA-motif combination was paired with its synthetic target site and a unique barcode. This pool of epegRNAs was then cloned into a lenti viral backbone containing a hU6 promoter to drive expression of the epegRNA as well as an EFlalpha promoter driving expression of a puromycin-T2A-BFP selection cassette. HEK293T cells were transduced with the epegRNA library at low multiplicity-of-infection (MOI &lt; 0.3) and enriched for transduced cells under puromycin selection for six days. To profile motif activity across prime editor variants, cells were transfected with expression plasmids encoding PEmax, PE6a-g, or PE7, genomic DNA was harvested 96 hours post-transfection, and high-throughput sequencing (HTS) of the integrated cassettes was performed to link each candidate 3' motif with its editing outcome for all epegRNAs (FIGs. ID and IE). Editing outcomes were quantified by aligning reads to the full reference amplicon and applying quality and depth thresholds (FIG. 6B, see Methods). After filtering, the vl PE-PRISM dataset comprised editing data for 457 motifs across nine target protospacers and nine prime editor constructs, with two biological replicates per condition.

0483PE-PRISM identifies motifs that support efficient editing comparable to tevopreQi [0149] To compare motifs across different prime editors and target protospacers in order to nominate the best generalist motif candidates for further validation and development, an aggregate score of motif performance was developed. The correlation between biological replicates and prime editors was assessed under the assayed conditions (average Pearson R = 0.77) (FIGs. 6C and 6D), and analysis of editing outcomes revealed variable PE efficiencies depending on the editor variant (11-22% average editing) and target protospacer (3.0-39%

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0486#14568046vl average editing, FIGs. 6E and 6F). For each unique combination of editor and target protospacer, z-scores were assigned to each motif. The average z-score was calculated as a composite metric across replicates, editors, and target protospacers. Finally, overall library representation of each motif was accounted for by counting the number of total z-scores assigned to each motif as a measure of library representation to account for post-filtering element dropout (FIG. IF, left). Negative controls included no motif, random sequence, or a variant of tevopreQi containing a single point mutation that disrupted pseudoknot folding (“dead”)³⁵. Among the top scoring motifs with the highest average normalized PE efficiencies (FIG. IF, right), a set of novel pseudoknot motifs was identified with performance comparable to that of tevopreQi (avg. z = 0.85). These pseudoknots included sequences derived from the human signal recognition particle (HSRP, avg. z = 0.94), wild cucumber mosaic virus (WCMV, avg. z = 0.86), human parechovirus 1 (HPeVl, avg. z = 0.75), soil-borne rye mosaic virus (SBRMV1, avg. z = 0.67), and hepatitis A virus (HAV, avg. z = 0.60), among many other identified candidates (FIG. 1G). Across other broad categories of structured RNAs evaluated in the vl PE- PRISM screen, designed G-quadruplexes and RT recruitment and initiation motifs did not generally result in improved PE outcomes compared to pseudoknots (FIGs. 6G-6I). Substitutions did not improve tevopreQi (FIG. 6J), suggesting that additional mutation types may be required to escape tevopreQi ’s local optimum in sequence space. Together, these data demonstrated that the design and application of the PE- PRISM approach rapidly nominated 3' structured motifs that supported efficient prime editing from a broad initial pool of candidate sequences.

0487[0150] To cross- validate the nominated motifs from the pooled lentiviral screen, a set of 16 motif pseudoknots was selected for further characterization. EpegRNA constructs were delivered by arrayed plasmid transfection in HEK293T cells, targeting six endogenous loci rather than synthetic lentiviral cassettes, and their performance was assayed with PEmax, PE6c, and PE7 three days post-transfection. Pooled self-targeting lentivirus and arrayed plasmid transfection editing outcomes were moderately correlated (r = 0.39, 0.64, and 0.53 for PEmax, PE6c, and PE7, respectively; FIG. 7A). Higher efficiencies following lentiviral delivery were attributed to more persistent epegRNA expression, higher local epegRNA concentration proximal to the adjacent target site, and preferential lentiviral integration at open chromatin⁶⁵.

0488[0151] Quantification of editing at endogenous sites revealed similar trends in PE efficiency to those observed in the pooled lentiviral context (FIGs. 7B and 7C). For treatment with PEmax or PE6c, it was found that the new pseudoknot candidates performed comparably to 72/115

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0490#14568046vl tevopreQi (FIGs. 7D and 7E). Interestingly, for PE7, although absolute editing levels remained similar to those of PEmax and PE6c, the structured motifs showed little benefit over the unstructured dead control (FIG. 7F), a result consistent with previous observations²⁹, as well as FIG. 1H and FIG. 7G. This result suggested there is no additional cooperative benefit between highly structured 3' RNA sequences and La- mediated stabilization of terminal poly-U tracts, where steric occlusion disrupted La binding to the 2' hydroxyl groups of polyuridylated termini, as previously demonstrated²⁹. Collectively, these experiments demonstrated that naturally occurring pseudoknots as 3 ' epegRNA motifs supported efficient prime editing of endogenous genomic targets by plasmid transfection despite diverse motif evolutionary origins and natural functions.

0491Targeted mutagenesis of pseudoknots improved PE outcomes

0492[0152] Having identified a diverse collection of natural RNA structural motifs that supported robust prime editing, their performance was further improved by introducing targeted mutations to sample local regions of RNA sequence space near these wild-type pseudoknots. Based on predicted secondary structural modeling⁵³, unpaired leader and trailing sequences were removed that were not anticipated to participate in pseudoknot folding. Into the resulting trimmed sequences, targeted single point deletions, substitutions, and insertions were introduced in unpaired loop regions, as well as paired double deletions, substitutions, and insertions in base-paired regions, prioritizing the preservation of pseudoknot structure to yield more active variants (FIG. 2A, left). This structure-guided targeted mutagenesis approach was applied to a set of 13 pseudoknots that included the top-scoring candidates nominated by the vl screen less than 40 nt in length. From these 13 starting points a collection of 2,044 sequence variants was generated, including 13 wild- type positive controls and 12 negative controls (FIG. 2A, right).

0493[0153] As before, these motifs were appended combinatorially to pegRNAs specifying five diverse prime edits, the resulting epegRNAs were paired with their cognate synthetic target sites in a v2 PE-PRISM lentiviral library, HEK293T cells were transduced with the resulting pool of 10,220 library members, and prime editor plasmid was delivered by transfection after six days of puromycin selection. After sequencing depth and quality control filtering, the v2 dataset included measurements of PE efficiencies for 2,043 motif variants in combination with five self-targeting edits (averaging 4.1% to 34% editing efficiency) by PEmax, PE6c, and PE7 (average 7.7%, 7.5%, and 11% editing, respectively) collected in two biological replicates (Pearson r = 0.97-0.98 between replicates; FIGs. 8A and 8B).

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0496#14568046vl [0154] To globally compare the activities of engineered variants, as described above, raw editing values were normalized for each combination of editor and target protospacer and for each biological replicate. The average z-score was calculated across all instances of each motif’s occurrence in the dataset (FIG. 2B). Among rank-ordered library elements, negative controls including random sequence and dead motifs yielded poor editing (rank > 1,507 out of 2,043, avg. z < -0.28), while the standard tevopreQi continued to support robust PE (rank 228, avg. z = 0.48). Within each set of motif variants that originated from a given starting point, 10 out of 14 families included a top variant that on average, outperformed tevopreQi (0.67 < avg. z < 1.20; FIG. 2B, right). Closer examination of the PE efficiencies associated with these top variants in comparison to those of tevopreQi revealed robust improvements in editing across the five target protospacers, with up to 2.4-fold improvement compared to tevopreQi (FIG. 8C). These findings indicated that specific pseudoknot variants supported substantially more efficient prime editing than the standard tevopreQi epegRNA motif used by the field.

0497[0155] A subset of v2 library motif variants were selected for testing in arrayed format, both to evaluate individual self-targeting elements delivered by lentiviral transduction, which closely resembled the pooled screening conditions, and to assay their activity at the endogenous protospacer target by plasmid transfection. A strong correlation between different prime editors and assay formats was observed (Pearson r = 0.98 - 0.99 between arrayed vs pooled for all data sets; FIG. 8D). Importantly, top-performing variants consistently supported PE efficiencies similar to or exceeding those of tevopreQi across the five edits (up to 1.5-fold average improved efficiency; FIG. 8E). Taken together, these findings demonstrated that the structure-based mutagenesis and screening approach yielded engineered pseudoknot variants with improved activities over their naturally occurring precursors (PEmax P = 0.0004; FIG. 2C).

0498[0156] The relationship between specific nucleotide changes and their functional impact on editing efficiencies was assessed, both in the context of a top-scoring motif variant and globally across the v2 PE-PRISM dataset. A reversion analysis of the top-performing motif variant was conducted across all families of novel pseudoknots, a variant of the FMDVA-2 pseudoknot containing a paired double nucleotide insertion (avg. z = 1.00). Variants that trim unpaired nucleotides and that insert alternative paired bases were investigated (FIG. 2D). Removal of one or two trailing nucleotides, which were not anticipated to engage in secondary structure⁵³, did not reduce activity (avg. z = 0.18 and 0.32, respectively) relative to the wild-type starting point (avg. z = 0.07); indeed, minimal perturbation was observed from 74/115

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0500#14568046vl unpaired base truncation across all trimmed library elements relative to their corresponding full-length sequences (FIG. 8F). Within this family of truncated variants, it was observed that the highest activity mutations were paired double nucleotide insertions at position 5, with a slight preference for the U: G wobble base pair (avg. z = 1.00) over A: U (avg. z = 0.91), as well as other alternative insertions or their complements (FIG. 2D). These findings established that the platform described herein can readily identify the effects of single- and double-nucleotide variants of structured motifs on prime editing efficiency, supporting the application of the v2 targeted mutagenesis workflow.

0501[0157] To assess the global features of the RNA sequence landscape among all mutagenized pseudoknots — including less-active motifs — changes in the AU versus GC nucleotide content of variant sequences were categorized and compared to their parental sequences, and the resulting average change in performance across the mutants in the library was quantified (FIG. 2E). Mutations that resulted in no net change in either AU or GC counts were detrimental to motif performance (avg. z = -0.14), and indeed most other combinations of nucleotide changes resulted in similarly detrimental or worse outcomes, with notable exceptions for (i) a net single insertion of a G or C ribonucleotide (avg. Az = -0.01) and (ii) a double substitution of an AU pair with a GC pair (avg. Az = -0.06). That a majority of variants underperformed relative to their progenitors — even among structure-based mutations that aimed to preserve both stem-loop substructures of the starting pseudoknots — underscored the complexity of the pseudoknot mutational landscape, suggesting that higher-order structural determinants play a critical role in the context of efficient PE. Together, these observations highlighted the importance of iterated high-throughput parallel screening with PE-PRISM in identifying successful RNA motif sequences.

0502Combinations of beneficial mutations further enhanced pseudoknot performance

0503[0158] Having demonstrated that pseudoknots are amenable to iterated screening and mutation via PE-PRISM, the present example contemplates whether combining mutations from multiple high-activity variants could further enhance prime editing efficiency. If high-performance motif variants improved prime editing by thermodynamically stabilizing the 3' pseudoknot, it was reasoned that combining several pseudoknot- stabilizing mutations might further increase epegRNA stability and editing efficiency. A set of top scoring variants were selected for further combinatorial screening and new candidate sequences were generated containing pairwise combinations of mutations derived from tevopreQi (N=130), SBRMV1 (N=92), HPeVl (N=84), FMDVA-2 (N=73), and HAV (N=38). Five motif variants and 75/115

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0505#14568046vl combinations from other lineages were selected to carry forward, despite their lower activities, to avoid unnecessary bottlenecking of sequence diversity, including Ec, FMDVO-3, FMDVO-4, PhyMV, TMEV, and WCMV. A v3 PE-PRISM library was assembled with 13 different prime edits (comprising 5,889 pegRNAs in total) and motif performances with PEmax, PE6c, or PE7 were measured (FIGs. 2F and 2G, FIG. 9A). Comparing the activities of top motifs for each of the five major motif families, it was observed that combinations of mutations conferred modest improvement over v2 variants (paired /-test, one-tailed P = 0.0098; FIG. 2H). Reversion analysis of the top scoring tevopreQi derivative (tevo2.0), which combined a double substitution (hplO UA> CG) and double insertion (hp2 UA), revealed an additive beneficial impact of combining mutations, as each mutation in isolation (avg. z = 0.17 and 0.50, respectively) underperformed relative to the combination (avg. z = 0.67; FIG. 9B). These observations suggest that beneficial effects of pseudoknot in some cases can be combined to boost PE outcomes beyond improvements from mutations in isolation.

0506[0159] Based on the enhanced performance of motifs in the v3 PE-PRISM pooled library, the improved activities were validated over tevopreQi in independent experiments. Eight motifs were selected for advancement into final evaluation studies on the combined basis of (i) aggregate normalized editing efficiencies, (ii) consistency of improvement across target protospacers in the v3 library, and (iii) diversity of precursor sequences. For each combination of prime editor, target edit, and replicate, motifs were ranked by editing efficiency, and the frequency of motif rankings was calculated. Motif performance was tested both by plasmid transfection (FIG. 21) and by lentiviral transduction (FIG. 2J), between which moderate correlation for raw editing values was observed (average Pearson r = 0.82 across replicates; FIGs. 9C-9E). It was found that tevopreQi was the best motif in plasmid transfection and lentiviral transduction only 6.3% (4/63) and 13% (9/72) of the time, respectively, with other engineered pseudoknot motifs outperforming tevopreQi in the remaining majority of cases. In particular, the combination mutant tevo2.0 demonstrated the highest performance across both experimental contexts, supporting the most efficient (rank 1) PE efficiencies both at synthetic and endogenous sites in 29% (18/63) and 28% (20/72) of cases, respectively, offering a substantial improvement over tevopreQi (FIGs. 21 and 2J). Across seven edits at endogenous genomic targets in HEK293T cells, a 1.2-fold average PE efficiency improvement was observed over tevopreQi for top v3 motifs eFMDVA-2.2 and eFMDVO-4 (FIG. 9F). In summary, these data indicate that engineered pseudoknot variants bearing combinations of beneficial mutations can further enhance PE activity, demonstrating 76/115

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0508#14568046vl how the iterative process of diversification and screening using the PE-PRISM platform can enable the directed evolution of RNA pseudoknots to identify variants with synergistic combinations of beneficial mutations.

0509New motifs improved PE efficiencies across cell lines and primary human cell types [0160] With a promising set of engineered v3 pseudoknot variants in hand, their potential improvements for prime editing across an array of target cell types and delivery modalities in cell culture was evaluated. To systematically explore potential cell line- dependent differences between motif activities, including due to differential expression of RNases or small RNA stabilization factors across cell types (FIG. 10A), 12 pegRNA-stabilizing motifs were tested with eight target protospacers, transduced human or mouse HEK293T, HeLa, K562, U2OS, or N2a cells, and editing with PEmax, PE6c, or PE7 editors was assessed. Normalizing raw editing values to account for differences in editor plasmid delivery efficiencies, a comparable correlation was observed between different editors for a given cell type (Pearson r = 0.58 -0.89) as between the same editor across cell types (Pearson r = 0.63 - 0.84; FIG. 10B).

0510Consistent trends in performance were observed at each of the eight assayed PE target protospacers between cell lines (FIG. 3A), where top v3 motif tevo2.0 (avg. z = 0.72) showed improved activity over tevopreQi (avg. z = 0.12). These data also revealed that at least one new motif surpassed tevopreQi at a majority of edits for each cell context (FIG. 10C). To identify the best performing motifs across all tested contexts, the frequency distributions of motif rankings were analyzed, which revealed similar trends in relative performance between cell lines that broadly favored the mutant tevo2.0 over tevopreQi among the top 3 motifs in the majority of cases (for tevo2.0 vs tevopreQi: 81% vs 46% in HEK293T, 81% vs 35% in HeLa, 71% vs 33% in U2OS, and 100% vs 35% in N2a cells; FIG. 3B). These data demonstrate that improvements to the PE system with engineered pseudoknots translated across a variety of different cell lines.

0511[0161] Next, the compatibility of 3' structural motifs was investigated with additional delivery modalities. All previous experiments — which had relied on U6-driven constitutive lentiviral integrants or plasmid transfection — produced relatively high intracellular epegRNA concentrations. To complement these data, contexts were sought in which editing reagents, due to the limited lifetimes of RNA and protein components, would be subjected to much more stringent kinetic requirements for efficient PE, which required many more bondforming and bond-breaking events than base editing or nuclease editing. In contrast to adeno-associated virus (AAV) or lipid nanoparticle (LNP) modalities that conventionally deliver 77/115

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0513#14568046vl DNA- or RNA-based payloads, respectively, engineered virus-like particles (eVLPs)⁶⁶ support efficient and transient delivery of ribonucleoprotein (RNP) complexes, including prime editors^67,68. To test the activity of the set of six leading pseudoknots under these highly limiting conditions alongside tevopreQi and negative control motifs, the v3b PE-eVLP architecture was used, which included a Com RNA aptamer in the ST2 loop of the scaffold to aid packaging to incorporate these motifs into epegRNAs targeting four highly optimized edits in human HEK293T and mouse N2a cells. PE-eVLPs in Gesicle 293T cells were produced, target cells were treated with increasing doses of v3b PE-eVLPs, and editing was quantified at each target locus. It was found that the top-performing engineered variants offered substantial improvement in editing over tevopreQi at Dnmtl (33% with tevo2.0, 12% with tevopreQi, 2.8-fold improvement), Coll2al (3.1% with eFMDVO-4, 2.2% with tevopreQi, 1.4-fold improvement), n HEK3 (9.4% with tevo2.0, 6.9% with tevopreQi, 1.4-fold improvement) and similar efficiencies at FANCF (1.5% with tevo2.0, 2.0% with tevopreQi) at the lowest tested dose (approximately 1.3 x 10⁸ eVLPs uL ¹; see Methods), with similar trends observed upon dose escalation (FIG. 3C, FIG. 11A). These data indicate that transient delivery formats based on prime editing RNPs, which lack persistent epegRNA expression, may benefit more substantially from enhanced structured motifs.

0514[0162] These eVLP-delivered PE outcomes were compared with those from low-dose plasmid transfection ( 1750^th of standard treatment) targeting the same four edits, and improvements in average prime editing efficiency were observed at Dnmtl (51% with eHAV, 46% with tevopreQi), Coll2al (33% with eFMDVO-4, 26% with tevopreQi), HEK3 (14% with eFMDVO-4, 12% with tevopreQi), and FANCF (34% with HAV, 29% with tevopreQi), representing a 1.2-fold average performance increase over previously optimized PE conditions (FIG. 3D, FIG. 11B). Despite good correlation between eVLP transduction and low-dose plasmid transfection for each edit (Pearson r = 0.78 - 0.93, FIG. 11C), the optimal motif choice varied across delivery formats, an outcome that could be due to additional packaging, trafficking, and structural components (by inclusion of the Com aptamer in eVLPs). Taken together, these results showed that novel pseudoknot motifs can enhance prime editing when transiently delivered as components of RNP prime editing complexes by PE-eVLPs.

0515[0163] Motif performance was assessed for therapeutically relevant PE correction strategies — extensively optimized in prior studies — for att site installation⁴⁷, sickle cell disease¹⁶, cystic fibrosis¹⁷, and alternating hemiplegia of childhood¹⁵ in human primary hematopoietic stem and progenitor cells (HSPCs), immortalized bronchial epithelial cells 78/115

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0517#14568046vl (16HBEge), and mouse primary fibroblasts. Among the most highly optimized edits to date, twin prime installation⁴⁵ of attP at the AAVS1 safe harbor locus was improved from 84% with tevopreQi to 91% with eSBRMVl-A (FIG. 11D)⁴⁷. Electroporation of in vitro transcribed PEmax mRNA and chemically synthesized guide RNA components incorporating a panel of motif candidates resulted in substantial editing improvements at the HBB locus in CD34+ HSPCs (23% with eSBRMVl-A, 12% with tevopreQi; FIG. 3E, FIG. HE), CFTR in 16HBEge cells (67% with eSBRMVl-A, 61% with tevopreQi; FIG. 3F, left) and Atpla3 in mouse dermal fibroblasts (86% with HAV, 82% with tevopreQi, FIG. HF) where baseline efficiencies were already quite high. Larger improvements (5.7% with tevopreQi, 15.3% with eSBRMVl-A, 2.7-fold improvement) were observed when prime editing was performed with RNP complexes at the CFTR locus delivered by lipofection into a model HEK293T cell line (FIG. 3F, right)⁶⁹. Notably, these results suggested that, even for editing strategies extensively optimized around the use of tevopreQi epegRNAs, these alternative 3 ' motifs can further elevate editing efficiencies beyond previous local maxima independent of other pegRNA parameters such as RTT length, PBS length, and silent edits. Collectively, these experiments demonstrated that these 3' motif variants boost prime editing efficiencies over tevopreQi across DNA, RNA, and RNP-based delivery into specialized cell types, including primary cells, when correcting clinically relevant pathogenic alleles.

0518High-throughput evaluation of motifs for correction of 847 pathogenic mutations [0164] Pseudoknot candidates were assessed to determine which would provide the most robust improvements in PE outcomes across a broad range of target edits, with an additional goal of identifying to what extent other pegRNA features may synergize with — or oppose — motif effects. Given the highly sequence- dependent nature of RNA secondary structure and its impact on prime editing⁴³, a v4 PE- PRISM library was designed with 19 top motifs and parent sequences targeting the reversion of 1,000 clinically relevant mutations from ClinVar, which had been previously validated in self-targeting PE screens³⁴ (FIG. 4A). These diverse edits targeted the full range of possible transformations encodable with prime editing, including all 12 possible base substitutions, as well as small insertions and deletions (FIG.

05194B). HEK293T cells were transduced with a lentiviral pool encoding this 19,000 element v4 library, prime editor was delivered by plasmid transfection, and editing was quantified using the established workflow. Normalized editing efficiencies were calculated on a per-target site basis (FIG. 4C), accounting for variable editing outcomes across the library (FIGs. 12A and 12B), and the rank order distribution of motifs was determined (FIG. 4D) at 847 therapeutic 79/115

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0521#14568046vl targets after filtering, where it was required that three (out of n=4) biological replicates surpassed minimum read and editing thresholds. While all negative controls performed poorly as expected (avg. z = -1.40 to -1.12 for no motif, scrambled, or dead tevopreQi), three top-performing motifs (eHAV, tevo2.0, and HAV; FIG. 4E) for editing with PEmax emerged consistently above average (z = 0.49, 0.49, and 0.64, respectively) and were frequently ranked among the top three motifs across target sites (n = 284, 288, and 336 times) in contrast to worse performance by tevopreQi (z = -0.20 and n = 79 among top three). Interestingly, despite the similar origin and proximity in RNA sequence space (differing by four nucleotides) of the motifs HAV and eHAV, their preferred usage cases were largely complementary sets of target edits (FIG. 4F), confirming that different 3 ' motifs were optimal for different prime edits. These top three motifs were also found to be the most beneficial for PE6c and PE7 (FIG. 4G, FIGs. 12C, and 12D). These data confirmed the superior average performance of the new 3 ' motifs compared to tevopreQi for the correction of many disease-associated mutations.

0522[0165] These outcomes were validated using a panel of eight therapeutically relevant prime edits by arrayed lenti viral transduction with self-targeting epegRNAs incorporating 12 motifs, including tevopreQi and negative controls (array vs. pooled library Pearson r = 0.85, FIG. 12E). Improvements ranging from 1.2- to 1.6-fold were observed with the best alternative motif over tevopreQi for correction of pathogenic variants in NLRP3, WAC, FBN1, APC, PYGL, FGF23, ATRX, and KMT2a (FIG. 12F). Mutations in these genes are linked to cyroprin-associated periodic syndrome, Desanto-Shinawi syndrome, Marfan syndrome, Familial adenomatous polyposis, glycogen storage disease, hypophosphatemia, ATRX syndrome, and Wiedermann-Steiner syndrome, respectively. In summary, these results illustrated the broad utility of novel pseudoknots in enhancing prime editing correction of therapeutic targets. Since no single motif was superior to all others across all tested sites, three motifs — eHAV, tevo2.0, and HAV — can be tested in standard prime editing optimization workflows.

0523Motifs improved in vivo PE using eVLP and LNP-based delivery

0524[0166] In vivo studies provide a more challenging context for precision genome editing by introducing additional bottlenecks associated with targeting, delivery, and release of therapeutic cargos, as well as modality-dependent editor lifetime and durability of expression⁷⁰ (FIG. 5A). Therefore, the performance of these motifs was assessed in the context of PE-eVLPs in vivo. Based on the improvement of tevo2.0 over tevopreQi that had 80/115

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0526#14568046vl been previously observed for in vitro editing at Dnmtl in cultured N2a cells, large-scale batches of these v3b PE-eVLPs were produced for in vivo injection experiments, eVLP titers were normalized based on MMLV p30 protein levels⁶⁷, and batch performance was validated in vitro in N2a cells (FIGs. 13A and 13B).

0527[0167] When C57BL/6J mice were treated with eVLPs delivered by P0 ICV administration (5 x 10¹⁰ eVLPs per mouse), tevo2.0 yielded a 2.6-fold improvement in Dnmtl +2 GTCG> CAAC prime editing efficiency over tevopreQi (bulk brain cortex editing efficiencies of 0.89% with tevopreQi and 2.28% with tevo2.0) (FIG. 5B). Among highly transduced nuclei marked by a co-delivered lentiviral eGFP: KASH vector, a modest increase from 37% to 43% for tevopreQi and tevo2.0 was observed, respectively, an outcome consistent with more highly transduced cells being less likely to be limited by epegRNA levels (FIG. 13C).

0528These observations suggest that under stringent kinetic regimes in vivo in which epegRNA lifetime and activity are bottlenecks for prime editing efficiency, the new engineered and evolved 3' motifs substantially improved outcomes for systems that express epegRNAs only transiently.

0529[0168] Among methods for delivering RNA-based cargos, lipid nanoparticles (LNPs) have emerged as a powerful and clinically validated approach for delivering genome editing agents into target cells, animals, and human patients⁷¹. LNPs offer several key advantages over viral scaffold-based modalities, including scalable production of entirely synthetic components^72,73; non- viral transient expression with reduced potential for off-target editing or integration events⁷⁴; and reduced immunogenicity that supports repeat dosing for enhanced editing efficiencies^75-77. To assess the potential benefit of these compact, structured motifs on prime editing with LNPs (PE-LNPs), a recently developed in-house LNP formulation relying on an OF-02 ionizable cationic lipid⁷⁸ was utilized to deliver an admixture of prime editor mRNA, epegRNA, and ngRNA components at an optimized ratio (see Methods) targeting prime edits at mouse Pcsk9 and Dnmtl loci in Hepal-6 cells (FIG. 13D) and in adult mouse liver administered as a single dose by retro-orbital (RO) injection (1.7 mg/kg, FIG. 5C).

0530[0169] While improvement was observed in cultured cells with the best engineered variants over tevopreQi (up to 1.1-fold and 2.0-fold improvements at Pcsk9 and Dnmtl, respectively), these differences were substantially magnified in vivo, in which the top performing motifs offered 2.1-fold and 4.6-fold improvements for Pcsk9 and Dnmtl, respectively (18% with eSBRMVl-A and 8.5% with tevopreQi for Pcsk9', 0.89% with eHAV and 0.19% with tevopreQi for Dnmll). Good correlation was also observed between in vitro and in vivo data (Pearson r = 0.79, P &lt; 0.0001; FIG. 13E), suggesting that in vitro evaluation may be 81/115

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0532#14568046vl sufficient for identifying optimal epegRNA structured motifs in an LNP context. In summary, these experiments demonstrated that the second-generation epegRNA 3 ' motifs supported more efficient prime editing, especially under pegRNA-limited conditions in cultured cells and in animals, such as when using preferred transient in vivo delivery modalities.

0533[0170] Overall, the pooled prime editing screens described herein were designed and applied to nominate wild-type structured motif candidates, RNA sequences were evolved through iterative mutagenesis and rescreening, and their performance in correcting 847pathogenic alleles was assessed. The evaluation of 2,858 structured RNA motifs resulted in the identification of a set of highly active pseudoknot variants that consistently outperformed previous state-of-the-art tevopreQi, improving prime editing across cell types and delivery modalities in cultured cells and in vivo.

0534[0171] In practice, these data demonstrated that testing three of the top-performing variants — eHAV, tevo2.0, and HAV — was sufficient to identify a pseudoknot motif that outperforms tevopreQi in the vast majority of cases. If the PE application requires the most efficient editing outcomes possible, six motif variants (eHAV, tevo2.0, HAV, eSBRMVl-A, FMDVII-A, and tevopreQi) can be tested to sample distinct regions of RNA sequence space, prioritized by likelihood of improvement (see FIG. 4D).

0535[0172] The recommended set of RNA motifs improved average editing outcomes without increasing the length of epegRNAs compared to tevopreQi and therefore will not complicate chemical synthesis or compromise compatibility with size-limited dual AAV architectures. PE7 constructs, which contributed an additional 600 bp, typically did not substantially increase PE efficiencies when using the new structured pseudoknot motifs across multiple independent experiments (FIGs. 6E, 7C, 8B, 9A, 12A). It was speculated that structured RNAs sterically antagonized La binding, in agreement with prior studies²⁹. Regardless, pseudoknot- or La-mediated stabilization seemed to offer complementary strategies for enhancing prime editing, and their adoption will likely be used case-specifically.

0536Methods

0537General molecular biology and cloning

0538[0173] Primers were ordered from Integrated DNA Technologies®. All plasmids, including self-targeting lentiviral elements, PE-PRISM libraries, epegRNA expression plasmids, and eVLP plasmids, were cloned by isothermal assembly using NEBuilder® HiFi DNA Assembly Master Mix (New England BioLabs®, E2621) with DNA inserts from Integrated DNA Technologies® (eBlocks or gBlocks) or Twist Bioscience® (gene fragments without adapters)

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0541#14568046vl and backbones amplified by PCR with Q5® Hot Start High-Fidelity 2X Master Mix (New England BioLabs®, M0494), unless otherwise indicated. For arrayed lentiviral transfer plasmids for self-targeting lentiviral elements, assemblies using PCR-amplified backbone from pSEP308 plasmid (PMID: QQQQ PERT) were transformed into New England BioLabs® 5-alpha competent cells (New England BioLabs®, C2987). For epegRNA plasmids for transfection, assemblies using PCR-amplified backbone from pU6-pegRNA-GG-acceptor plasmid (Addgene®, 132777) were transformed into One Shot® MaehlTM cells (Invitrogen®, C862003). Plasmids were purified by Qiagen® Plasmid Plus 96 Miniprep Kits (16181), Qiagen® Plasmid Plus Midi Kits (12943), or Qiagen® Plasmid Plus Maxi Kits (12963).

0542General mammalian cell culture conditions

0543[0174] HEK293T (ATCC CRL-3216), N2a (CCL-131), HeLa (CCL-2), K562 (CCL-243), and U2OS (HTB-96) cell lines were purchased from ATCC®. Gesicle 293T cells were purchased from Takara® (632617). HEK293T, Gesicle 293T, HeLa, and N2a cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAX® (Gibco®, 10569044) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco®, 16000044). K562 and U2OS cells were cultured in RPMI 1640 medium with GlutaMAX® (Gibco®, 61870127) and McCoy’s 5A (Modified) medium (Gibco®, 16600082), respectively, supplemented with 10% (v/v) FBS (Gibco®, 16000044). G-CSF-mobilized human CD34+ HSPCs from unidentified healthy adult donors (Fred Hutchinson Research Center) were cultured in X-VIVO-10 (Lonza®, 04-380Q) medium supplemented with 100 ng pL ¹ human SCF (Peprotech, 300-07), lOOng pL ¹ human TPO (Peprotech, 300-18) and 100 ng pL ¹ human Fit- 3 ligand (Peprotech, 300-19) at a density of 1-2 x 10⁶ cells mL as previously described¹⁶. Primary mouse fibroblasts were isolated from tail dissections and cultured in DMEM with GlutaMAX® (Gibco®, 10569044) supplemented with 20% (v/v) FBS (Gibco®, 16000044) and IX penicillin- streptomycin (Gibco®, 15070063), as previously described¹⁵. Immortalized bronchial epithelial 16HBEge-F508del cells (16HBE14o- cells homozygous for F508del and M470; gifts from the Cystic Fibrosis Foundation Therapeutics Lab) were cultured in minimum essential medium (Gibco®, 11095-072) supplemented with 10% (v/v) FBS (Gibco®, 16000044) and 1% penicillin- streptomycin (Gibco®, 15070063); tissue culture dishes were pre-treated at 37 °C 5% CO2 for 2 hours with LHC-8 basal medium (Gibco®, 12678017) supplemented with 1.34 pL 111L ¹ of 7.5% (v/v) bovine serum albumin (BSA; Gibco®, 15260037), 10 pL 111L ¹ bovine collagen solution type 1 (Advanced BioMatrix, 5005-100ML), and 10 pL 111L ¹ fibronectin from human plasma (Thermo Fisher 83/115

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0545#14568046vl Scientific®, 33016015), as previously described¹⁷. All cell lines tested negative for mycoplasma and identity-authenticated by their suppliers. All cell lines were cultured at 37 °C with 5% CO₂.

0546Design ofvl screen

0547[0175] Pseudoknot sequences were extracted from PseudoBase⁵³. G-quadruplexes were selected from previously reported sequences or designed according to the pattern (63X1)4, (63X2)4, or (63X3)4, in which three-guanine tracts are separated by randomized loop sequences of 1-3 nt^55-57. Reverse transcriptase-specific motifs were manually designed from subsets of secondary structures (stem-loop and hairpin motifs) from the murine proline tRXA⁵⁹, the msDXA RXA-DXA hybrid of the Ec48 retron, and the self-priming region of the Tfl mRXA⁶⁰. Finally, all possible single nucleotide variants of tevopreQi were designed and included in the vl library. Xext, motifs were filtered to (1) exclude sequences with 6C content exceeding the range 20-80%, (2) enforce a maximum pseudoknot length of 50 nt, and (3) remove elements with oligo-U tracts > 4 nt that would terminate RXA polymerase III transcription. Including five random controls, final selected motif designs (n=460) were paired combinatorially with pegRXAs specifying 9 previously developed prime edits^27,28 and one non-targeting control pegRXA (targeting mouse DnmtD) to arrive at a list of 4,600 epegRXAs. These assembled epegRXAs were paired with an adjacent synthetic target site (43-44 nucleotides in length); the negative control was paired with a scrambled target site. Each epegRXA-target site element was assigned a unique 10 nt barcode, which was split into 5 nt halves that flanked the synthetic target site that immediately followed the U6 terminator. The length of library elements was standardized using randomized variable length linkers with balanced GC content and excluded homopolymers, such that inclusion of homology arms (24 nt each) resulting in a uniform library length of 300 nt.

0548Design ofv2 screen

0549[0176] Motif variants were generated from 13 top vl sequences and tevopreQi as follows. Based on the expected secondary structure (e.g., dot-bracket annotation included in the PseudoBase reference⁵³), unpaired nucleotides were iteratively trimmed from both ends to generate shortened sequences (n=61) with minimally disrupted secondary structures. Xext, from each maximally trimmed sequence or tevopreQi as the starting point, targeted mutagenesis was performed to maintain secondary structure as follows: (1) double mutations in paired regions (n=562), (2) double deletions in paired regions (n=99), (3) double insertions 84/115

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0551#14568046vl in paired regions (n=792), (4) single deletions in unpaired regions (n=85), (5) single substitutions in unpaired regions (n=366), and (6) double deletions in unpaired regions (n=54). Pairing interactions in generated sequences included canonical Watson-Crick and the G»U wobble base pair. Along with 11 negative controls (dead or random variants) and 14 positive controls (unmodified vl sequences), the final list of 2,044 motif variants was paired with five top pegRNAs from the vl screen (targeting EXMI, PAH, ADA, SCN1A, and FBNE) to generate 10,220 assembled epegRNAs. Randomized, GC-balanced linkers were installed between the epegRNA and reverse-complemented synthetic target site (45 nt) to result in a uniform length of 246 nt, after which a 12 nt barcode was added. The assembled insert was flanked by 21 nt homology arms for Gibson assembly.

0552Design ofv3 screen

0553[0177] Top variants from the v2 dataset from motif families for Ec (n=3), FMDVA-2 (n=ll), FMDVO-3 (n=3), FMDVO-4 (n=3), HAV1 (n=8), and HPeVl (n=15), PhyMV (n=3), SBRMV1 (n=16), tevopreQi (n=20), TMEV (n=3), and WCMV (n=3) were included as positive controls (n=88). Within each family, all possible pairwise combinations of mutations present in the selected variants were generated (n=314 total across all families).

0554Combinations were also generated from three of the top variants for FMDVA-2 (n=13), HAV (n=3), HPeVl (n=4), SBRMV1 (n=9), tevopreQi (n=16). Including dead and random negative controls (n=9), the final list of motifs (n=453) was paired with 13 prime edits from previous screens or reports⁸¹ (to generate 5,889 epegRNAs). Each epegRNA was assigned a unique 20 nt barcode, which was split into 10 nt halves that flanked a 46 nt reverse-complemented synthetic target site. Balancing linkers were introduced between the epegRNA and 66 nt barcode-flanked synthetic target site to have uniform insert length of 258 nt, such that 21 nt cloning homology arms brought the full library length to 300 nt.

0555Design ofv4 screen

0556[0178] From the v3 library, 19 motifs including n=9 top motif sequences were advanced, n=7 parental sequences were advanced from the vl library, and n=3 controls (dead, scrambled, none). From Eibrary ClinVar³⁴, n=976 edits were randomly selected after applying these filters: (1) exclusion of oligo-U tracts of length > 4 nt, (2) maximum RTT length of 28 nucleotides, (3) maximum edit length of 1 nt (for insertions/deletions), and (4) minimum 5% reported published editing efficiency. Edits from previous screens (n=14) and negative control edits using the S. auri scaffold sequence (n=10) were also included. As before,

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0559#14568046vl epegRNAs (n=19,000) were combinatorially generated incorporating these edits and motifs, a variable-length GC-balanced linker was appended, and a reverse complemented target site (49 nt) was flanked by two halves of a unique 16 nt barcode. Flanking 23 nt homology arms resulted in a uniform library of 300 nt.

0560General library cloning protocol

0561[0179] All high-throughput screening libraries were ordered as single- stranded oligo pools from Twist Bioscience® and amplified to prepare a double- stranded library using primers 0HSOO8I and OHS0082 according to recommended protocols using Q5® Hot-Start High-Fidelity 2X Master Mix (New England BioLabs®, M0494) under the following conditions: initial denature at 98 °C for 30 seconds; 11-13 cycles of (98 °C for 10 seconds, 68 °C for 30 seconds, and 72 °C for 20 seconds). Amplified inserts were purified on MinElute® PCR Purification Kit (Qiagen® 28004). Isothermal assembly was performed with NEBuilder® HiFi DNA Assembly Master Mix (New England BioLabs® E2621) using amplified doublestranded inserts (12 ng) and DpnLtreated (New England BioLabs®, R0176) PCR-amplified lentiviral backbone (50 ng; 10-fold molar excess) with incubation at 50 °C for 1 hour.

0562Assemblies were purified using QIAquick® PCR purification kit (Qiagen® 28104) into a minimum volume of ddH2O, electroporated into New England BioLabs® 10-beta Electrocompetent E. coli (New England BioLabs®, C3020) according to manufacturer’s instructions and grown on carbenicillin- supplemented (50 pg 111L ¹) LB agar plates at lOOOx coverage, based on serial dilution plating. After 12-16 hours, transfer plasmids were isolated from scraped colonies with the Qiagen® Plasmid Plus Maxi Kit (12963).

0563Lentivirus packaging and transduction in cell culture

0564[0180] For production of lentivirus for self-targeting epegRNA constructs, HEK293T cells were seeded in six-well plates (Coming®, 0720083) at a density of 9 x 10⁵ cells per well. At 16 hours after seeding, cells were transfected with Lipofectamine® 2000 (12 pL;

0565Invitrogen®, 11668019) following the manufacturer’s protocol to deliver transfer plasmid (1,333 ng), pCMV-dR8.2 dvpr (1,000 ng; Addgene®, 8455), and pMD2. G (667 ng;

0566Addgene®, 12259) in Opti-MEM® I reduced serum medium (250 pL; Gibco®, 31985070). Media was changed 12-18 hours after transfection, and viral supernatant was collected at 48 hours post-transfection, centrifuged at 500 x g for 5 minutes to remove debris, and filtered through 0.45-pm polyvinylidene difluoride (PVDF) filter (MilliporeSigma®, SLHVM33RS) and directly used without further concentration. For PE-PRISM library pools, lentivirus 86/115

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0568#14568046vl packaging was scaled up 6x in 10-cm dishes for virus production to ensure sufficient virus for library coverage. Cells were transduced aiming for an MOI of 0.3 and >1000x coverage of transduced cells.

0569[0181] For transduction of HEK293T cells, the cell culture was supplemented with lentivirus-containing supernatant. For transduction of K562, U2OS, HeLa, and N2a cells, spinfection was performed in six-well plates (Coming, 0720083) with 2 x 10⁶ cells in media supplemented with lentivirus-containing supernatant and 10 pg mL polybrene (MilliporeSigma®, TR-1003-G), and centrifugation at 900 x g was performed for 90 minutes at 33 °C. The cells were then transferred to T75 flasks (Coming®, 353136) for recovery at 37 °C, 5% CO2 atmosphere. Transduced cells were passaged after 24 hours. To select for transduced cells, media was supplemented with puromycin at 1 pg mL (InvivoGen®, #ant-pr-1) at 48 hours post-transduction, and cells were cultured in selective media for six days until >95% of live cells were BFP-positive measured by CytoFlex® S Flow Cytometer (Beckman Coulter®). To quantify transduction efficiency, BFP fluorescence was measured at >72 hours post-transduction in cells passaged under non-selective conditions to ensure functional multiplicity of infection of less than 0.3.

0570Prime editing by transfection and electroporation for plasmid DNA delivery

0571[0182] For prime editing in pooled PE-PRISM screens, transduced and selected HEK293T cell libraries were plated in 15-cm dishes (Celltreat Scientific Products®, 229651) at a density of 6 x 10⁶ cells, seeding as many plates as required to ensure >l,000x library coverage per biological replicate. After 16 hours, each 15-cm dish was transfected with a mixture of Lipofectamine® 2000 (120 pL) and prime editor plasmid (50 pg) in 2 mL Opti-MEM® I reduced semm medium (Gibco®, 31985070), following the manufacturer’s recommended protocol. Cells were passaged after 48 hours and cultured for 96 hours for gDNA extraction using QIAamp® DNA blood maxi kit (Qiagen®, 51192) following the manufacturer’s protocol.

0572[0183] For transfection of editors with pooled self-targeting panels in alternative cell types (FIGs. 3A, 3B, and 10B), transduced HeLa and U2OS cells were seeded at 2 x 10⁵ cells per well in six-well plates (Corning®, 0720083) and transfected after 16 hours with Lipofectamine® 3000 (6 pL; Invitrogen®, L3000015), P3000 enhancer reagent (8 pL), and prime editor plasmid (4,000 ng) in Opti-MEM® I reduced serum medium (250 pL; Gibco®, 31985070), following the manufacturer’s protocol. Transduced N2a and HEK293T cells were seeded at 2 x 10⁵ cells per well in six-well plates (Corning®, 0720083) and transfected after 87/115

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0574#14568046vl 16 hours with Lipofectamine® 2000 (10 pL; Invitrogen®, 11668019) and prime editor plasmid (4,000 ng) in Opti-MEM® I reduced serum medium (250 pL; Gibco®, 31985070), following the manufacturer’s protocol. Transduced K562 cells (200,000 per reaction, two reactions per biological replicate) were electroporated in 20 pL complete SE Nucleofector® solution supplemented with 1 pg prime editor plasmid in 16-well strip of SE 4D-Nucleofector® X Kit S (Lonza®, V4XC-1032) and electroporated (program FF-120) with the 4D-Nucleofector® device (Lonza®, AAF-1003X); cells were resuspended in 80 pL of prewarmed medium and transferred into 1 mL of pre- warmed culture in 24-well plates (Coming®, 3527). After 72 hours, gDNA was collected from HeLa, U2OS, N2a, HEK293T, and K562 cells using the QIAamp® DNA mini kit (Qiagen®, 51304) following the manufacturer’s protocol.

0575[0184] For arrayed validation experiments, HEK293T cells were plated in 96-well plates (Falcon, 353075) at a density of 10⁴ cells per well and cultured for 16 hours. For prime editing in cells with pre-installed self-targeting lentiviral constructs, cells were treated with Lipofectamine® 2000 (0.5 pL) and prime editor plasmid (200 ng) in Opti-MEM® I reduced serum medium (10 pL; Gibco®, 31985070), according to the manufacturer’s protocol. For prime editing of endogenous genomic targets, cells were treated with Lipofectamine® 2000 (0.5 pL), prime editor plasmid (200 ng), (e)pegRNA plasmid (50 ng), and ngRNA plasmid (15 ng if PE3 was optionally used) in Opti-MEM® I reduced serum medium (10 pL), according to the manufacturer’s protocol. Low-dose transfection conditions (FIG. 3D, FIG.

05769D) used 200 ng prime editor plasmid and 2 ng epegRNA plasmid. For twin prime editing experiments, cells were treated with Lipofectamine® 2000 (0.5 pL), prime editor PE6c plasmid (50 ng), and both epegRNA plasmids (10 ng each) in Opti-MEM® (10 pL). Cells were harvested 72 hours after transfection, culture medium was removed, and cells were lysed in 100 pL lysis buffer (10 mM Tris-HCl pH 8.0, 0.05% SDS, and 25 pg mL proteinase K) with incubation at 37 °C for 1 hour, followed by incubation at 55 °C for 30 minutes. The resulting crude mixture was used directly as input for targeted amplicon sequencing.

0577High-throughput sequencing of PE-PRISM screens

0578[0185] Genomic DNA from each replicate was subjected to a first round of PCR (PCR1) using Q5 Hot-Start High-Fidelity 2X Master Mix (New England BioLabs®, M0494L) to amplify the integrated lentiviral cassette and append sequencing adapters. Each 50 pL reaction contained up to 5 pg genomic DNA. PCR1 products were purified using the 88/115

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0580#14568046vl QIAquick® PCR Purification Kit (Qiagen®, 28104), and 2 pL (corresponding to 5-10 ng) of purified DNA was used as input for a second PCR (PCR2), which incorporated unique sample indices and flow cell adapters. Final PCR products were bead-purified with a 0.7x ratio of SPRI beads, assessed for quality using a TapeStation® High-Sensitivity DI 000 ScreenTape® assay (Agilent®), and quantified with the Qubit® dsDNA High Sensitivity Assay Kit (Invitrogen®, Q33231). Libraries were then sequenced with a custom Readl primer (oHS0139) on an Illumina® MiSeq®, Illumina® NextSeq®, or Element BiosciencesTM AVITITM platform for 190 cycles for the R1 read, 8 cycles for the i7 index read, and 8 cycles for the i5 index read. Reads were demultiplexed with Bases2Fastq (Element BiosciencesTM) or Generate FASTQ analysis module (Illumina®). For further demultiplexing of individual elements, reads were separated into individual fastq files by alignment to a corresponding member of the library using bowtie2⁸² and trimming to exclude scaffold bases. Editing outcomes for each library element were analyzed using CRISPresso2⁸³, as described below.

0581High-throughput sequencing for arrayed experiments

0582[0186] Target amplicon sequencing was performed as previously described². Briefly, the first round of PCR (PCR1) using primers containing Illumina® sequencing adapters and targeting the genomic locus of interest was templated with 1 pL of cell lysate using Phusion U Green Multiplex PCR Master Mix (Thermo Fisher Scientific®, F564L) under the following conditions 98 °C for 30 seconds; 30 cycles of (98 °C for 10 seconds, 58-68 °C (optimized experimentally) for 20 seconds, and 72 °C for 30 seconds); and 72 °C for 1 minute. A second round of PCR (PCR2) to append unique Illumina® barcodes was performed with 1 mL of PCR1 as template, under the following conditions: initial denature at 98 °C for 30 seconds; 7 cycles of (98 °C for 10 seconds, 61 °C for 20 seconds, and 72 °C for 30 seconds); and 72 °C for 1 minute. PCR2 amplicons from the same target were pooled and purified using QIAquick® Gel Extraction Kit (Qiagen®, 28704), and eluted libraries were quantified by Qubit® dsDNA High Sensitivity Assay Kit (Invitrogen®, Q33231) and sequenced by singleend sequencing with AVITITM Sequencing Kit Cloudbreak FreestyleTM Low Output (Element BiosciencesTM, 860-00011) or MiSeq® 300 v2 Kit (Illumina®, MS- 102-2002) for 150-300 cycles for the R1 read, 8 cycles for the i7 index read, and 8 cycles for the i5 index read.

0583Reads were demultiplexed with Bases2Fastq (Element BiosciencesTM) or Generate FASTQ analysis module (Illumina®).

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0586#14568046vl Analysis of prime editing outcomes

0587[0187] Demultiplexed sequencing reads were aligned to a reference sequence using CRISPResso2 (v2.2.10)⁸³ in batch analysis mode. For all analyses, the discard_indel_reads parameter was set as ‘TRUE’, the q parameter set as ‘30’, and the expected_hdr_amplicon_seq provided as the amplicon sequence containing the intended editing outcome. The quantification window coordinates (“-qwc”) were set to minimally encompass the pegRNA nick site, the ngRNA nick site (if applicable), the dead sgRNA putative nick site (if applicable; FIG. 3F, FIG. 11F), and the end of the extended 3' flap generated by reverse transcription. The frequency of prime editing without indels was calculated as (non-discarded reads aligning to HDR amplicon)/(total reads aligning to all amplicons) and is referred to as ‘editing’ throughout. The frequency of indels was calculated as (total discarded reads)/(total reads aligning to all amplicons).

0588[0188] For analysis of pooled PE-PRISM screens to identify top elements, raw data were filtered to exclude library elements that had dropped out (&lt;50 reads) and elements that showed undetectable editing (&lt;5 reads). For each unique combination of motif, target protospacer, and prime editor, a z-score was computed to normalize editing outcomes and account for differences in absolute editing percentage across these variables. Average z-scores for each motif were calculated across occurrences throughout the library dataset and biological replicates.

0589Chemically synthesized guide RNA generation and in vitro transcription

0590[0189] Synthetic epegRNAs were ordered from Integrated DNA Technologies® and contained 2'-O-methyl modifications at the first three and last three nucleotides and phosphorothioate linkages between the three first and last three nucleotides. Synthetic ngRNAs were ordered from Synthego® and contained 2'-O-methyl modifications at the first three and last three nucleotides and 3 '-phosphorothioate linkages between the first three and last two nucleotides.

0591[0190] Prime editor mRNA was generated by in vitro transcription as previously described². Briefly, a linear IVT substrate was prepared by PCR amplification with primers that repaired an inactive T7 promoter and installed a 119-nt poly (A) tail from a plasmid template using New England BioLabs® Next High-Fidelity 2x PCR Master Mix (New England BioLabs®, M0541). The PCR product was purified using QIAquick® PCR purification kit (Qiagen®, 28104) and in vitro transcribed with HiScribe® T7 high-yield RNA synthesis kit (New England BioLabs®, E2040) following the manufacturer’s protocol with two modifications:

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0594#14568046vl (1) co-transcriptional capping with CleanCap® reagent AG (Trilink®, N-7113) and (2) complete substitution of -methylpseudouridine-S'-triphosphate (Trilink®, N-1081) for uridine triphosphate. Reactions were incubated at 37 °C for 2-4 hours, treated with DNase I (New England BioLabs®, M0303), purified on Monarch® spin RNA cleanup kit (T2050), reconstituted in nuclease-free water, and stored at -80 °C.

0595Electroporation of RNA in mouse fibroblasts, 16HBEge cells, and HSPCs

0596[0191] Electroporation of human primary CD34+ HSPCs from healthy donors was performed as previously described¹⁶. Cells (200,000 per reaction) were washed once with 1 mL phosphate-buffered saline and electroporated in 20 pL complete P3 Nucleofector® solution (Lonza®, V4XP-3032) supplemented with 1 pg PEmax mRNA, 180 pmol epegRNA, and 120 pmol ngRNA. The cell-RNA mixture was transferred to a 16- well strip of P34D-Nucleofector® X Kit S (Lonza®, V4XP-3032) and electroporated (program DS- 130) with the 4D-Nucleofector® device (Lonza®, AAF-1003X). Cells were suspended in 80 pL of pre- warmed growth medium and incubated for 72 hours.

0597[0192] Electroporation of Atpla3 E815K mouse primary fibroblasts was performed as previously described¹⁵. Cells (200,000 per reaction) were dissociated with TrypLE® Express Enzyme (Gibco®, 12605036), washed once with 1 mL phosphate-buffered saline, and resuspended in 20 pL complete P2 Nucleofector® solution (Lonza®, 197189) supplemented with 1 pg PEmax mRNA, 90 pmol epegRNA, 30 pmol ngRNA, and 30 pmol dead sgRNA. The cell-RNA mixture was transferred to a 16- well strip of P24D-Nucleofector® X Kit S (Lonza®, 197189) and electroporated (program DS- 150) with the 4D-Nucleofector® device (Lonza®, AAF-1003X). Cells were suspended in 80 pL of pre-warmed growth medium, transferred into 1 mL of pre- warmed culture in 24- well plates, and incubated for 72 hours.

0598[0193] Electroporation of 16HBEge-F508del immortalized epithelial cells was performed as previously described¹⁷. Cells (200,000 per reaction) were dissociated with TrypLE® Express Enzyme (Gibco®, 12605036), washed once with 1 mL phosphate-buffered saline, and resuspended in 20 pL complete SG Nucleofector® solution (Lonza®, 197175) supplemented with 1 pg PEmax mRNA, 90 pmol epegRNA, 30 pmol ngRNA, and 30 pmol dead sgRNA. The cell-RNA mixture was transferred to a 16- well strip of SG 4D-Nucleofector® X Kit S (Lonza®, 197175) and electroporated (program CM- 137) with the 4D-Nucleofector® device (Lonza®, AAF-1003X). Cells were suspended in 80 pL of pre-warmed growth medium, transferred into 500 pL of pre- warmed culture in 24- well plates, and incubated for 6 days. On

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0601#14568046vl day 4, cells were washed with 200 |aL phosphate-buffered saline and incubated with fresh growth media.

0602Prime editor protein expression and lipofection of HEK293T CFTR F508del cells [0194] Recombinant PEmax with a C-terminal non-cleavable 8xHis-tag was transformed into E. coli BL21 Star (DE3) (ThermoFisher Scientific, C601003). Single colonies were picked into 5 mL of TB media and incubated at 37 °C for 16 hours with continuous shaking at 220 rpm. The overnight cultures were diluted 1:50 in 250 mL TB media and incubated at 37 °C with continuous shaking at 220 rpm until OD 0.5-1.0. The cultures were cold-shocked on ice for 1 hour, induced with a final concentration of 0.8% rhamnose (w/v), and shaken overnight at 220 rpm at 16 °C for 24 hours. Cultures were centrifuged at 5,000 x g, and the cell pellets were resuspended in 25 mL lysis buffer (20% glycerol, 100 mM Tris*HCl pH 8.0, 5 mM TCEP, 1 M NaCl supplemented with two tablets of completeTM EDTA-free Protease Inhibitor Cocktail [Roche, 11873580001] and 1:1000 DNase I Solution [ThermoFisher Scientific, 90083]). Cells were lysed by sonication (3% power, 6 minutes total; 3 seconds on, 6 seconds off; Qsonica CL-334 tip with Fisher Scientific FB705 power module) at 4 °C on ice and clarified by centrifugation at 18,000 x g for 30 min at 4 °C.

0603[0195] To purify PEmax, clarified lysates were incubated with 2.0 mL of HisPurTM Ni-NTA Resin (ThermoFisher Scientific, 88221) and 40 mM imidazole for 2 hours at 4 °C. The Ni-NTA resin was then loaded onto gravity flow columns (G-Biosciences, 82021-346), washed twice with 10 mL wash buffer (20% glycerol (w/v), 100 mM Tris*HCl pH 8.0, 5 mM TCEP, 1 M NaCl) and once with 5 mL size-exclusion buffer (200 mM NaCl, 1 mM TCEP, 10% glycerol, 50 mM Tris*HCl pH 7.5). The protein was eluted in size-exclusion buffer with imidazole gradient (4 x 1000 uL fractions at 100-400 mM). Pure protein fractions were concentrated (100 kDa MWCO Amicon Ultra Centrifugal filters, Millipore, UFC910008) and analyzed on NuPAGETM Bis-Tris Mini Protein Gels, 4-12%, 1.0-1.5 mm (Invitrogen, NP0321BOX) using the PierceTM BCA Protein Assay Kit (ThermoFisher Scientific, 23225) and Quick Start Bovine Serum Albumin Standard Set (BioRad, 5000207) to quantify protein concentration.

0604[0196] HEK293T cells bearing the CFTR F508del mutation were seeded at 5 x 10³ cells per well in 96-well plates 18-24 hours before transfection. Immediately prior to transfection, medium was replaced with Opti-MEM I reduced serum medium (100 pL; Gibco, 31985070). Prime editor protein (4 pmol), epegRNA (8 pmol), ngRNA (0.5 pmol), and dsgRNA (0.5 pmol) was incubated with Cas9 Plus Reagent (1.0 pL) and CRISPRMAX reagent (0.9 pL;

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0607#14568046vl Invitrogen, CMAX00015) in Opti-MEM I reduced serum medium (10 pL; Gibco, 31985070) and delivered to cells following the manufacturer’s recommended protocol. At 12 hours posttransfection, an additional 100 pL complete medium was added. Cells were cultured for 72 hours and harvested for high-throughput sequencing.

0608eVLP production and transduction

0609[0197] PE-eVLPs were produced as previously described⁶⁷. Gesicle 293T cells were plated in T75 flasks (Coming®, 353136) at a density of 5 x 10⁶ cells per flask. After 18 hours, a mixture of plasmids was transfected with jetPRIME® transfection reagent (Polyplus® 101000001), following the manufacturer’s protocol, to deliver plasmids expressing VSV-G (400 ng; Addgene®, 8454), wild-type MMLV Gag-Pol (2,813 ng; Addgene®, 35614), Gag-COM-Pol (2,000 ng; Addgene®, 211373), Gag-P3-Pol (422 ng; Addgene®, 211374), P4-PE (422 ng; Addgene®, 211375), COM-epegRNA (3,520 ng; Addgene®, 211376), and COM-ngRNA (880 ng). After 48 hours, supernatant was collected, centrifuged at 500 x g for 5 minutes, and the supernatant was filtered through 0.45-pm polyvinylidene difluoride (PVDF) filter (MilliporeSigma®, SE1M003M00).

0610[0198] For cell culture transduction, 5x PEG-it Virus Precipitation Solution (System Biosciences, LV825A-1) was added to precipitate eVLPs at 4 °C for 18 hours, eVLPs were pelleted at 1,500 x g for 30 minutes at 4 °C, and resuspended in Opti-MEM® I reduced serum medium (Gibco®, 31985070) at lOOx concentration (100 pL from initial 10 mL medium); all cell culture experiments were performed with eVLPs concentrated uniformly as above to directly compare PE-eVLP potency per volume transduced. Target cells were plated at 30,000 cells per well in 48-well plates (Corning®, 356509), and PE-eVLPs were added directly to the media 18 hours later. Cells were harvested 72 hours after transfection and gDNA was isolated by crude lysis, as described above for arrayed validation experiments.

0611[0199] To prepare eVLPs for in vivo injections, PE-eVLPs were ultracentrifuged (SW28 rotor, OptimaTM XPN Ultracentrifuge, Beckman Coulter®) at 26,000 rpm (141,000 x g for an rAV of 118.2 mm) for 2 hours at 4 °C over a 20% (w/v) sucrose in phosphate-buffer saline (PBS) cushion solution. The eVLP pellet was hydrated in PBS for 2 hours at 4 °C at 3,000x concentration and centrifuged again at 1,000 x g for 5 minutes to remove debris on a fixed-angle tabletop centrifuge. Concentrated eVLPs were stored at 4 °C for up to seven days before use. Lentivirus containing expression constructs for eGFP-KASH was produced for co-injection with eVLPs as a marker of transduction as previously described⁶⁷.

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0614#14568046vl LNP formulation and characterization

0615[0200] LNPs were prepared using a microfluidic mixing device⁸⁴. The ethanol phase was generated by dissolving OF-02 ionizable lipid (Cayman Chemicals, 37652), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti Research®, 850725), cholesterol (Sigma-Aldrich®, C8667), and l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] ammonium salt (C14-PEG2000, Avanti Research®, 880150) at a molar ratio of 35:16:46.5:2.5 (OF-02: DOPE: Chol: C14-PEG2000). The aqueous phase comprised a 10 mM citrate buffer containing the desired RNA cargo (mRNA, epegRNA, or ngRNA). The ethanol and aqueous phases were combined at a 3:1 ratio (aqueous:ethanol) using syringe pumps, yielding a final mRNA concentration of 0.15 mg/mL.

0616[0201] Formulations were dialyzed overnight at 4 °C against lx PBS using a 20 kDa MWCO dialysis cassette (Thermo Fisher Scientific®, 66003). After dialysis, ENPs were concentrated at 4 °C with 100 kDa MWCO Amicon Ultra centrifugal filters (Millipore Sigma®, UFC210024). Total RNA concentration in the resultant ENP solution was measured by Stunner® (Unchained Labs®). LNPs were stored frozen in a 10% sucrose solution at -80 °C until further use.

0617In vitro transfection of LNPs

0618[0202] Hepal-6 cells (ATCC®, CRL-1830) were seeded 24 hours prior to transfection in 48-well poly-D-lysine-coated plates (Coming®, 356509) at a density of 30,000 cells per well in 300 pL of DMEM with GlutaMAX® (Thermo Fisher Scientific®, 10566016) supplemented with 10% FBS. PE-LNPs were prepared at 50 ng per well (1:0.9:0.1 mRNA-:epegRNA-:ngRNA-LNP by total RNA concentration) and incubated for 10 minutes at 37 °C with recombinant apolipoprotein E3 (7 pg/mL; R&amp; D Systems®, 4144- AE). A total of 50 pL of the PE-LNP mixture was then added to each well. 72 hours after transfection, culture medium was removed, and cells were washed once with IxPBS. Cells were lysed in 100 pL of lysis buffer (10 mM Tris-HCl, pH 8.0; 0.05% SDS; 25 pg/mL proteinase K) and incubated for 1 hour at 37 °C. Lysates were then heat-inactivated at 80 °C for 30 minutes and used directly for high-throughput sequencing (HTS) library preparation.

0619Animal use

0620[0203] Timed pregnant C57BL/6J mice (Charles River Laboratories®, #027) and adult C57BL/6J mice (#000664) were housed in facilities at 20-22 °C, 30-50% humidity, on a 12-hour light/12-hour dark cycle with ad libitum access to standard diet and water. All mouse 94/115

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0622#14568046vl protocols were approved by the Broad Institute Institutional Animal Care and Use Committee (D16-00903; 0048-04-15-2).

0623LNP administration to mice and tissue processing

0624[0204] LNPs were administered to 6-7 week old, female C57BL/6J mice via RO injections. Mice were anesthetized with 4% isoflurane. After induction, the right eye was gently protruded, and an insulin syringe was inserted into the retrobulbar sinus. The LNP solution (1.7 mg/kg total RNA dose; 1:0.9:0.1 mRNA-:epegRNA-:ngRNA-LNP by total RNA concentration) was then slowly injected. Immediately following injection, one drop of 0.5% proparacaine hydrochloride ophthalmic solution (Patterson Veterinary®, 07-892-9554) was applied to the eye for analgesia.

0625[0205] Mice were euthanized by CO2 asphyxiation 1 week after LNP injection and perfused with IxPBS via the right ventricle. For gDNA extraction and downstream HTS sample preparation, bulk tissues from mice were harvested and minced with scissors. gDNA was isolated from the minced tissue using the DNAdvance® Kit (Beckman Coulter®, A48705) according to the manufacturer’s instructions, and the purified gDNA was used as input for HTS library preparation, as described above.

0626eVLP administration to mice and tissue processing

0627[0206] Microinjection pipettes were generated from PCR micropipettes (Drummond®, 5-000-1001-X10) using a Sutter® Pl 000 puller to obtain a tip diameter of approximately 100 pm. The injection mixture contained 4.6 x 10¹⁰ PE-eVLP particles, diluted in 0.9% NaCl (Covetrus®, 061758) to a final volume of 4 pL with the addition of 0.3 uL concentrated eGFP: KASH lentivirus and 0.1 pL Fast Green. A total of 4 pL of the prepared solution was loaded into the micropipette syringe. Neonatal mice were anesthetized on ice, and 2 pL was administered into each ventricle. Successful delivery was confirmed by visualization of Fast Green dispersion using head transillumination. Mice were euthanized by CO2 asphyxiation 3 weeks after eVLP injection, and tissues were immediately harvested without perfusion. Samples were snap-frozen in liquid nitrogen.

0628[0207] Nuclei were extracted using the Miltenyi Biotec® Nuclei Extraction Buffer (Miltenyi Biotec®, #130-128-024) in combination with the gentleMACS® Octo Dissociator with Heaters (Miltenyi Biotec®), as described previously¹⁵. All procedures were performed at 4 °C. Dissected brain tissue was resuspended in 2 mL of complete nuclei extraction buffer (Nuclei Extraction Buffer supplemented with Murine RNase Inhibitor [New England 95/115

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0630#14568046vl BioLabs®, M0314S] at a final concentration of 0.2 U/pL), transferred into gentleMACS® C tubes (Miltenyi Biotec®, 130-093-237), and homogenized using the manufacturer’s default program “4C_nuclei_l.” The resulting homogenate was passed through a 100 pm MACS® SmartStrainer into a 15 mL tube, after which 2 mL of additional complete extraction buffer was added. The 4 mL filtrate was centrifuged at 500 x g for 5 minutes at 4 °C, and the supernatant was discarded. The pellet was resuspended in 4 mL of ice-cold nuclei suspension buffer (PBS containing 100 pg/pL albumin [New England BioLabs®, B9200S], 3.33 pM Vybrant® DyeCycleTM Ruby Stain [Thermo Fisher Scientific®, V10309], and 0.2 U/pL Murine RNase Inhibitor [New England BioLabs®, M0314S]) and centrifuged again under the same conditions. The supernatant was removed, and the pellet was resuspended in 1 mL of nuclei suspension buffer before being passed through a 35 pm cell strainer (Corning, 352235).

0631[0208] Nuclei were then flow-sorted on a Sony® MA900 Cell Sorter (Sony® Biotechnology) at the Broad Institute Flow Cytometry Core. Gating was performed using forward and side scatter to identify nuclei, followed by singlet discrimination via DyeCycleTM Ruby intensity. Sorting was subsequently carried out based on GFP fluorescence to isolate bulk and GFP-positive nuclei. Sorted nuclei were collected into RLT Plus buffer (Qiagen®, AllPrep® DNA/RNA Mini Kit), and DNA was purified following the manufacturer’s instructions as input for HTS library preparation, as described above.

0632[0209] Table 1. PegRNA Motifs

0633Motif Motif_ Motif_name Motif_ Motif-Vari Motif_ Motif Moti Mo _ID abbrev parent ant_ sequen f_ tif_ full type ce SEQ dotb len ID rack gth NO: et motifO tevo2.0 tevopreql_D tevopreql COMBO CGTC 6 (((((: 39 1 OUBLEMU GGTT:::::[[ T_hplOT35A CTAC [[[[[: _to_10C35G CTAG ))))): _HPEXTEN TTAC::::::] D_ins_hp2T GACG ]]]]]] 20A TTAA

ACCA ACTA

0635<img file="WO2026106945A2_D0023.tif" />

GGA

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0639#14568046vl motifO eHAV HAV_PK1_ HAV_PK1 COMBO AGGC 7:((((: 31 2 DOUBLEM CATG:[[[[[ UT_hp3T17 GTGA [[)))) A_to_3G17C GGGG

0640_HPDEL_del CCTG ]]]]]: _hp2A20T ATACC

TCAC CG

0642motifO eFMD tFMDV_A_P FMDV_A HPEXTEN CCGC 8:((((( 27 3 VA-2.1 KII_HPEXT _PKII D CTTGT::[[[[ END_ins_hp CCCG ))))): 5T11G GGCG

0643TTAA ]]:

AGGG AA

0645motifO eHPeV HPeVl_HPE HPeVl COMBO CCCA 9:((((( 39 4 1 XTENDJns GACC ((::[[ _hplOA33T_ TTGA [[[[)) HPEXTEND GGGT ))))): _ins_hp5A16 GGTC

0646T TGGT:]]]]] CAAT ]:

AAAA ACCC TCC

0648motifO eFMD tFMDVjDl FMDV_O HPEXTEN CCTTC 10:((((: 25 5 VO-4 K_PKIV_HP 1K_PKIV D GCGT:[[[[) EXTENDJn CGGA )))::: s_hp7G22C AGTA::]]]] AAAC GACC

0649motifO eSBRM SBRMV1JJ SBRMV1 COMBO ACGG 11:((((: 31 6 VI PD_PKb_HP _UPD_PK TCGT [[[[[[ EXTENDJn b GCAG [[)))) s_hp3G13T_ TCATC

0650HPEXTEND GGTA ]]]]]] Jns_hpl2Cl AGAC

9G TGCA

CA

0653motifO eEc Ec_PKl_HP Ec_PKl HPEXTEN CGAG 12 (((((: 32 7 EXTENDJn D GGTG [[[[[[ s_hp6T30A CGGT [::))) TGGC ))::::: CTCG ]]]]]] TAAA ]

AAGC

0655<img file="WO2026106945A2_D0024.tif" />

CGCA

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0659#14568046vl motifO eFMD tFMDV_A_P FMDV_A HPEXTEN CCGC 13:((((( 27 8 VA-2.2 KII_HPEXT _PKII D CATGT::[[[[ END_ins_hp CCCT ))))): 5A11T GGCG

0660TTAA ]]:

AGGG AA

0662motifO Dead tevopreql_de CONTRO NEG AGCG 14 ((((:: 37 9 ad L GTTCT

ATCTA CTTAC ))::::: GCGT TAAA CCAA CTAG AA

0664motifl Scramb tevopreql_sc CONTRO NEG TTTGC 15 37 0 le rambled L GTCC

AAGT CAGA GTCA ACCT ATAA CCTTA AGA

0666motifl None none CONTRO NEG 0 1 L

0667motifl HAV HAV_PK1 HAV_PK1 PARENT AAGT 16:((((( 33 2 CCAT::[[[[ GGTG [[[))) AGGG ))::::: GACT ]]]]]] TGATA ]:

CCTC ACCG

0669motifl HPeVl HPeVl HPeVl PARENT CCCA 17:((((( 35 3 GCCT (::[[[ TGGG [[)))) GTGG ))::::: CTGG TCAAT ]]]:

AAAA ACCC

0671<img file="WO2026106945A2_D0025.tif" />

CC

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0675#14568046vl motifl tevopre tevopreql tevopreql PARENT CGCG 77 ((((:: 37 4 Ql GTTCT

0676ATCTA [[[[[: GTTA )))):: CGCG TTAA ]]]]]: ACCA ACTA GAA

0677motifl FMDV FMDVJD1K FMDV_O PARENT CCTTC 18:((((: 23 5 0-4 _PKIV 1K_PKIV GCTC:[[[)) GGAA ))::::: GTAA ]]]:

AACG AC

0679motifl SBRM SBRMV1JJ SBRM VI PARENT ACGT 19:(((:[ 27 6 VI PD_PKb _UPD_PK CGTG [[[[[[ b CAGT ))):::

0680ACGG:]]]]] TAAA ]]:

CTGC ACA

0682motifl FMDV FMDV_A_P FMDV_A PARENT CCGC 20:((((: 25 7 A-2 KII _PKII CTGT:[[[[) CCCG )))::: GCGT::]]]] TAAA GGGA

A

0684motifl Ec Ec_PKl Ec_PKl PARENT CGAG 21 (((((: 30 8 GGGC [[[[[[ GGTT::)))) GGCC ):::::] TCGT ]]]]] AAAA AGCC GC

0685motifl eSBRM SBRMV1JJ SBRM VI DOUBLEM ACGT 22:(((:[ 27 9 Vl-A PD_PKb_D _UPD_PK UT CGTG [[[[[[ OUBLEMU b CCGT )))::: T_hp9A21T_ ACGG:]]]]] to_9C21G TAAA ]]:

CGGC

0687<img file="WO2026106945A2_D0026.tif" />

ACA

0689[0210] Table 2. Additional PegRNA Motifs

0690Motif ID Motif Sequence SEQ ID NO:

0691Hs_SRP GCCGGGCGCGGTGGCGCGTGCCTGTAGTCCC 1

0692<img file="WO2026106945A2_D0027.tif" />

AGCTACTCGGGAGGCT

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0696#14568046vl antiHIVl-RT_1.3a ATCTTCCGAAGCCGAACGGGAAAACCGGCAT 2

C WCMV CGGGTGCAACCCCCCCTCCCCCCGTAGGTTA 3

ACGGGACCA CYVV TGGGTGCAACCCCCCCGTCCATCTCGAACGT 4

CATCGAGACCA

0700<img file="WO2026106945A2_D0028.tif" />

0701EMCV-R_PKC AGGGCGGGTACTGCCGTAAGTGCCA 5

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EQUIVALENTS AND SCOPE

0789[0211] In the articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Embodiments or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

0790[0212] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that 106/115

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0792#14568046vl is dependent on another claim can be modified to include one or more limitations found in any other claims that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

0793[0213] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the embodiments. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any embodiment, for any reason, whether or not related to the existence of prior art.

0794[0214] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended embodiments. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following embodiments.

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