Prime editing and base editing of the ATP1a3 gene for the treatment of alternating hemiplegia of childhood

Abstract

The present disclosure relates to compositions and methods for editing a ATP1A3 gene for the treatment of alternating hemiplegia of childhood (AHC). In some embodiments, the compositions comprise a prime editor and a pegRNA for editing a ATP1A3 D801N c.2401A, ATP1A3 E815K C.2443A, ATP1A3 G947R c.2839C, or ATP1A3 L839P c.2516C mutation. The compositions may further comprise a nicking guide RNA and / or a dead single guide RNA. In other embodiments, the compositions comprise a base editor and a single guide RNA for editing a ATP1A3 D801N c.2401A, ATP1A3 E815K c.2443A, ATP1A3 G947R c.2839C, or ATP1A3 L839P c.2516C mutation. Further provided herein are methods for editing a ATP1A3 gene using prime editing and / or base editing. Polynucleotides, vectors, cells, and kits comprising said compositions are also disclosed.

Description

1 / 895 PRIME EDITING AND BASE EDITING OF THE ATP1A3 GENE FOR THE TREATMENT OF ALTERNATING HEMIPLEGIA OF CHILDHOOD RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application, U.S.S.N. 63 / 633,613, filed April 12, 2024, which is incorporated herein by reference. GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant Nos. HG009490, GM118062, and EB031172 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0003] Alternating hemiplegia of childhood (AHC) is a neurodegenerative disease that emerges in the first 18 months of life and is characterized by regular and trigger-induced attacks of paralysis affecting one side of the body (hemiplegia), involuntary muscle contractions (dystonia), and poor coordination (ataxia). AHC patients experience developmental delays, cognitive deficits, and the inability to live independently; approximately half of all AHC patients will develop epilepsy as adults1. AHC is exceptionally rare with an approximate incidence of 1 in 1,000,000 and is often misdiagnosed as intractable epilepsy2. Patients have no effective treatment options and obtain only partial symptomatic relief with off-label use of antimigraine medication or sedative drugs that facilitate relaxation and sleep1.

[0004] Approximately 80% of AHC cases are the result of aberrant Na+ / K+ATPase function caused by mutations in the ATP1A3 gene3,4, which codes for the α3 subunit of the Na+ / K+ATPase. There are four distinct isoforms of the catalytic α subunit (α1, α2, α3, and α4, from the genes ATP1A1, ATP1A2, ATP1A3, and ATP1A4, respectively) and isoforms α1 and α3 are neuron-specific5–9. Functionally, α1 and α3 are distinct: α3 has a lower affinity for Na+and K+that is believed to facilitate its function as a supplementary pump for α1 at higher Na+concentrations; this allows neurons to quickly recover from periods of rapid firing9,10. Notably, α1 and α3 are differentially expressed throughout neuron subtypes in the CNS: while many neurons express both α1 and α3, some GABAergic neurons, such as B1195.70198WO00 13849370.1 (April 11, 2025)2 / 895 parvalbumin-expressing neurons in the hippocampus and cerebral cortex and Purkinje cells in the cerebellum, highly express α3 but not α17. This differential expression of α3 in mouse brains aligns with observations from human infant brains, where α3, but not α1, is enriched in parvalbumin-expressing neurons of the postnatal neocortex, and suggests a possible conserved vulnerability of these cell types to ATP1A3 mutations in mice and humans11.

[0005] While over 59 ATP1A3 mutations have been linked to AHC, three – D801N, E815K, and G947R – collectively account for 70% of AHC cases, with respective prevalences of approximately 40%, 20%, and 10%3,4,12–17. The symptomatic severity of AHC varies with each ATP1A3 mutation and, interestingly, subsets of ATP1A3 mutations can cause disorders phenotypically distinct from AHC1. Milder ATP1A3 mutations produce different neurological syndromes including rapid-onset dystonia parkinsonism (RDP)18,19and fever- induced disorders with symptoms including cerebellar ataxia and encephalopathy20–22, whereas more severe mutations cause catastrophic epilepsy, microcephaly in infants23, and brain malformations in utero11,24. ATP1A3 mutations are autosomal dominant, always heterozygous, almost always de novo, and nearly entirely represented by missense mutations rather than loss-of-function variants such as frameshifts or premature truncations3,4,12–17. Electrophysiological studies in Xenopus laevis oocytes of ATP1A3 D801N, E815K, or G947R have demonstrated dominant-negative suppression of wild-type ATP1A3 activity by the co-expressed mutant variants25, and hemizygous Atp1a3 mice have a milder phenotype compared to mice heterozygous for Atp1a3 mutations homologous to ATP1A3 D801N and E815K9,26–29. It remains unsettled within the AHC research community whether ATP1A3 mutation pathology is caused by haploinsufficiency or a dominant-negative mechanism1,9,30,31.

[0006] The AHC research and patient communities are immensely interested in the development of therapeutic strategies to mitigate or reverse the pathology of AHC. The most promising effort reported to date has been a gene therapy approach by Hunanyan, Mikati, and co-workers in 2021, in which an Atp1a3 D801N mouse that faithfully recapitulates hallmarks of AHC was treated with an AAV9-delivered ATP1A3 transgene. Median survival of the Atp1a3 D801N mice improved after treatment, but rescue of behavioral phenotypes was modest and inconsistent between adolescent mice evaluated at postnatal day (“P”) 40, P40, and adult mice evaluated at postnatal day 70, P7030. Thus, improvements are needed. SUMMARY B1195.70198WO00 13849370.1 (April 11, 2025)3 / 895

[0007] The present disclosure relates to compositions, systems, and methods for editing the ATP1A3 gene for the treatment of alternating hemiplegia of childhood (AHC) using prime editing and base editing. In some embodiments, the compositions comprise a prime editor and a pegRNA for editing a ATP1A3 D801N c.2401A, ATP1A3 E815K c.2443A, ATP1A3G947R c.2839C, or ATP1A3 L839P c.2516C mutation. Prime editors (PE) are fusion proteins comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a reverse transcriptase (RT) domain. The compositions may further comprise a nicking guide RNA (ngRNA) and / or a dead single guide RNA (dsgRNA). In other embodiments, the compositions comprise a base editor and a single guide RNA (sgRNA) for editing a ATP1A3 G947R c.2839A mutation. Base editors (BE) are fusion proteins comprising a napDNAbp domain and a deaminase domain. Further provided herein are methods for improving the editing efficiency of said mutations and methods of treating a subject suspected of having said one or more mutations. Polynucleotides, complexes, vectors, and cells comprising said compositions are also disclosed.

[0008] In some embodiments, the compositions disclosed herein comprise a prime editor and a pegRNA for editing a ATP1A3 D801N c.2401A mutation, a ATP1A3 E815K c.2443A mutation, a ATP1A3 G947R c.2839, C mutation, a ATP1A3 L839P c.2516C mutation, or a ATP1A3 G947R c.2839A mutation. In other embodiments, the compositions disclosed herein comprise a base editor and a sgRNA for editing a ATP1A3 D801N c.2401A mutation, a ATP1A3 E815K c.2443A mutation, a ATP1A3 G947R c.2839C mutation, a ATP1A3 L839P c.2516C mutation, or a ATP1A3 G947R c.2839A mutation. The choice of editor, and accompanying RNA, for inclusion in the composition depends on the gene sequence, which may vary depending on the genetic species being edited (e.g., human versus mouse). For example, in some embodiments, BE approaches are preferred when the targeted sequences are well-isolated from potential bystander edits; likewise, PE approaches are preferred when using a BE strategy would result in undesired bystander edits. However, either editing approach can be used to edit a desired mutation.

[0009] In some embodiments, the compositions comprise a pegRNA, for example, to program a prime editor to edit a ATP1A3 D801N c.2401A mutation, a ATP1A3 E815K c.2443A mutation, a ATP1A3G947R c.2839C mutation, or a ATP1A3 L839P c.2516C mutation. In some cases, a pegRNA may also be used to program a prime editor to edit a ATP1A3 G947R c.2839A mutation. In some cases, the pegRNA used to program the prime editor to edit the one or more mutations may be the same or different. Those of skill in the B1195.70198WO00 13849370.1 (April 11, 2025)4 / 895 art will understand that the pegRNA comprises various elements that may be individually tailored to direct a prime editor to the target mutation. For example, pegRNAs, according to some embodiments, comprises a spacer sequence, a scaffold sequence, a reverse transcriptase template (RTT) sequence, and a primer binding site (PBS) sequence.

[0010] In some embodiments, a pegRNA spacer sequence, a RTT sequence, and / or a PBS sequence may comprise one or more translationally silent edits. In some cases, the one or more silent edits is designed to disrupt a pegRNA protospacer adjacent motif (PAM) sequence, disrupt pegRNA protospacer sequence (which has same sequence as the spacer sequence), and / or install one or more silent edits proximal to the corrective edit. Those of skill in the art will appreciate and understand that inclusion of such silent edits within the pegRNA architecture may permit a contiguous or semi-contiguous tract of edits that collectively evade cellular mismatch repair, and therefore, enhance prime editing efficiency. In some embodiments, the one or more translationally silent edits is identified using a screening assay. Any suitable assay known to the skilled artisan may be used to identify the translationally silent edits, such as those methods disclosed in International Patent Application Number PCT / US2023 / 065947, filed on April 19, 2023 and published on October 26, 2023 with International Publication Number WO2023 / 205687, the contents of which are hereby incorporated by reference in its entirety. Such assays are useful, for example, for identifying spacer sequences, RTT sequences, and PBS sequences.

[0011] In some cases, compositions disclosed herein comprise pegRNA spacer sequences that are truncated, relative to the standard pegRNA spacer sequences commonly used in prime editing. For example, in some embodiments, the compositions comprise pegRNA spacer sequences comprising 19 nucleotides (nt), compared to the standard 20 nt length used in prime editing. This allows inclusion of a guanine (G) at a 5′-end of the pegRNA, which may be useful for increasing prime editing efficiency in a target-dependent manner42.

[0012] In some embodiments, the pegRNAs may further comprise one or more linkers. Such linkers may be used, for example, to fuse one or more elements of the pegRNA (e.g., PBS sequences-linker-RTT sequence). Additionally, in some cases, the pegRNA may further comprise a 3′ structural motif (e.g., tevopreQ1). Such motifs are useful, for example, for reducing exonucleolytic degradation of the pegRNA, and thus, enhancing the overall prime editing efficiency. B1195.70198WO00 13849370.1 (April 11, 2025)5 / 895

[0013] As mentioned above, compositions disclosed herein, according to some embodiments, comprise a prime editor. Any suitable prime editor known to one of ordinary skill in the art may be used in the compositions disclosed herein. Exemplary embodiments include, but are not limited to PE2, PE3, PE3b, PE4, PE5, PE5b, PEmax, PE3max, PE3bmax, PE4max, PE4max, PE5bmax, PE6, PE6b, PE6c, PE6d. The prime editor may comprise one or more additional elements, for example, nuclear localization signals (NLS) and / or linkers. In some embodiments, the various elements of a prime editor are arranged in one or more configurations (e.g., architectures). For example, in some embodiments, the prime editor comprises the following architecture (from the N-terminal to C-terminal direction): [bipartite NLS]-[Cas9:(R221K+N394K+H840A)+(D1135V+G1218R+R1335Q+T1337R)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)]-[bipartite NLS]-[NLS]. In other embodiments, the prime editor comprises the following architecture: [bipartite NLS]- [Cas9(R221K+N394K+H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)]-[bipartite NLS]-[NLS]. Those of skill in the art will understand and appreciate that the compositions disclosed herein may comprise any suitable prime editor known in the art, such as those disclosed elsewhere herein.

[0014] The compositions, as disclosed herein, may further comprise one or more additional elements, for example, to enhance prime editing efficiency. For example, in some embodiments, the compositions further comprise a nicking guide RNA (ngRNA). Those skilled in the art will understand and appreciate that ngRNAs may be useful, for example, for directing the napDNAbp domain (e.g., Cas9 nickase) of a fusion protein to nick the unedited nucleic acid strand (e.g., DNA strand). Nicking the non-edited strand causes the cell’s natural repair system to copy the information in the edited strand to the complementary strand, thus permanently installing the edit (e.g., nicking helps to minimize lost edits due to MMR). Additionally, or alternatively, the compositions disclosed herein may comprise a catalytically “dead” single guide RNA (dsgRNA). dsgRNAs are art recognized sgRNAs that disrupt the local ATP1A3 chromatin state. It is believed, according to some embodiments, that compositions comprising dsgRNAs improve the accessibility of the prime editor- pegRNA complex to the target gene, thus improving prime editing efficiency.

[0015] In some embodiments, one or more compositions disclosed herein may further comprise a one or more polynucleotides encoding a prime editor, a pegRNA, a ngRNA, and / or a dsgRNA. In some embodiments, the compositions comprise one or more vectors comprising the one or more polynucleotides. For example, in some embodiments, the B1195.70198WO00 13849370.1 (April 11, 2025)6 / 895 compositions comprise a first recombinant adeno associated virus (rAAV) vector comprising a first nucleotide sequence encoding a N-terminal portion of the prime editor fused at its C- terminus to an intein-N. In some embodiments, the N-terminal portion of the prime editor comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 15. In some embodiments, the compositions comprise a second recombinant adeno associated virus (rAAV) vector comprising a second nucleotide sequence encoding an intein-C fused to the N- terminus of a C-terminal portion of the prime editor. In some embodiments, the C-terminal portion of the prime editor comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 33.

[0016] Other aspects of the disclosure relate to compositions comprising a single guide RNA (sgRNA), for example, to program a base editor to edit a ATP1A3 G947R c.2839A mutation. The sgRNA, like pegRNAs, may be tailored to maximize base editing efficiencies. For example, sgRNAs comprise spacer sequences and scaffold sequences that can be individually tailored to enhance base editing efficiencies. In some embodiments, the sgRNA spacer sequence comprises a nucleic acid sequence of any one of SEQ ID NOs: 77- 96, 919-1066, 1229-1317, 1408-1409. Additionally, or alternatively, in some embodiments, the sgRNA scaffold sequence comprises a nucleic acid sequence of anyone of SEQ ID NOs: 97-102, 1067.

[0017] In some embodiments, compositions comprising a sgRNA further comprise a base editor. As described above, base editors are fusion proteins comprising a napDNAbp domain and a deaminase domain. The deaminase domain may comprise an adenosine deaminase domain or a cytosine deaminase domain. In some embodiments, the deaminase domain has been evolved, for example, directed evolution techniques such as phage-assisted continuous evolution (PACE) or phage-assisted non-continuous evolution (PANCE). In some embodiments, the evolved deaminase comprises a TadA-8e deaminase that has been evolved from TadA7.10, a deoxyadenosine deaminase that was previously evolved from an E. coli tRNA adenosine deaminase (ecTadA, or TadA) to act on single-stranded DNA. In some embodiments, the base editor comprising a napDNAbp domain comprising SpCas9 (D10A) (SEQ ID NO: 50) and an adenosine deaminase domain comprising TadA-8e (SEQ ID NO: 48) (herein ‘ABE8e’). Those of skill in the art will understand and appreciate that the compositions disclosed herein may comprise any one of the prime editors disclosed in the various issued patents, published patent applications, scientific journal articles, and other publications described in the section titled “Incorporation by Reference.” B1195.70198WO00 13849370.1 (April 11, 2025)7 / 895

[0018] In some embodiments, one or more compositions disclosed herein may further comprise a one or more polynucleotides encoding a base editor and an sgRNA. In some embodiments, the compositions comprise one or more vectors comprising the one or more polynucleotides. For example, in some embodiments, the compositions comprise a first recombinant adeno associated virus (rAAV) vector comprising a first nucleotide sequence encoding a N-terminal portion of the base editor fused at its C-terminus to an intein-N. In some embodiments, the N-terminal portion of the base editor comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 15. In some embodiments, the compositions comprise a second recombinant adeno associated virus (rAAV) vector comprising a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the base editor. In some embodiments, the C-terminal portion of the prime editor comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 33.

[0019] Additional aspects of the present disclosure relate to compositions for installing a ATP1A3 D801N c.2401G>A mutation, a ATP1A3 E815K c.2443G>A, ATP1A3 G947R c.2839G>A, a ATP1A3 G947R c.2839G>C mutation or a ATP1A3 G947R c.2839G>A, into a cell line. Such compositions are useful, for example, to create monoclonal cells lines expressing said mutations, which can be used, for example, as a screening tool to identify therapeutics capable of either reversing said mutations and / or treating one or more phenotypes associated with said mutations As above, the compositions, according to some embodiments may comprise a PE (SEQ ID NOs: 1413,1414, 1418-1421, 1515-1522), a pegRNA (SEQ ID NOs: 283-918, 1573-1578), a ngRNA (SEQ ID NOs: 1068-1228, 1579- 1583), and / or a dsgRNA (SEQ ID NOs: 1318-1407, 1585-1601). In some embodiments, the pegRNA comprises a spacer sequence (SEQ ID NOs: 77-96, 919-1066, 1229-1317, 1408- 1409, 1523-1525), a scaffold sequence (SEQ ID NOs: 97-102, 1067), a RTT sequence (SEQ ID NOs: 103-223, 1526-1528), and a PBS sequence (SEQ ID NOs: 224-280, 1529-1531). Alternatively, in some embodiments, the compositions comprise a BE (SEQ ID NOs: 33, 39, 41, 47-48, 52, 56, 1443-1514) and an sgRNA (SEQ ID NOs: 1410, 1411).

[0020] Remaining aspects of the disclosure relate to one or more methods. In some embodiments, the methods are directed toward methods of optimizing prime editing efficiency of a ATP1A3 D801N c.2401A>G edit, a ATP1A3 E815K c.2443A>G edit, a ATP1A3G947R c.2839C>G edit, or a ATP1A3 L839P c.2516C>T edit. The methods, according to some embodiments, comprise testing the efficiency of installation of a target edit by a PE using two or more pegRNAs. In some cases, each pegRNAs comprises a different B1195.70198WO00 13849370.1 (April 11, 2025)8 / 895 RTT length and / or PBS length. In some embodiments, the methods further comprise performing high throughput sequencing to select the pegRNA (e.g., RTT / PBS combination) with the highest editing efficiency. The method may further comprise optimizing one or more additional parameters of the pegRNA and / or PE to identify an optimal prime editing system. For example, in some instances, the one or more additional parameters comprise optimization of a ngRNA, optimization of a dsgRNA, introduction of one or more nucleotide mismatches in the RTT (e.g., silent mutations) of the pegRNA relative to the sequence of the target ATP1A3 edit of interest, introduction of a flip and extension (F+E) scaffold into the pegRNA, and / or the use of PE2, PE3, PE3b, PE4, PE5, PE5b, PEmax, PE3max, PE3bmax, PE4max, PE4max, PE5bmax, PE6, PE6b, PE6c, or PE6.

[0021] Other methods relate to methods for editing a gene suspected of having ATP1A3 D801N c.2401A mutation, a ATP1A3 E815K c.2443A mutation, a ATP1A3G947R c.2839C mutation edit, or a ATP1A3 L839P c.2516C mutation. In some embodiments, the methods comprise contacting the ATP1A3 gene at the desired location with a PE system, the PE system comprising a napDNAbp domain, and RT domain, and a pegRNA. Further, upon contacting the target gene, the PE system installs two or more nucleotide changes in the target ATP1A3 sequence, thereby modifying the ATP1A3 gene.

[0022] Other aspects of the disclosure relate to a prime editing guide RNA (pegRNA) for targeting a D801N (c.2401A) mutation in ATP1A3, comprising a nucleotide sequence of SEQ ID NOs 355-450 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. Other aspects of the disclosure relate to a prime editing guide RNA (pegRNA) for targeting a E815K (c.2443A) mutation in ATP1A3, comprising a nucleotide sequence of SEQ ID NOs 355-450 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. Other aspects of the disclosure relate to a prime editing guide RNA (pegRNA) for targeting a L839P (c.2516C) mutation in ATP1A3, comprising a nucleotide sequence of SEQ ID NOs 733-827, 1574, 1577 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. Other aspects of the disclosure relate to a prime editing guide RNA (pegRNA) for targeting a G947R (c.2839C) mutation in ATP1A3, comprising a nucleotide B1195.70198WO00 13849370.1 (April 11, 2025)9 / 895 sequence of SEQ ID NOs 639-732, 1578 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleotide sequences of SEQ ID NOs 639-732, 1578.

[0023] Other aspects of the present disclosure relate to a complex comprising any one of the prime editors and pegRNAs described herein. In some embodiments, in any one of the complexes described herein, the prime editors are a fusion protein comprising a napDNAbp domain and a reverse transcriptase (RT) domain.

[0024] In some embodiments, in any one of the complexes described herein, the prime editor is selected from the group consisting of PE2, PE3, PE3b, PE4, PE5, PE5b, PEmax, SpCas9(VRQR)-PEmax, PE3max, PE3bmax, PE4max, PE4max, PE5bmax, PE6, PE6b, PE6c, PE6d, SpCas9(VRQR)-PE6b, SpCas9(VRQR)-PE6c, and SpCas9(VRQR)- PE6d.

[0025] In some embodiments, in any one of the complexes described herein, the prime editor is selected from the group consisting of: PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)- PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)- PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

[0026] Other aspects of the present disclosure relate to a composition comprising the any one of the pegRNAs, any one of the ngRNAs, and / or any one of the dsgRNAs described herein. In some embodiments, the composition further comprises a prime editor. In some embodiments, the prime editor selected from the group consisting of: PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

[0027] Other aspects of the present disclosure relate to a system comprising any one of the pegRNAs described herein and a prime editor. In some embodiments, the prime editor B1195.70198WO00 13849370.1 (April 11, 2025)10 / 895 selected from the group consisting of: PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

[0028] In some embodiments, the system further comprises a dsgRNA. In some embodiments, the system further comprising a ngRNA. In some embodiments, the dsgRNA is any one of the dsgRNA described herein. In some embodiments, the ngRNA is any one of the ngRNA described herein.

[0029] Other aspects of the present disclosure relate to a recombinant adeno- associated viral (rAAV) vector comprising one or more nucleotide sequences encoding any one of the pegRNA described herein and a prime editor. In some embodiments, the prime editor is selected from the group consisting of: PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

[0030] In some embodiments, in any one of the rAAV vectors described herein the prime editor is a split prime editor encoded by a first and a second nucleotide sequence. In some embodiments, the first nucleotide sequence encodes a N-terminal portion of the prime editor fused at its C-terminus to an intein-N. In some embodiments, the first nucleotide sequence encodes an intein-C fused to the N-terminus of a C-terminal portion of the prime editor.

[0031] Other aspects of the present disclosure relate to a polynucleotide encoding any one of the pegRNA described herein. Other aspects of the present disclosure relate to one or more polynucleotides encoding any one of the pegRNA described herein, any one of the complexes described herein, any one of the compositions described herein, or any one of the systems described herein. Other aspects of the present disclosure relate to a vector comprising B1195.70198WO00 13849370.1 (April 11, 2025)11 / 895 the polynucleotide described herein. Other aspects of the present disclosure relate to a vector comprising the one or more polynucleotides described herein.

[0032] Other aspects of the present disclosure relate to a pharmaceutical composition comprising any one of the pegRNAs described herein, any one of the ngRNAs described herein, any one of the dsgRNAs described herein, any one of the sgRNAs described herein, any one of the complexes described herein, any one of the compositions described herein, any one of the systems described herein, any one of the rAAV vectors described herein, any one of the polynucleotides described herein, and / or any one of the vectors described herein, and a pharmaceutically acceptable excipient.

[0033] Other aspects of the present disclosure relate to a cell comprising any one of the pegRNAs described herein, any one of the ngRNAs described herein, any one of the dsgRNAs described herein, any one of the sgRNAs described herein, any one of the complexes described herein, any one of the compositions described herein, any one of the systems described herein, any one of the rAAV vectors described herein, any one of the polynucleotides described herein, and / or any one of the vectors described herein.

[0034] Other aspects of the present disclosure relate to a medicinal kit comprising any one of the pegRNAs described herein, any one of the ngRNAs described herein, any one of the dsgRNAs described herein, any one of the sgRNAs described herein, any one of the complexes described herein, any one of the compositions described herein, any one of the systems described herein, any one of the rAAV vectors described herein, any one of the polynucleotides described herein, and / or any one of the vectors described herein, and optionally one or more containers, delivery devices, and / or set of instructions.

[0035] Other aspects of the present disclosure relate to a method of prime editing an ATP1A3 D801N c.2401A mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 D801N c.2401A mutation with a prime editor and a prime editing guide RNA (pegRNA) comprising a nucleotide sequence of SEQ ID NOs 355-450 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the method further comprises providing a ngRNA. In some embodiments, the ngRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 929-939 or 1050-1065. In some embodiments, the ngRNA B1195.70198WO00 13849370.1 (April 11, 2025)12 / 895 comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211-1227. In some embodiments, the method further comprises providing a dsgRNA. In some embodiments, the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1229-1252 or 1277-1316. In some embodiments, the dsgRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406. In some embodiments, the prime editor is selected from the group consisting of: PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

[0036] Other aspects of the present disclosure relate to a method of prime editing an ATP1A3 L839P c.2516C mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 L839P c.2516C mutation with a prime editor and a prime editing guide RNA (pegRNA) comprising a nucleotide sequence of SEQ ID NOs 733-827, 1574, 1577 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the method further comprising providing a ngRNA. In some embodiments, the ngRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 929-939 or 1050-1065. In some embodiments, the ngRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211- 1227. In some embodiments, the method further comprises providing a dsgRNA. In some B1195.70198WO00 13849370.1 (April 11, 2025)13 / 895 embodiments, the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1229- 1252 or 1277-1316. In some embodiments, the dsgRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406. In some embodiments, the prime editor is selected from the group consisting of: PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

[0037] Other aspects of the present disclosure relate to a method of prime editing an ATP1A3 G947R c.2839C mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 G947R c.2839C mutation with a prime editor and a prime editing guide RNA (pegRNA) comprising a nucleotide sequence of SEQ ID NOs 639-732, 1578 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleotide sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the method further comprises providing a ngRNA. In some embodiments, the ngRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 929-939 or 1050-1065. In some embodiments, the ngRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211-1227. In some embodiments, the method further comprises providing a dsgRNA. In some embodiments, the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1229-1252 or 1277-1316. In some embodiments, the dsgRNA comprises a sequence comprising a nucleic acid B1195.70198WO00 13849370.1 (April 11, 2025)14 / 895 sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406. In some embodiments, the prime editor is selected from the group consisting of: PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

[0038] Other aspects of the present disclosure relate to a method of prime editing an ATP1A3 E815K c.2443A mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 E815K c.2443A mutation with a prime editor and a prime editing guide RNA (pegRNA) comprising a nucleotide sequence of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the method further comprises providing a ngRNA. In some embodiments, the ngRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 929-939 or 1050-1065. In some embodiments, the ngRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211-1227. In some embodiments, the method further comprises providing a dsgRNA. In some embodiments, the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1229-1252 or 1277-1316. In some embodiments, the dsgRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406. In some embodiments, the prime editor is selected from the group consisting of: PEmax (SEQ ID NO: B1195.70198WO00 13849370.1 (April 11, 2025)15 / 895 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

[0039] It should be appreciated that 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 and claims. The details of one or more embodiments of the present disclosure are set forth herein. BRIEF DESCRIPTION OF DRAWINGS

[0040] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0041] FIGs.1A-1B show the optimization of PE strategy to install the ATP1A3 D801N mutation in HEK293T cells. FIG. 1A illustrates an exemplary PE strategy to install the ATP1A3 D801N c.2401A mutation into HEK293T cells, a panel of 24 epegRNAs with variable spacers (NGA1, NGA2, or NGA3), PBS lengths, and RTT lengths was transfected into HEK293T cells. PE was completed using SpCas9(VRQR)-PEmax33with MLH1dn34and epegRNAs35. One of the most efficient epegRNA designs, NGA1 PBS12 RTT18, was selected to generate a monoclonal HEK293T cell line for the ATP1A3 D801N c.2401A mutation. FIG. 1B is a schematic of the ATP1A3 locus edited to create the D801N c.2401A mutation. Protospacers used for epegRNAs NGA1, NGA2, and NGA3 are superimposed on the wild-type (WT) ATP1A3 sequence. For FIG. 1A, PBS and RTT lengths are listed as nucleotides lengths and data collected from one biological replicate.

[0042] FIGs.2A-2D show optimization of PE strategies to install multiple ATP1A3 mutations in HEK293T cells. To identify PE strategies to install multiple ATP1A3 mutation into HEK293T cells, panels of epegRNAs with variable PBS lengths, RTT lengths, and B1195.70198WO00 13849370.1 (April 11, 2025)16 / 895 nicking sgRNAs were transfected into HEK293T cells for each desired ATP1A3 mutation to be installed. PE was completed using PEmax34for epegRNAs with NGG-PAM spacers (FIGs. 2A-2C) or SpCas9(VRQR)-PEmax33for NGA epegRNAs with NGA-PAM spacers (FIG.2D). MLH1dn34was used to enhance editing in these experiments. FIG. 2A shows the PE data to optimize installation of ATP1A3 E815K c.2443A (top data plot) and a schematic of the ATP1A3 locus edited to create the E815K c.2443A mutation with the installation epegRNA protospacer sequence annotated above the wild-type (WT) ATP1A3 sequence (bottom sequence schematic). For each read total group in FIG. 2A, E815K G^A installation is on the left and the indels are on the right. One of the most efficient epegRNA + nick strategies, PBS13 RTT13 +93 ngRNA, was selected to generate a monoclonal HEK293T cell line for the ATP1A3 E815K c.2443A mutation. FIG. 2B shows the PE data to optimize installation of ATP1A3 G947R c.2839A (top data plot) and a schematic of the ATP1A3 locus edited to create the G947R c.2839A mutation with the installation epegRNA protospacer sequence annotated above the WT ATP1A3 sequence (bottom sequence schematic). For each read total group in FIG. 2B, G947R G^A installation is on the left and the indels are on the right. One of the most efficient epegRNA + nick strategies, PBS13 RTT14 +53 ngRNA, was selected to generate a monoclonal HEK293T cell line for the ATP1A3 G947R c.2839A mutation. FIG. 2C shows the PE data to optimize installation of ATP1A3 G947R c.2839C (top data plot) and a schematic of the ATP1A3 locus edited to create the G947R c.2839C mutation with the installation epegRNA protospacer sequence annotated above the WT ATP1A3 sequence (bottom sequence schematic). For each read total group in FIG. 2C, G947R G^C installation is on the left and the indels are on the right. One of the most efficient epegRNA + nick strategies, PBS13 RTT14 +53 ngRNA, was selected to generate a monoclonal HEK293T cell line for the ATP1A3 G947R c.2839C mutation. FIG. 2D shows the PE data to optimize installation of ATP1A3 L839P c.2516C (top data plot) and a schematic of the ATP1A3 locus edited to create the L839P c.2516C mutation with the installation epegRNA protospacer sequence annotated above the WT ATP1A3 sequence (bottom sequence schematic). For each read total group in FIG. 2D, L839P T^C installation is on the left and the indels are on the right. One of the most efficient epegRNA + nick strategies, PBS13 RTT26 +58 ngRNA, was selected to generate a monoclonal HEK293T cell line for the ATP1A3 L839P c.2516C mutation. For FIGs. 2A-2D, PBS and RTT lengths are listed as nucleotides lengths and data collected from one biological replicate. All ngRNAs are identified by their nicking position relative to the epegRNA nick (in base pairs). B1195.70198WO00 13849370.1 (April 11, 2025)17 / 895

[0043] FIGs. 3A-3E show the spacer, PBS, and RTT screen for correction of ATP1A3 D801N c.2401A. Monoclonal HEK293T cells heterozygous for the ATP1A3 D801N c.2401A mutation were transfected with epegRNA plasmids to optimize epegRNA spacer selection, PBS and RTT lengths, and the selection of silent edit installation strategies. Based on HTS analysis of unedited ATP1A3 D801N HEK293T cells (see NEG x-axis label in each plot), monoclonal ATP1A3 D801N HEK293T cells were presumed triploid for ATP1A3 with two alleles bearing the D801N c.2401A mutation and one allele bearing the wild-type D801 c.2401G allele. As such, PE sequencing data is shown as “percent specified genotype” and all samples start from a baseline of approximately 33% D801 c.2401G genotype. This baseline genotype is displayed on plots as a dotted line. FIG. 3A is a sequence schematic showing the pathogenic ATP1A3 D801N c.2401A mutation targeted for correction (c.2404A) and the two epegRNA protospacers (NGA1 and NGA2) used for PE. SpCas9(VRQR)-PEmax was used to target epegRNA protospacers with NGA PAMs due to an absence of mutation-proximal NGG PAMs. MLH1dn was not used to enhance editing in these experiments. FIG. 3B is sequence (top) schematic of the D801 c.2401G corrective edit (c.2401G) and accompanying silent editing strategy 1 (SE1) edits (ACTGAAT, where the C corresponds to c.2401G and the italicized nucleotides are the silent edits). The bottom is a bar chart of HTS-quantified D801N c.2401A genotype following editing with the protospacer NGA1, SE1, and variable PBS and RTT lengths. For each read total group in FIG. 3B, D801 c.2401G genotype is on the left and the indels are on the right. FIG. 3C is a sequence (top) schematic of the D801 c.2401G corrective edit (c.2401G) and accompanying silent editing strategy 2 (SE2) edits (CTGA, where the C corresponds to c.2401G and the italicized nucleotides are the silent edits). The bottom is a bar chart of HTS-quantified D801N c.2401A genotype following editing with the protospacer NGA1, SE2, and variable PBS and RTT lengths. For each read total group in FIG. 3C, D801 c.2401G genotype is on the left and the indels are on the right. FIG. 3D is a sequence schematic of the D801 c.2401G corrective edit (c.2401G) and accompanying silent editing strategy 3 (SE3) edits (ACTGA, where the C corresponds to c.2401G and the italicized nucleotides are the silent edits). Bottom: Bar chart of HTS- quantified D801N c.2401A genotype following editing with the protospacer NGA1, SE3, and variable PBS and RTT lengths. For each read total group in FIG. 3D, D801 c.2401G genotype is on the left and the indels are on the right FIG. 3E is a sequence (top) schematic of the D801 c.2401G corrective edit (c.5401G) and accompanying SE3 edits (ACTGA, where the C corresponds to c.2401G and the italicized nucleotides are the silent edits). The bottom B1195.70198WO00 13849370.1 (April 11, 2025)18 / 895 is a bar chart of HTS-quantified D801N c.2401A genotype following editing with the protospacer NGA2, SE3, and variable PBS and RTT lengths. The positioning of protospacer NGA2 prevented the use of SE1 or SE2. For each read total group in FIG. 3E, D801 c.2401G genotype is on the left, and the indels are on the right. For FIGs. 3B-3E, data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as dots). RTT and PBS values are sequence lengths measured in nucleotides.

[0044] FIGs. 4A-4E show the spacer, PBS, and RTT screen for correction of ATP1A3 E815K c.2443A. Monoclonal HEK293T cells heterozygous for the ATP1A3 E815K c.2443A mutation were transfected with epegRNA plasmids to optimize epegRNA spacer selection, PBS, and RTT lengths. A single silent edit installation strategy (SE1) was tested for all combinations of spacers, PBS lengths, and RTT lengths. Based on HTS analysis of unedited ATP1A3 E815K c.2443A HEK293T cells (see NEG x-axis label in each plot), monoclonal ATP1A3 E815K HEK293T cells were presumed triploid for ATP1A3 with one allele bearing the E815K c.2443A mutation and two alleles bearing the wild-type E815K c.2443G allele. As such, PE sequencing data is shown as “percent specified genotype” and all samples start from a baseline of approximately 66% E815 c.2443G genotype. This baseline genotype is displayed on plots as a dotted line. FIG. 4A is a sequence schematic showing the pathogenic ATP1A3 E815K c.2443A mutation targeted for correction (c.2443A) and the four epegRNA protospacers (NGG120nt, NGG119nt, NGG2, and NGG3) used for PE. PEmax was used to target epegRNA protospacers with NGG PAMs. MLH1dn was not used to enhance editing in these experiments. FIG. 4B is a sequence (top) schematic of the E815 c.2443G corrective edit (c.2443G) and accompanying SE1 edits (ACTT, where the C corresponds to c.2443G and the italicized nucleotides are the silent edits). Bottom: Bar chart of HTS-quantified E815 c.2443G genotype following editing with the protospacer NGG120nt, SE1, and variable PBS and RTT lengths. For each read total group in FIG. 4B, E815 c.2443G genotype is on the left and the indels are on the right. FIG. 4C is a sequence (top) schematic of the E815 c.2443G corrective edit (c.2443G) and accompanying SE1 edits (ACTT where the C corresponds to c.2443G and the italicized nucleotides are the silent edits). The bottom is a bar chart of HTS- quantified E815 c.2443G genotype following editing with the protospacer NGG119nt, SE1, and variable PBS and RTT lengths. For each read total group in FIG. 4C, E815 c.2443G genotype is on the left and the indels are on the right. FIG. 4D is a sequence (top) schematic of the E815 c.2443G corrective edit (c.2443G) and accompanying SE1 edits (ACTT where B1195.70198WO00 13849370.1 (April 11, 2025)19 / 895 the C corresponds to c.2443G and the italicized nucleotides are the silent edits). The bottom is a bar chart of HTS-quantified E815 c.2443G genotype following editing with the protospacer NGG2, SE1, and variable PBS and RTT lengths. For each read total group in FIG. 4D, E815 c.2443G genotype is on the left and the indels are on the right. FIG. 4E is a sequence (top) schematic of the E815 c.2443G corrective edit (c.2443G) and accompanying SE1 edits (ACTT where the C corresponds to c.2443G and the italicized nucleotides are the silent edits). The bottom is a bar chart of HTS-quantified E815 c.2443G genotype following editing with the protospacer NGA3, SE1, and variable PBS and RTT lengths. For each read total group in FIG. 4E, E815 c.2443G genotype is on the left and the indels are on the right. For FIGs. 4B-4E, data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as dots). RTT and PBS values are sequence lengths measured in nucleotides.

[0045] FIGs. 5A-5C show the spacer, PBS, and RTT screen for correction of ATP1A3 G947R c.2839C. Monoclonal HEK293T cells homozygous for the ATP1A3 G947R c.2839C mutation were transfected with epegRNA plasmids to optimize epegRNA spacer selection, PBS, and RTT lengths. A single silent edit installation strategy (SE1) was tested for all combinations of spacers, PBS lengths, and RTT lengths. Based on HTS analysis of unedited ATP1A3 G947R c.2839C HEK293T cells (see NEG x-axis label in each plot), monoclonal ATP1A3 G947R c.2839C HEK293T cells were presumed homozygous for the G947R c.2839C mutation. As such, PE sequencing data is shown as “percent specified genotype” and all samples start from a baseline of 0% G947 c.2839G genotype. FIG. 5A is a sequence schematic showing the pathogenic ATP1A3 G947R c.2839C mutation targeted for correction (c.2839C) and the two epegRNA protospacers (NGG1 and NGG2) used for PE. PEmax was used to target epegRNA protospacers with NGG PAMs. MLH1dn was not used to enhance editing in these experiments. FIG. 5B is a sequence (top) schematic of the G947 c.2839G corrective edit (c.2839G) and accompanying SE1 edits (ACCGA, where the C corresponds to c.2839G and the italicized nucleotides are the silent edits). The bottom is a bar chart of HTS- quantified G947 c.2839G genotype following editing with the protospacer NGG1, SE1, and variable PBS and RTT lengths. For each read total group in FIG. 5B, G947 c.2839G genotype is on the left and the indels are on the right. FIG. 5C is a sequence (top) schematic of the G947 c.2839G corrective edit (c.2839G) and accompanying SE1 edits (ACCGA, where the C corresponds to c.2839G and the italicized nucleotides are the silent edits). The bottom is a bar chart of HTS-quantified G947 c.2839G genotype following editing with the B1195.70198WO00 13849370.1 (April 11, 2025)20 / 895 protospacer NGG2, SE1, and variable PBS and RTT lengths. For each read total group in FIG. 5B, G947 c.2839G genotype is on the left and the indels are on the right. For FIG. 5B and FIG. 5C, data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as dots). RTT and PBS values are sequence lengths measured in nucleotides.

[0046] FIGs. 6A-6D show the spacer, PBS, and RTT screen for correction of ATP1A3 L839P c.2516C. Monoclonal HEK293T cells homozygous for the ATP1A3 L839P c.2516C mutation were transfected with epegRNA plasmids to optimize epegRNA spacer selection, PBS, and RTT lengths. A single silent edit installation strategy (SE1) was tested for all combinations of spacers, PBS lengths, and RTT lengths. Based on HTS analysis of unedited ATP1A3 L839P c.2516C HEK293T cells (see NEG x-axis label in each plot), monoclonal ATP1A3 L839P c.2516C HEK293T cells were presumed homozygous for the L839P c.2516C mutation. As such, PE sequencing data is shown as “percent specified genotype” and all samples start from a baseline of 0% L839 c.2516T genotype. FIG. 6A is a sequence (top) schematic showing the pathogenic ATP1A3 L839P c.2516C mutation targeted for correction (c.2516C) and the three epegRNA protospacers (NGG120nt, NGG119nt, and NGG2) used for PE. PEmax was used to target epegRNA protospacers with NGG PAMs. MLH1dn was not used to enhance editing in these experiments. FIG. 6Bis a sequence (top) schematic of the L839 c.2516T corrective edit (c2516T) and accompanying SE1 edits (GTTG, where the T corresponds to c.2516T and the italicized nucleotides are the silent edits). Bottom: Bar chart of HTS-quantified L839 c.2516T genotype following editing with the protospacer NGG1 20nt, SE1, and variable PBS and RTT lengths. For each read total group in FIG. 6B, L839 c.2516T genotype is on the left and the indels are on the right. FIG. 6C is a sequence (top) schematic of the L839 c.2516T corrective edit (c.2516T) and accompanying SE1 edits (GTTG, where the T corresponds to c.2516T and the italicized nucleotides are the silent edits). Bottom: Bar chart of HTS-quantified L839 c.2516T genotype following editing with the protospacer NGG119nt, SE1, and variable PBS and RTT lengths. For each read total group in FIG. 6C, L839 c.2516T genotype is on the left and the indels are on the right. FIG. 6Dis a sequence (top) schematic of the L839 c.2516T corrective edit (c.2516T) and accompanying SE1 edits (GTTG, where the T corresponds to c.2516T and the italicized nucleotides are the silent edits). Bottom: Bar chart of HTS-quantified L839 c.2516T genotype following editing with the protospacer NGG2, SE1, and variable PBS and RTT lengths. For each read total group in FIG. 6D, L839 c.2516T genotype is on the left and the indels are on B1195.70198WO00 13849370.1 (April 11, 2025)21 / 895 the right. For FIGs. 6B-6D, data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as dots). RTT and PBS values are sequence lengths measured in nucleotides.

[0047] FIG. 7 shows the correction of ATP1A3 D801N c.2401A with a panel of ngRNAs. Monoclonal HEK293T cells heterozygous for the ATP1A3 D801N c.2401A mutation were transfected with ngRNA plasmids to optimize correction of D801N c.2401A using three different epegRNAs each optimized for silent edit strategies SE1-SE3 (detailed in FIGs. 3A- 3E). Numeric x-axis labels identify different ngRNAs by their nicking position relative to the epegRNA nick (in base pairs). Silent editing strategy and the epegRNA design used for each ngRNA screen are indicated below the x-axis. The “No Nick” x-axis label specifies a control PE condition in which the specified epegRNA was transfected without an ngRNA. PE was completed using SpCas9(VRQR)-PEmax. MLH1dn was not used to enhance editing in these experiments. Based on HTS analysis of unedited ATP1A3 D801N HEK293T cells (see NEG x-axis), monoclonal ATP1A3 D801N HEK293T cells were presumed triploid for ATP1A3 with two alleles bearing the D801N c.2401A mutation and one allele bearing the wild-type D801 c.2401G allele. As such, PE sequencing data is shown as “percent specified genotype” and all samples start from a baseline of approximately 33% D801 c.2401G genotype. This baseline genotype is displayed on the plot as a dotted line. For each read total group in FIG. 7, D801 c.2401G genotype is on the left and the indels are on the right. Data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as dots).

[0048] FIG. 8 shows the correction of ATP1A3 E815K c.2443A with a panel of ngRNAs. Monoclonal HEK293T cells heterozygous for the ATP1A3 E815K c.2443A mutation were transfected with ngRNA plasmids to optimize correction of E815K c.2443A using the best epegRNA design identified for each protospacer tested in FIGs. 4A-4E (listed as the title above each plot and excluding NGG1’s alternative 20 nt spacer variant). Numeric x-axis labels identify different ngRNAs by their nicking position relative to the epegRNA nick (in base pairs). The “No Nick” x-axis label specifies a control PE condition in which the specified epegRNA was transfected without an ngRNA. PE was completed using PEmax. MLH1dn was not used to enhance editing in these experiments. Based on HTS analysis of unedited ATP1A3 E815K c.2443A HEK293T cells (see NEG x-axis label in each plot), monoclonal ATP1A3 E815K HEK293T cells were presumed triploid for ATP1A3 with one allele bearing the E815K c.2443A mutation and two alleles bearing the wild-type E815K B1195.70198WO00 13849370.1 (April 11, 2025)22 / 895 c.2443G allele. As such, PE sequencing data is shown as “percent specified genotype” and all samples start from a baseline of approximately 66% E815 c.2443G genotype. This baseline genotype is displayed on the plot as a dotted line. For each read total group in FIG. 8, E815 c.2443G genotype is on the left and the indels are on the right. Data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as dots).

[0049] FIG. 9 shows the correction of ATP1A3 G947R c.2839C with a panel of ngRNAs. Monoclonal HEK293T cells homozygous for the ATP1A3 G947R c.2839C mutation were transfected with ngRNA plasmids to optimize correction of G947R c.2839C using the best epegRNA design identified for each protospacer tested in FIGs. 5A-5C (listed as the title above each plot). Numeric x-axis labels identify different ngRNAs by their nicking position relative to the epegRNA nick (in base pairs). The “No Nick” x-axis label specifies a control PE condition in which the specified epegRNA was transfected without an ngRNA. PE was completed using PEmax. MLH1dn was not used to enhance editing in these experiments. Based on HTS analysis of unedited ATP1A3 G947R c.2839C HEK293T cells (see NEG x- axis label in each plot), monoclonal ATP1A3 G947R c.2839C HEK293T cells were presumed homozygous for the G947R c.2839C mutation. As such, PE sequencing data is shown as “percent specified genotype” and all samples start from a baseline of 0% G947 c.2839G genotype. For each read total group in FIG. 9, G947 c.2839G genotype is on the left and the indels are on the right. Data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as dots).

[0050] FIG. 10 shows the correction of ATP1A3 L839P c.2516C with a panel of ngRNAs. Monoclonal HEK293T cells homozygous for the ATP1A3 L839P c.2516C mutation were transfected with ngRNA plasmids to optimize correction of L839P c.2516C using the best epegRNA design identified for each protospacer tested in FIGs. 5A-5C (listed as the title above each plot and excluding NGG1’s alternative 20 nt spacer variant). Numeric x-axis labels identify different ngRNAs by their nicking position relative to the epegRNA nick (in base pairs). The “No Nick” x-axis label specifies a control PE condition in which the specified epegRNA was transfected without an ngRNA. PE was completed using PEmax. MLH1dn was not used to enhance editing in these experiments. Based on HTS analysis of unedited ATP1A3 L839P c.2516C HEK293T cells (see NEG x-axis label in each plot), monoclonal ATP1A3 L839P c.2516C HEK293T cells were presumed homozygous for the L839P c.2516C mutation. As such, PE sequencing data is shown as “percent specified B1195.70198WO00 13849370.1 (April 11, 2025)23 / 895 genotype” and all samples start from a baseline of 0% L839P c.2516T genotype. For each read total group in FIG. 10, L839 c.2516T genotype is on the left and the indels are on the right. Data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as dots).

[0051] FIG. 11 shows the correction of ATP1A3 D801N c.2401A with a panel of dsgRNAs. Monoclonal HEK293T cells heterozygous for the ATP1A3 D801N c.2401A mutation were transfected with dsgRNA plasmids to optimize correction of D801N c.2401A using the D801N NGA1 SE1 PBS12 RTT19 epegRNA and the +85 ngRNA. X-axis labels identify different dsgRNAs by their putative nicking position and protospacer strand relative to the epegRNA nick (in base pairs) and PAM, respectively. The “No dsgRNA” x-axis label specifies a control PE condition in which the epegRNA and ngRNA were transfected without a dsgRNA. PE was completed using SpCas9(VRQR)-PEmax. MLH1dn was not used to enhance editing in these experiments. Based on HTS analysis of unedited ATP1A3 D801N HEK293T cells (see NEG x-axis), monoclonal ATP1A3 D801N HEK293T cells were presumed triploid for ATP1A3 with two alleles bearing the D801N c.2401A mutation and one allele bearing the wild-type D801 c.2401G allele. As such, PE sequencing data is shown as “percent specified genotype” and all samples start from a baseline of approximately 33% D801 c.2401G genotype. This baseline genotype is displayed on the plot as a dotted line. For each read total group in FIG. 11, D801 c.2401G genotype is on the left and the indels are on the right. Data and error bars represent mean and standard deviation, respectively, collected from at least two independent biological replicates (shown as dots).

[0052] FIGs. 12A-12D show the summary of optimized PE correction strategies for four AHC ATP1A3 mutations. Summary of the best PE editing strategies for (FIG. 12A) ATP1A3 D801N c.2401A (summarized from FIG. 11), (FIG. 12B) ATP1A3 E815K c.2443A (summarized from FIG. 8), (FIG. 12C) ATP1A3 G947R c.2839C (summarized from FIG. 9), and (FIG. 12D) ATP1A3 L839P c.2516C (summarized from FIG. 10). For FIGs. 12A-12D, “PE-corrected (mutation) alleles” is equal to the difference of percent HTS-measured genotype and percent unedited genotype (“No Edit”) divided by the difference of 100% and percent unedited genotype.

[0053] FIGs. 13A-13B show the ABE correction of ATP1A3 G947R c.2839A. Monoclonal HEK293T cells homozygous for the ATP1A3 G947R c.2839A mutation were transfected with SpCas9-ABE8e and ABE sgRNA plasmid G947R ABE NGG1 to evaluate correction of G947R c.2839A. The “ABE8e” x-axis label specifies the sample treated with B1195.70198WO00 13849370.1 (April 11, 2025)24 / 895 SpCas9-ABE8e and the G947R ABE NGG1 sgRNA. The “No Edit” x-axis label specifies a control PE condition in which no editing reagents were transfected. Based on HTS analysis of unedited ATP1A3 G947R c.2839A HEK293T cells (see “No Edit” x-axis label), monoclonal ATP1A3 G947R c.2839A HEK293T cells were presumed homozygous for the G947R c.2839A mutation. As such, PE sequencing data is shown as “percent specified genotype” and all samples start from a baseline of 0% G947 c.2839G genotype. Data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as dots).

[0054] FIGs. 14A-14B show BE and PE correction of ATP1A3 mutations in AHC- patient-derived iPSCs. AHC-patient derived iPSCs were electroporated with editor mRNA and synthetic guide RNAs to evaluate correction of ATP1A3 G947R c.2839A and ATP1A3 E815K c.2443A. FIG. 14A shows in the “ABE8e” x-axis label, iPSCs from three AHC patients with the G947R c.2839A mutation were electroporated with SpCas9-ABE8e mRNA and synthetic G947R ABE NGG1 sgRNA. The “No Edit” x-axis label specifies a control BE condition in which no editing reagents were transfected into the same cells. Data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as dots). FIG. 14B, shows, in the “PE” x-axis label, iPSCs from an AHC patient were electroporated with PEmax mRNA, MLH1dn mRNA, and synthetic RNA versions of the epegRNA and ngRNA specified in FIG. 12B. The “No Edit” x-axis label specifies a control PE condition in which no editing reagents were transfected. E815K data is from a single replicate. In FIGs. 14A and 14B iPSCs from AHC patients are heterozygous for pathogenic ATP1A3 mutations. As such, PE and BE sequencing data is shown as “percent specified genotype” and all samples start from a baseline of 50% non- pathogenic genotype.

[0055] FIGs 15A-15B show the comparison of ATP1A3 and Atp1a3 sequence around D801N and E815K mutations. Sequence schematics comparing (FIG. 15A) ATP1A3 D801N c.2401A and Atp1a3 D801N c.2401A and (FIG. 15B) ATP1A3 E815K c.2443A and Atp1a3 E815K c.2443A. AHC-causing pathogenic mutations are colored in red. Sequence disagreements between human and mouse sequences are shown in Atp1a3 schematics with text and linked to corresponding human sequence base pairs with black lines. Optimized epegRNA sequence elements for the ATP1A3 D801N and E815K mutations (summarized in FIG. 12) are annotated on their respective sequence schematics. For conciseness, encoded silent edits are omitted from RTT annotations. B1195.70198WO00 13849370.1 (April 11, 2025)25 / 895

[0056] FIGs. 16A-16C show the development of a Atp1a3 E815K c.2443A PE strategy. Mouse primary fibroblasts derived from E815K mice were electroporated with prime editor mRNA and synthetic guide RNAs to evaluate correction of Atp1a3 E815K c.2443A. (FIG. 16A) Correction of Atp1a3 E815K c.2443A with PEmax, the Atp1a3 E815K NGG1 SE1 PBS13 RTT25 epegRNA, and the Atp1a3 +49 ngRNA. FIG. 16B shows the correction of Atp1a3 E815K c.2443A with PEmax and PE6 prime editor variants. The Atp1a3 E815K NGG1 SE1 PBS13 RTT25 epegRNA and the Atp1a3 +49 ngRNA were used with each prime editor. FIG. 16C shows a screen of dsgRNAs to enhance correction of Atp1a3 E815K c.2443A with PE6c, the Atp1a3 E815K epegRNA, and the Atp1a3 +49 nsgRNA. X-axis labels identify different dsgRNAs by their putative nicking position and protospacer strand relative to the epegRNA nick (in base pairs) and PAM, respectively. The “No dsgRNA” x- axis label specifies a control PE condition in which PE6c, the epegRNA, and ngRNA were co-electroporated with a non-targeting dsgRNA. For each read total group in FIG. 16C, E815 c.2443G genotype is on the left and the indels are on the right. For FIGs. 16A-16C, the “No Edit” x-axis label specifies HTS data from untreated cells. E815K mice primary fibroblasts are heterozygous for Atp1a3 E815K c.2443A. As such, PE and BE sequencing data is shown as “percent specified genotype” and all samples start from a baseline of 50% non-pathogenic genotype. This baseline genotype is displayed on plots as a dotted line. Data collected from one biological replicate. MLH1dn was not used in these experiments.

[0057] FIGs. 17A-17C show the optimization of PE strategies to install Atp1a3 D801N c.2401A in N2A cells. FIG. 17A is a sequence schematic of the PE strategy to introduce the Atp1a3 D801N c.2401A mutation into N2A cells. In addition to the D801N c.2401A mutation (shown in red), a translationally silent L802 c.2406A mutation was introduced into the N2As to match the Atp1a3 sequence present in the D801N mice. In FIG. 17B, to a identify PE strategy to install Atp1a3 D801N c.2401A in N2A cells, panels of pegRNAs with variable PBS lengths and RTT lengths were transfected into N2A cells. For each read total group in FIG. 17B, the following groups should be read left to right: D801N c.2401A genotype, L802 c.2406A genotype, and Indels. In FIG. 17C, using two of the best PBS and RTT lengths identified in (FIG. 17B) to generate new installation epegRNAs, a screen of nicking sgRNAs to enhance installation of Atp1a3 D801N c.2401A and L802 c.2406A was transfected into N2A cells. One of the most efficient epegRNA + nick strategies, PBS13 RTT23 with the +58 ngRNA, was selected to generate a monoclonal N2A cell lines with the Atp1a3 D801N c.2401A and L802 c.2406A mutations. For each read total group in FIG. 17C, B1195.70198WO00 13849370.1 (April 11, 2025)26 / 895 the following groups should be read left to right: D801N c.2401A genotype, L802 c.2406A genotype, and Indels. For FIGs. 17B-17C, PBS and RTT lengths are listed as nucleotides lengths and data collected from one biological replicate. PE was completed using PE2. Two monoclonal cell lines were isolated, one heterozygous for the Atp1a3 D801N c.2401A and L802 c.2406A mutations and one homozygous for the Atp1a3 D801N c.2401A and L802 c.2406A mutations. For FIG. 17C, numeric x-axis labels identify different ngRNAs by their nicking position relative to the epegRNA nick (in base pairs). MLH1dn was not used in these experiments.

[0058] FIGs.18A-18D show the development of an Atp1a3 D801N c.2401A PE correction strategy with epegRNA, ngRNA, and dsgRNA screens. Monoclonal N2A cells with the Atp1a3 D801N c.2401A mutation were transfected with plasmids to develop an Atp1a3 D801N c.2401A PE correction strategy. FIG. 18Ais a sequence (top) schematic showing pathogenic Atp1a3 D801N c.2401A (in red) and the Atp1a3 D801N NGG1 protospacer used for correction. The bottom portion is a sequence schematic of the Atp1a3 D801N c.2401G corrective edit and accompanying SE1 edits. FIG. 18B shows monoclonal N2A cells heterozygous for the Atp1a3 D801N c.2401A mutation were transfected with epegRNA plasmids to optimize PBS and RTT lengths. PEmax and the +58 ngRNA from (FIG.17) were used in the transfection. A single silent edit installation strategy (SE1, FIG. 18A) was tested for all combinations of PBS and RTT lengths. Based on HTS analysis of the unedited Atp1a3 D801N c.2401A N2A cells used in this experiment (see NEG x-axis label in plot), monoclonal Atp1a3 D801N c.2401A cells were presumed triploid for Atp1a3 with one allele bearing the D801N c.2401A mutation and two alleles bearing the wild-type D801N c.2401G allele. As such, PE sequencing data is shown as “percent specified genotype” and all samples start from a baseline of approximately 66% D801N c.2401G genotype. This baseline genotype is displayed on the plot as a dotted line. MLH1dn was used to enhance editing in this experiment. For each read total group in FIG. 18B, D801n c.2401A genotype is on the left and the indels are on the right. Data collected from one biological replicate. FIG. 18C shows monoclonal N2A cells homozygous for the Atp1a3 D801N c.2401A mutation were transfected with ngRNA plasmids to optimize correction of D801N c.2401A using PEmax and the Atp1a3 D801N NGG1 SE1 PBS9 RTT23 epegRNA (see FIG. 18B). Numeric x-axis labels identify different ngRNAs by their nicking position relative to the epegRNA nick (in base pairs). The “No Nick” x-axis label specifies a control PE condition in which PEmax and the epegRNA were transfected without an ngRNA. MLH1dn was not used to B1195.70198WO00 13849370.1 (April 11, 2025)27 / 895 enhance editing in this experiment. Data collected from one biological replicate. FIG. 18D shows monoclonal N2A cells homozygous for the Atp1a3 D801N c.2401A mutation were transfected with dsgRNA plasmids to optimize correction of D801N c.2401A using PEmax, the Atp1a3 D801N NGG1 SE1 PBS9 RTT23 epegRNA, and the +7 PE3b ngRNA (see FIG. 18C). X-axis labels identify different dsgRNAs by their putative nicking position and protospacer strand relative to the epegRNA nick (in base pairs) and PAM, respectively. The “No dsgRNA” x-axis label specifies a control PE condition in which the epegRNA and ngRNA were transfected without a dsgRNA. For each read total group in FIG. 18D, D801N c.2401A genotype is on the left and the indels are on the right. Data and error bars represent mean and standard deviation, respectively, collected from at least three independent biological replicates (shown as dots). MLH1dn was not used to enhance editing in these experiments. For FIGs. 18C and 18D, based on HTS analysis of the unedited Atp1a3 D801N c.2401A N2A cells used for these experiments (see NEG x-axis label in both plots), monoclonal Atp1a3 D801N c.2401A N2A cells were presumed homozygous for the D801N c.2401A mutation. As such, PE sequencing data is shown as “percent specified genotype” and all samples start from a baseline of 0% D801N c.2401A genotype.

[0059] FIG. 19 shows Atp1a3 D801N c.2401A PE correction in D801N mouse primary fibroblasts. Mouse primary fibroblasts derived from D801N mice were electroporated with prime editor mRNA, and synthetic guide RNAs to evaluate correction of Atp1a3 D801N c.2401A with various prime editor variants and the -52 dsgRNA. All conditions completed with the Atp1a3 D801N NGG1 SE1 PBS9 RTT23 epegRNA and the +7 PE3b ngRNA. MLH1dn was not included in this experiment. The “No Edit” x-axis label specifies HTS data from untreated cells. D801N mice primary fibroblasts are heterozygous for Atp1a3 D801N c.2401A. As such, PE sequencing data is shown as “percent specified genotype” and all samples start from a baseline of 50% non-pathogenic genotype. This baseline genotype is displayed on plots as a dotted line. Data collected from one biological replicate. The Y-axis is split into three segments to better compare indel rates, display the “No Edit” data, and display data from all other conditions.

[0060] FIGs.20A-20B show the summary of optimized PE correction strategies for two AHC Atp1a3 mutations. Summary of PE editing strategies for (FIG. 20A) Atp1a3 D801N c.2401A (summarized from FIG. 19), (FIG. 20B) Atp1a3 E815K c.2443A (summarized from FIG. 16). For FIGs. 20A-20B, “PE-corrected (mutation) alleles” is equal to the difference of B1195.70198WO00 13849370.1 (April 11, 2025)28 / 895 percent HTS-measured genotype and percent unedited genotype (“No Edit”) divided by the difference of 100% and percent unedited genotype.

[0061] FIGs. 21A-21F shows how D801N-PE-AAV9 prime editing modifies DNA, RNA, and protein in P28 mice. FIG. 21A shows D801N-PE-AAV9 dual AAV9 system encoding split-intein PE6c prime editor halves, the Atp1a3 D801N NGG1 SE1 PBS9 RTT23 epegRNA (hU6-epegRNA), the +7 PE3b ngRNA (mU6-ngRNA), and the -52 dsgRNA (hU6- dsgRNA). P0 intracerebroventricular (ICV) injections (5 × 1010vg of each AAV, 1 × 1011vg total) were administered to each mouse, in addition to an eGFP AAV (1 × 1010vg) to fluorescently mark transduced cells for sorting. Labeled as “PE-injected” or “PE-treated” sub-figures. ITR: inverted terminal repeats; Npu: Nostoc punctiforme; EFS: elongation factor 1α short; hU6: human U6 polymerase III promoter; mU6: mouse U6 polymerase III promoter. Schematic modified from49FIG. 21B shows precise HTS-measured Atp1a3 D801N c.2401G genotypes from gDNA of bulk and GFP-positive (GFP+) nuclei from specified brain regions of mice injected with (FIG. 21A) and collected for necropsy at P28. FIG. 21C shows indels from editing in (FIG. 21B). FIG. 21D shows precise HTS-measured Atp1a3 D801N c.2401G genotypes from cDNA of bulk and GFP-positive (GFP+) nuclei from specified brain regions of mice injected with (FIG. 21A) and collected for necropsy at P28. FIG. 21E shows indels from editing in (FIG. 21C). Note that indel analysis from cDNA is imperfect due to the close proximity of D801N c.2401 to an intron. FIG. 21F shows specific ATP hydrolysis activity of Atp1a3 measured from hippocampal homogenates of mice with specified genotype, injected with (FIG. 21A) and collected for necropsy at P28. P-values, determined by two-sided unpaired T-test, are shown. For FIGs. 21B and 21D, D801N mice are heterozygous for Atp1a3 D801N c.2401A. As such, PE sequencing data is shown as “percent specified genotype” and all samples start from a baseline of approximately 50% D801N c.2401G genotype. This baseline genotype is displayed on plots as a dotted line. For FIGs. 21B-21F, data and error bars represent mean and standard deviation, respectively, and were collected from at least three mice (shown as dots). Vehicle treatment was an equivalent volume injection of phosphate-buffered saline. For FIGs. 21B-21E bulk and GFP+nuclei subpopulations were sorted by fluorescence-activated cell sorting (FACS).

[0062] FIGs.22A-22D show the survival and bodyweight or D801N mice treated with D801N-PE-AAV9. WT and D801N mice were treated with D801N-PE-AAV9 as described in FIG. 21A or vehicle (phosphate-buffered saline). FIG. 22A shows a Kaplan-Meier survival curve for male WT and D801N mice treated with vehicle or D801N-PE-AAV9. The size of B1195.70198WO00 13849370.1 (April 11, 2025)29 / 895 each cohort is displayed in the plot’s legend. FIG. 22B shows a Kaplan-Meier survival curve for female WT and D801N mice treated with vehicle or D801N-PE-AAV9. The size of each cohort is displayed in the plot’s legend. FIG. 22C shows weights of male WT and D801N mice treated with vehicle or D801N-PE-AAV9. The size of each cohort is displayed in the plot’s legend. Data points and error bars represent mean and standard deviation, respectively, for the number of surviving mice at each time point. FIG. 22D shows weights of female WT and D801N mice treated with vehicle or D801N-PE-AAV9. The size of each cohort is displayed in the plot’s legend. Data points and error bars represent mean and standard deviation, respectively, for the number of surviving mice at each time point. For (a-d), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0063] FIGs. 23A-23H shows the behavioral assays characterize effect of D801N-PE- AAV9 treatment on D801N mice. FIG. 23A shows rotarod performance of WT and D801N mice at 12 weeks old. FIG. 23B shows vertical activity of WT and D801N mice during open field test at 14 weeks old. FIG. 23C shows paroxysmal event score of WT and D801N mice subject to HIP test at 10 weeks old. Scoring criteria outlined in Table A. FIG. 23D shows quantification of convulsive seizures observed in WT and D801N mice during HIP test at 10 weeks old. Convulsive seizures defined as tonic-clonic-like seizures. FIG. 23E shows quantification of convulsive seizure frequency in WT and D801N mice during HIP test at 10 weeks old. Convulsive seizures defined as tonic-clonic-like seizures. FIG. 23F shows the figure legend for FIGs. 23A-23E and the groups should be read from left to right in each graph as: WT (vehicle), D801N (vehicle), WT (D801N-PE-AAV9), and D801N (D801N-PE- AAV9). FIG. 23G shows quantification of latency to regain righting / balance in WT and D801N mice following HIP test at 10 weeks old. Righting / balance is defined as the mouse’s ability to stay balanced and upright, but mice do not yet return to normal voluntary movement. (FIG. 23H) Quantification of latency to regain mobility in WT and D801N mice following HIP test at 10 weeks old. Mobility is defined as the return to normal, voluntary movement, including licking, grooming, moving, and walking. For FIGs. 23A-23H, all dots represent individual mice, significance assessed by two-way ANOVA with Tukey correction for multiple comparison, and *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. D801N-PE-AAV9 treatments described in FIG.21A. Vehicle treatment was injection of phosphate-buffered saline.

[0064] FIG. 24 shows the design of a PE3b / PE5b nicking sgRNA. To use the PE3b or PE5b systems, a PAM needs to be present on the non-edited strand close to the edit. A B1195.70198WO00 13849370.1 (April 11, 2025)30 / 895 nicking sgRNA can then be designed such that it can only bind and direct nicking of the non- edited strand after reverse transcription and flap equilibration have occurred. Such a PE3b / PE5b nicking sgRNA has a spacer that is perfectly complementary to the edited DNA sequence, but contains mismatches with the unedited sequence.

[0065] FIGs. 25A-25N show BE and PE efficiently correct AHC mutations in engineered HEK293T cells and iPSCs from patients with AHC. FIG. 25A shows the frequency of common ATP1A3 mutations in AHC cases, adapted from reference.6FIG. 25B shows base editing by Cas9 HNH nickase fused to a ssDNA-deaminase (PDB: 6VPC) directs targeted deamination in complex with a sgRNA. FIG. 25C shows prime editing by Cas9 RuvC nickase fused to a reverse transcriptase (PDB: 8WUT) directs precise editing in complex with an epegRNA. The nicked intermediate hybridizes to the PBS and the RTT specifies the desired edit. FIG. 25D shows the assessment of editing precision upon ABE treatment (D801N c.2401A, E815K c.2443A, G947R c.2839A) or CBE treatment (L839P c.2516C) via transfection in HEK293T clonal cell lines. The y-axis shows the absolute percent change in sequencing reads that correspond to the precise WT allele, and the x-axis shows the cumulative sum of all bystander editing events within the protospacer sequence consistent with ABE- or CBE-dependent single point mutations. Each dot represents the outcome of base editing with a unique combination of target protospacer and deaminase variant with the appropriate PAM-compatible Cas9 variant as the average of n = 3 independent biological replicates. See FIGs. 30A-30D for more details. FIGs. 25E-25I Top: Sequence schematic demonstrating the correction of ATP1A3 mutations to the wild-type base and the co-installation of benign silent edits. Bottom: Subset of optimization experiments in a monoclonal HEK239T cell line bearing the target mutation. Conditions selected for use in further experiments are highlighted in a box. All data represent the mean of n = 3 independent biological replicates. FIG.25E shows ATP1A3 D801N c.2401A correction with NGA2 20nt SE1 epegRNAs in D801N c.2401A (+ / – / –) cell line, highlighting NGA220nt SE1 PBS12 RTT19, related to FIG. 31B and 31C. FIG. 25F shows ATP1A3 E815K c.2443A correction with NGG119nt SE1 epegRNAs in E815K c.2443A (+ / + / –) cell line, highlighting NGG1 19nt SE1 PBS13 RTT25, related to FIG. 32B and 32C. FIG. 25G shows ATP1A3 L839P c.2516C correction with NGG119nt SE1 epegRNAs in L839P c.2516C (– / – / –) cell line, highlighting NGG119nt SE1 PBS12 RTT19, related to FIG. 33B. FIG. 25H shows ATP1A3 G947R c.2839C correction with NGG120nt SE1 epegRNAs in G947R c.2839C (– / – / –) cell line, highlighting NGG120nt SE1 PBS14 RTT17, related to FIG. 34B. FIG. 25I B1195.70198WO00 13849370.1 (April 11, 2025)31 / 895 shows ATP1A3 G947R c.2839A correction with ABE sgRNAs in G947R c.2839A (– / – / –) cell line, highlighting G947R BE-3, related to FIG. 30D. (J-N) Monoclonal iPSCs derived from patients with AHC with heterozygous ATP1A3 mutations were electroporated to evaluate correction with chemically synthesized guide RNA component(s) and in vitro transcribed editor mRNA. For PE (J-M), X-axis labels identify conditions with and without MLH1dn mRNA supplementation (“MLH1dn +” and “MLH1dn –”, respectively). The “No edit” x-axis label represents a control condition in which cells were electroporated without RNA editing reagents. Because iPSCs derived from patients with AHC are heterozygous for pathogenic ATP1A3 mutations, sequencing data are presented as “percent specified genotype” and all samples start from approximately 50% wild-type genotype. Data represent the mean of n = 3 independent biological replicates. Dots show individual replicate values. FIG. 25J shows PE correction of D801N c.2401A with NGA220nt SE1 PBS12 RTT19 epegRNA, +49 ngRNA, and –44 non-PAM dsgRNA VRQR-PE6c mRNA, with replicates from three donors. FIG. 25K shows PE correction of E815K c.2443A with NGG119nt SE1 PBS13 RTT25 epegRNA, +49 ngRNA, and –42 PAM dsgRNA and PE6b mRNA, with replicates from three donors. FIG. 25L shows PE correction of L839P c.2516C with NGG119nt SE1 PBS12 RTT19 epegRNA, –1 ngRNA, and –36 PAM dsgRNA and PE6b mRNA, with replicates from one donor (using two different monoclonal lines derived from a single donor: one line was edited twice, and the other line was edited once). FIG. 25M shows PE correction of G947R c.2839C with NGG120nt SE1 PBS14 RTT17 epegRNA, –1 ngRNA, and –49 PAM dsgRNA and PE6b mRNA, with replicates from two donors (using two different monoclonal lines from one donor and one monoclonal line from a second donor). FIG. 25N shows BE correction of G947R c.2839A with G947R BE-3 sgRNA and ABE8e-SpCas9 mRNA, with replicates from three donors.

[0066] FIGs.26A-26F show off-target editing analysis in iPSCs derived from patients with AHC (FIGs. 26A and 26B) Off-target editing at 457 CIRCLE-seq nominated sites shown as the ratio of indels (FIG. 26A) or substitutions (FIG. 26B) in treated versus untreated samples. All substitutions were quantified for PE, and A•T-to-G•C substitutions at protospacer nucleotides 4–10 were quantified for BE. Each dot represents editing at an off- target site, shown as the ratio of the mean of editor-treated versus mock controls (n = 3 independent biological replicates) in monoclonal AHC patient-derived iPSCs assayed via rhAmpSeq. Sites surpassing 1.4-fold increase (dashed line) are highlighted. The top 32 sites by CIRCLE-seq read counts are shown for the pegRNA, ngRNA, and dsgRNA for each PE B1195.70198WO00 13849370.1 (April 11, 2025)32 / 895 correction strategy, and the top 96 sites by CIRCLE-seq read counts are shown for the sgRNA for the ABE correction strategy. FIG. 26C shows the summary of off-target editing at the 24 validated genomic loci with off-target substitutions or indels exceeding the 0.5 log2- unit threshold. Editing is calculated as the genetic background-corrected increase in the percentage of sequencing reads with the specified mutation type. FIGs. 26D and 26E show tabulated genomic context of 24 verified off-target sites and 10 additional sites approaching the 0.5 log2-unit cutoff. Off-target (OT) epegRNA, ngRNA, and dsgRNA site are identified by numbers with the prefixes epegOT, ngOT, and dgOT, respectively. Gene annotations are provided where available. In FIG. 26E the horizontal bars represent the mean of n = 3 independent biological replicates for PE- or BE-treated samples (labeled with A) and untreated controls (labeled with B). Dots show individual replicate values. Sites are grouped by indels (FIG. 26D) and substitutions (FIG. 26E). FIG. 26F shows monoclonal iPSCs derived from patients with AHC with heterozygous ATP1A3 G947R c.2839A mutations were electroporated to evaluate correction with chemically synthesized guide RNA component(s) and in vitro transcribed editor mRNA. (Left) Sequencing data are presented as “percent specified genotype” and all samples start from approximately 50% wild-type genotype as the heterozygous baseline (dotted line). PE correction using NGG120nt SE1 PBS14 RTT17 epegRNA, –1 ngRNA, and –49 PAM dsgRNA and PE6b mRNA, with and without MLH1dn mRNA supplementation. BE correction using G947R BE-3 sgRNA with ABE8e-SpCas9, ABE8e(V106W)-SpCas9, or ABE7.10-SpCas9 mRNA. (Right) Ratio of edit-to-indels. FIG. 26G shows off-target indels (left) and substitutions (right) characterized as in (FIG. 26A) and (fig. 26B), where each dot represents editing at an off-target site, shown as the ratio of the mean of editor-treated versus mock controls (n = 3 independent biological replicates) in monoclonal AHC patient-derived iPSCs assayed via rhAmpSeq™. For the BE off-targets associated with G947R BE-3 sgRNA, connecting lines between identical off-target genomic loci are shown for the different base editor conditions. All PE off-targets are shown for NGG1 20nt SE1 PBS14 RTT17 epegRNA, –1 ngRNA, and –49 PAM dsgRNAs.

[0067] FIGs. 27A-27K show PE efficiently corrects Atp1a3 D801N c.2401A in vitro and in vivo (FIGs. 27A and 27B) Top: Sequence schematic demonstrating correction of (FIG. 27A) Atp1a3 D801 c.2401A to the wild type nucleotide (c.2401G) with co-installation of benign silent edits (CGATTTG, where the G corresponds to c.2401G and the italicized nucleotides are the silent edits); and (FIG. 27B) E815 c.2443A to the wild type nucleotide (c.2443G) with co-installation of benign silent edits (TGAA, where the G corresponds to B1195.70198WO00 13849370.1 (April 11, 2025)33 / 895 c.2401G and the italicized nucleotides are the silent edits). Bottom: Primary fibroblasts were electroporated with chemically synthesized guide RNA component(s) and IVT PE6c mRNA without MLH1dn (see “PE” x-axis label). The “No edit” x-axis label represents a control condition in which cells were electroporated without RNA editing reagents. Because fibroblasts were heterozygous for the pathogenic mutation, sequencing data are presented as “percent specified genotype” and all samples start from approximately 50% wild-type genotype. Data represent the mean of n = 3 independent biological replicates. Dots show individual replicate values. FIG. 27A shows correction in C57BL / 6J Atp1a3 D801N c.2401A mouse primary fibroblasts with NGG120nt SE1 PBS9 RTT23 epegRNA, +7 PE3b ngRNA, and –52 PAM dsgRNA. FIG. 27B shows correction in E815K mouse primary fibroblasts with NGG1 19nt SE1 PBS13 RTT25 epegRNA, +49 ngRNA, and –32 non-PAM dsgRNA. FIG. 27C shows D801N-PE-AAV9 system epegRNA, ngRNA, and dsgRNA components for Atp1a3 D801N c.2401A correction (top) and E815K-PE-AAV9 system epegRNA, ngRNA, and dsgRNA components for Atp1a3 E815K c.2443A correction (bottom). FIG. 27D shows PE-AAV9 dual AAV9 system encoding split-intein PE6c prime editor halves, epegRNA, ngRNA, and dsgRNA. P0 intracerebroventricular (ICV) injections (5×1010vg of each AAV, 1×1011vg total) were administered to each mouse, in addition to an eGFP AAV (1×1010vg) to fluorescently mark transduced cells for sorting. NpuNor NpuC: Nostoc punctiforme intein N-terminal or C-terminal halves, respectively; PEFS: Promoter, elongation factor 1α short; PhU6: Promoter, human U6 polymerase III; PmU6: Promoter, mouse U6 polymerase III; W3: minimized gamma portion of the woodchuck hepatitis virus post-transcriptional regulatory element; bGH: bovine growth hormone polyadenylation signal. FIG. 27E shows the study design included sets of molecular assays, behavioral assays, and biometric readouts. FIG. 27F-27I shows HTS-measured Atp1a3 genotypes or and indels from gDNA (FIGs. 27F and 27G) or cDNA (FIGs. 27H and 27I) of bulk and GFP-positive (GFP+) nuclei from specified brain regions of D801N mice (FIGs. 27F and 27H) or E815K mice (FIGs. 27G and 27I) injected with PE-AAV9 (“PE”) or an equivalent volume injection of phosphate-buffer saline (PBS, “vehicle”). Tissue collected for necropsy at P28. All samples start from a heterozygous baseline of approximately 50% genotype (dotted line), and sequencing data is shown as “percent specified genotype”. Dots show values from individual mice. Data represent the mean of n = 4-7 mice. FIGs. 27J and 27K show specific activity of Atp1a3 measured from hippocampal homogenates of mice with specified genotypes, treated with PE-AAV9 (“PE”) or PBS (“vehicle”). Tissue collected at P28.Data represent the mean of at least (J) n = 3-16 B1195.70198WO00 13849370.1 (April 11, 2025)34 / 895 mice and (K) n = 5-12 mice. Dots shows values from individual mice. Two-way ANOVA followed by Tukey's multiple comparisons test was used to determine P-values between groups. ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0068] FIGs. 28A-28J show D801N-PE-AAV9 improves survival and rescues multiple patient-relevant behavioral phenotypes (FIG. 28A) Kaplan-Meier survival curve for male WT and D801N mice treated with phosphate-buffered saline (vehicle) or D801N-PE- AAV9. Two-way ANOVA followed by Tukey's multiple comparisons test was used to determine P-values between groups. Kaplan-Meier survival curve for female WT and D801N mice treated with vehicle or D801N-PE-AAV9. Two-way ANOVA followed by Tukey's multiple comparisons test was used to determine P-values between groups. FIG. 28C shows hypothermia-induced paroxysmal episode assay. Hypothermia was induced by submersion in a 5 ºC water bath for a body weight-normalized duration, and mice were observed for paroxysmal events and recovery. Mice scored by blinded assessors according to standardized criteria on a scale from 0-5. FIGs. 28D-28G show results of hypothermia-induced paroxysmal episode assay for WT and D801N mice treated with PBS (Vehicle) or D801N-PE-AAV9 (PE-treated) for males (M, left) and females (F, right). Paroxysmal event scores (FIG. 28D) and number of convulsive-like events (FIG. 28E) were measured upon hypothermia induction. Recovery period was monitored for latency to regain righting / balance (FIG. 28F) and mobility (FIG. 28G). FIG. 28H shows latency to fall off an accelerating rotarod was measured for D801N and WT mice treated with PBS (vehicle) or D801N-PE-AAV9 for males (M, left) and females (F, right). FIGs. 28I and 28J show D801N and WT mice treated with vehicle or PE-AAV9 were subjected to open field testing to interrogate exploratory and / or locomotor activity in males (M, left) and females (F, right). (I) Vertical episodes count. FIG. 28J shows total distance traveled. For FIGs. 28D-28J, each dot represents data for a single mouse. For (FIGs. 28A-28B and 28D-28J), the size of each cohort is displayed in the plot’s legend. Two-way ANOVA followed by Tukey's multiple comparisons test was used to determine P-values between groups. ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0069] FIGs. 29A-29I show E815K-PE-AAV9 improves survival and rescues multiple patient-relevant behavioral phenotypes (FIGs. 29A-29D) results of hypothermia- induced paroxysmal episode assay for WT and E815K mice treated with phosphate-buffered saline (vehicle) or E815K-PE-AAV9 (PE-treated) for males (M, left) and females (F, right). Paroxysmal event scores (FIG. 29A) and number of convulsive-like events (FIG. 29B) were B1195.70198WO00 13849370.1 (April 11, 2025)35 / 895 measured upon hypothermia induction. Recovery period was monitored for latency to regain righting / balance (FIG. 29C) and mobility (FIG. 29D). FIG. 29E shows latency to fall off an accelerating rotarod was measured for E815K and WT mice treated with PBS (vehicle) or E815K-PE-AAV9 for males (M, left) and females (F, right). FIGs. 29F-29I show E815K and WT mice treated with vehicle or E815K-PE-AAV9 were subjected to open field testing to interrogate exploratory and / or locomotor activity in males (M, left) and females (F, right). FIG. 29F shows total distance traveled. FIG. 29G shows total movement time. FIG. 29H shows stereotypic episode count. FIG. 29I shows total rest time. For FIGs. 29A-29I, each dot represents data for a single mouse. The size of each cohort is displayed in the plot’s legend. Two-way ANOVA followed by Tukey's multiple comparisons test was used to determine P- values between groups. ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0070] FIGs. 30A-30D show BE of AHC-causing ATP1A3 mutations, related to FIGs. 30A-30L. In FIG. 30A the top is a schematic of the ATP1A3 D801N c.2401A mutation with protospacers of sgRNAs D801N BE-1 through D801N BE-6 annotated. and the bottom shows ABE editing with sgRNAs D801N BE-1 through D801N BE-6 in a clonal ATP1A3 D801N c.2401A HEK293T cell line. ABE editing was conducted with combinations of the ABE deaminases ABE7.1043, ABE8e, ABE8e(V106W)83, and ABE9163 and the Cas9(D10A) DNA binding domains SpRY112, SpCas9164, and SpCas9-NG165. ABE8e(V106W) is listed as 8e(V106w). Based on HTS analysis of the unedited ATP1A3 D801N c.2401A HEK293T cells (see “No edit” x-axis label), monoclonal ATP1A3 D801N c.2401A HEK293T cells are presumed triploid for ATP1A3 with two alleles bearing the D801N c.2401A mutation and one allele bearing the WT D801 c.2401G allele. As such, all samples start from a baseline of approximately 33% “Precise WT (c.2401G)” genotype, which is displayed on plots as a dotted line. Data and error bars represent the mean ± SD of n = 3 independent biological replicates. In FIG. 30B the top is a schematic of the ATP1A3 E815K c.2443A mutation with protospacers of sgRNAs E815K BE-1 through E815K BE-6 annotated and the bottom shows ABE editing with sgRNAs E815K BE-1 through E815K BE- 6 in a clonal ATP1A3 E815K c.2443A HEK293T cell line. ABE editing was conducted with combinations of the ABE deaminases ABE7.10, ABE8e, ABE8e(V106W), and ABE9 and the Cas9(D10A) DNA binding domains SpRY, VRQR90, NRCH, and NRTH166. ABE8e(V106W) is listed as 8e(V106w). Based on HTS analysis of the unedited ATP1A3 E815K c.2443A HEK293T cells (see “No edit” x-axis label), monoclonal ATP1A3 E815K B1195.70198WO00 13849370.1 (April 11, 2025)36 / 895 c.2443A HEK293T cells are presumed triploid for ATP1A3 with one allele bearing the E815K c.2443A mutation and two alleles bearing the WT E815K c.2443AG allele. As such, all samples start from a baseline of approximately 66% “Precise WT (c.2443G)” genotype, which is displayed on plots as a dotted line. Data and error bars represent the mean ± SD of n = 3 independent biological replicates. In FIG. 30C the top is a schematic of the ATP1A3 L839P c.2516C mutation with protospacers of sgRNAs L839P BE-1 through L839P BE-6 annotated and the bottom shows CBE editing with sgRNAs L839P BE-1 through L839P BE- 6 in a clonal ATP1A3 L839P c.2516C HEK293T cell line. CBE editing was conducted with combinations of the CBE deaminases BE4max167, YE1-BE3168, TadCBEd169, and CBE6b170 and the Cas9(D10A) DNA binding domains SpRY, SpCas9, and SpCas9-NG. Based on HTS analysis of the unedited ATP1A3 L839P c.2516C HEK293T cells (see “No edit” x-axis label), monoclonal ATP1A3 L839P c.2516C HEK293T cells are presumed homozygous for the ATP1A3 L839P c.2516C allele. As such, all samples start from a baseline of approximately 0% “Precise WT (c.2516T)” genotype. Data and error bars represent the mean ± SD of n = 3 independent biological replicates. In FIG. 30D, the top is a schematic of the ATP1A3 G947R c.2839A mutation with protospacers of sgRNAs G947R BE-1 through G947R BE-6 annotated and the bottom shows ABE editing with sgRNAs G947R BE-1 through G947R BE-6 in a clonal ATP1A3 G947R c.2839A HEK293T cell line. ABE editing was conducted with combinations of the ABE deaminase ABE8e and the Cas9(D10A) DNA binding domains SpRY and SpCas9. Based on HTS analysis of the unedited ATP1A3 G947R c.2839A HEK293T cells (see “No edit” x-axis label), monoclonal ATP1A3 G947R c.2839A HEK293T cells are presumed homozygous for the ATP1A3 G947R c.2839A allele. As such, all samples start from a baseline of approximately 0% “Precise WT (c.2839G)” genotype. Data and error bars represent the mean ± SD of n = 3 independent biological replicates.

[0071] FIGs. 31A-31F show optimization of PE strategies to correct ATP1A3 D801N c.2401A, related to FIGs. 25A-25N. FIG. 31A is a schematic (left) showing the pathogenic ATP1A3 D801N c.2401A mutation targeted for PE correction (c.2401A) and the two epegRNA protospacers (NGA120nt and NGA220nt) used for PE. On the right, three schematics of the D801 c.2401G corrective edit (c.2401G) with alternate concomitantly installed silent editing strategies 1 – 3 (SE1 – SE3). (SE1 = TGACTTA, where the G corresponds to c.2401G and the italicized nucleotides are the silent edits; SE2 = GACT, where the G corresponds to c.2401G and the italicized nucleotides are the silent edits; and B1195.70198WO00 13849370.1 (April 11, 2025)37 / 895 SE3 = TGACT, where the G corresponds to c.2401G and the italicized nucleotides are the silent edits). FIGs. 31B-31C shows monoclonal HEK293T cells heterozygous for the ATP1A3 D801N c.2401A mutation were transfected with epegRNA expression plasmids to optimize spacer selection, PBS and RTT lengths, and silent edit strategies to correct D801N c.2401A. SpCas9(VRQR)-PEmax was used to target epegRNA protospacers with NGA PAMs due to an absence of mutation-proximal NGG PAMs. Bar charts show HTS-quantified D801 c.2401G genotype and indels after editing with epegRNAs with different combinations of PBS and RTT lengths (in nucleotides), with protospacers and silent edit strategies listed above each chart. Three top performing epegRNAs (NGA220nt SE1 PBS12 RTT19, NGA2 20nt SE2 PBS12 RTT19, and NGA220nt SE3 PBS12 RTT19) were selected for subsequent editing optimization experiments. FIG. 31D shows monoclonal HEK293T cells heterozygous for the ATP1A3 D801N c.2401A mutation were transfected with ngRNA expression plasmids to optimize D801N c.2401A correction using SpCas9(VRQR)-PEmax and the epegRNAs NGA220nt SE1 PBS12 RTT19, NGA220nt SE2 PBS12 RTT19, and NGA220nt SE3 PBS12 RTT19. X-axis labels identify different ngRNAs by their nicking position relative to the epegRNA nick (in base pairs). The “No nick” x-axis label represents a control PE condition where an epegRNA was transfected without an ngRNA. A top performing epegRNA-ngRNA combination (NGA220nt SE1 PBS12 RTT19 and ngRNA +85) was selected for subsequent editing optimization experiments. FIG. 31E shows monoclonal HEK293T cells heterozygous for the ATP1A3 D801N c.2401A mutation were transfected with various prime editor plasmids to optimize D801N c.2401A correction with NGA220nt SE1 PBS12 RTT19 and ngRNA +85. A top performing prime editor (VRQR-PE6c) was selected for subsequent optimization experiments. FIG. 31F shows monoclonal HEK293T cells heterozygous for the ATP1A3 D801N c.2401A mutation were transfected with dsgRNA expression plasmids to optimize D801N c.2401A correction using NGA220nt SE1 PBS12 RTT19, ngRNA +85, and SpCas9(VRQR)-PE6c. Numeric x-axis labels identify different dsgRNAs by their putative nicking position relative to the epegRNA nick (in base pairs). The “No dsgRNA” x-axis label represents a control where no dsgRNA plasmid was transfected with the epegRNA, ngRNA, and prime editor plasmids. A top performing dsgRNA (–44 non- PAM) was selected for subsequent editing experiments. For FIGs. 31B-31F, based on HTS analysis of unedited ATP1A3 D801N cells (see “No edit” x-axis label), monoclonal HEK293T cells were presumed triploid for ATP1A3, with two alleles carrying the D801N c.2401A mutation and one allele with wild-type D801 c.2401G. Therefore, PE sequencing B1195.70198WO00 13849370.1 (April 11, 2025)38 / 895 data is shown as “percent specified genotype,” with all samples starting at approximately 33% wild type D801 c.2401G genotype, marked by a dotted line on each plot. Data represent the mean of n = 3 independent biological replicates, with dots showing individual replicate values.

[0072] FIGs. 32A-32F show optimization of PE strategies to correct ATP1A3 E815K c.2443A, related to FIGs. 25A-25N. FIG. 32A, is a schematic (left) showing the pathogenic ATP1A3 E815K c.2443A mutation targeted for PE correction (c.2443A) and the four epegRNA protospacers (NGG120nt, NGG119nt, NGG220nt, and NGG320nt) used for PE. On the right, is a schematic of the E815 c.2443G corrective edit (c.2443G) with the concomitantly installed silent editing strategy 1 (SE1, TGAA, where the C corresponds to c.2443G and the italicized nucleotides are the silent edits). FIGs. 32B-32C show monoclonal HEK293T cells heterozygous for the ATP1A3 E815K c.2443A mutation were transfected with epegRNA expression plasmids to optimize spacer selection, PBS lengths, and RTT lengths to correct E815K c.2443A with PEmax. Bar charts show HTS-quantified E815 c.2443G genotype and indels after editing with epegRNAs with different combinations of PBS and RTT lengths (in nucleotides), with protospacers and silent edit strategies listed above each chart. Three top performing epegRNAs (NGG119nt SE1 PBS13 RTT25, NGG2 20nt SE1 PBS11 RTT22, and NGG320nt SE1 PBS10 RTT25) were selected for subsequent editing optimization experiments. FIG. 32D shows monoclonal HEK293T cells heterozygous for the ATP1A3 E815K c.2443A mutation were transfected with ngRNA expression plasmids to optimize E815K c.2443A correction using PEmax and the epegRNAs NGG119nt SE1 PBS13 RTT25, NGG220nt SE1 PBS11 RTT22, and NGG320nt SE1 PBS10 RTT25. X-axis labels identify different ngRNAs by their nicking position relative to the epegRNA nick (in base pairs). The “No nick” x-axis label represents a control PE condition where an epegRNA was transfected without an ngRNA. A top performing epegRNA-ngRNA combination (NGG119nt SE1 PBS13 RTT25 and ngRNA +49) was selected for subsequent editing optimization experiments. FIG. 32E shows monoclonal ATP1A3 E815K c.2443A iPSCs derived from patients with AHC were electroporated with various prime editor mRNAs to optimize E815K c.2443A correction with chemically synthesized NGG119nt SE1 PBS13 RTT25 pegRNA and +49 ngRNA. MLH1dn mRNA was used to suppress MMR. A top performing prime editor (PE6b) was selected for subsequent optimization experiments. The “No edit” x-axis label specifies a control in which no editing reagents were transfected. FIG. 32F shows monoclonal ATP1A3 E815K c.2443A iPSCs derived from patients with AHC B1195.70198WO00 13849370.1 (April 11, 2025)39 / 895 were electroporated with chemically synthesized dsgRNAs to optimize E815K c.2443A correction with chemically synthesized NGG119nt SE1 PBS13 RTT25 pegRNA and +49 ngRNA, and PE6b mRNA. MLH1dn mRNA was used to suppress MMR. X-axis labels identify different dsgRNAs by their putative nicking position and protospacer strand relative to the pegRNA nick (in base pairs) and PAM, respectively. The “No dsgRNA” x-axis label represents a control PE condition in which a non-targeting dsgRNA was electroporated with the pegRNA, ngRNA, and prime editor. A top performing dsgRNA (–42 PAM) was selected for subsequent editing experiments. The “No edit” x-axis label specifies a control in which no editing reagents were transfected. For FIGs. 32B-32D, based on HTS analysis of unedited ATP1A3 E815K cells (see “No edit” x-axis label), monoclonal HEK293T cells were presumed triploid for ATP1A3, with one allele carrying the E815K c.2443A mutation and two alleles with wild-type E815 c.2443G. Therefore, PE sequencing data is shown as “percent specified genotype,” with all samples starting at approximately 66% wild type E815 c.2443G genotype, marked by a dotted line on each plot. Data represent the mean of n = 3 independent biological replicates, with dots showing individual replicate values. For FIGs. 32E and 32F, because iPSCs derived from patients with AHC are heterozygous for pathogenic ATP1A3 mutations, sequencing data is shown as “percent specified genotype” and all samples start from approximately 50% wild type E815 c.2443G genotype. Data represent the mean of n = 3 independent biological replicates from three ATP1A3 E815K c.2443A donors, with dots showing individual replicate values.

[0073] FIGs. 33A-33E show optimization of PE strategies to correct ATP1A3 L839P c.2516C, related to FIGs. 25A-25N. FIG. 33A is a schematic (left) showing the pathogenic ATP1A3 L839P c.2516C mutation targeted for PE correction (c.2516C) and the three epegRNA protospacers (NGG119nt, NGG120nt, and NGG220nt) used for PE. Right: Schematic of the L839 c.2516T corrective edit (c.2516T) with the concomitantly installed silent editing strategy 1 (SE1, GTTG, where the T corresponds to c.2516T and the italicized nucleotides are the silent edits)). FIG. 33B shows monoclonal HEK293T cells homozygous for the ATP1A3 L839P c.2516C mutation were transfected with epegRNA expression plasmids to optimize spacer selection, PBS lengths, and RTT lengths to correct L839P c.2516C with PEmax. Bar charts show HTS-quantified L839 c.2516T genotype and indels after editing with epegRNAs with different combinations of PBS and RTT lengths (in nucleotides), with protospacers and silent edit strategies listed above each chart. Two top performing epegRNAs (NGG119nt SE1 PBS12 RTT19 and NGG220nt SE1 PBS11 RTT20) B1195.70198WO00 13849370.1 (April 11, 2025)40 / 895 were selected for subsequent editing optimization experiments. FIG. 33C shows monoclonal HEK293T cells homozygous for the ATP1A3 L839P c.2516C mutation were transfected with ngRNA expression plasmids to optimize L839P c.2516C correction using PEmax and the epegRNAs NGG119nt SE1 PBS12 RTT19 and NGG220nt SE1 PBS11 RTT20. X-axis labels identify different ngRNAs by their nicking position relative to the epegRNA nick (in base pairs). The “No nick” x-axis label represents a control PE condition where an epegRNA was transfected without an ngRNA. A top performing epegRNA-ngRNA combination (NGG119nt SE1 PBS12 RTT19 and ngRNA –1) was selected for subsequent editing optimization experiments. FIG. 33D shows monoclonal ATP1A3 L839P c.2516C iPSCs derived from a patient with AHC were electroporated with various prime editor mRNAs to optimize L839P c.2516C correction with chemically synthesized NGG119nt SE1 PBS12 RTT19 pegRNA and –1 ngRNA. MLH1dn mRNA was used to suppress MMR. A top performing prime editor (PE6b) was selected for subsequent optimization experiments. The “No edit” x-axis label specifies a control in which no editing reagents were transfected. FIG. 33E shows monoclonal ATP1A3 L839P c.2516C iPSCs derived from a patient with AHC were electroporated with chemically synthesized dsgRNAs to optimize L839P c.2516C correction with chemically synthesized NGG119nt SE1 PBS12 RTT19 epegRNA and –1 ngRNA, and PE6b mRNA. MLH1dn mRNA was used to suppress MMR. X-axis labels identify different dsgRNAs by their putative nicking position and protospacer strand relative to the epegRNA nick (in base pairs) and PAM, respectively. The “No dsgRNA” x-axis label represents a control PE condition in which a non-targeting dsgRNA was electroporated with the epegRNA, ngRNA, and prime editor. A top performing dsgRNA (–36 PAM) was selected for subsequent editing experiments. The “No edit” x-axis label specifies a control in which no editing reagents were transfected. For FIGs. 33B and 33C, (FIG. 33A) based on HTS analysis of unedited ATP1A3 L839P cells (see “No edit” x-axis label), monoclonal HEK293T cells were presumed homozygous for the ATP1A3 L839P c.2516C mutation. Therefore, PE sequencing data is shown as “percent specified genotype,” with all samples starting at approximately 0% wild type L839 c.2516T genotype. Data represent the mean of n = 3 independent biological replicates, with dots showing individual replicate values. For D and E, because iPSCs derived from patients with AHC are heterozygous for pathogenic ATP1A3 mutations, sequencing data is shown as “percent specified genotype” and all samples start from approximately 50% wild type L839 c.2516T genotype. Data represent the mean of n = 3 independent biological replicates from one ATP1A3 L839P c.2516C donor (Using two B1195.70198WO00 13849370.1 (April 11, 2025)41 / 895 donor-derived monoclonal iPSC lines, one line was edited twice, and the other line was edited once). Dots show individual replicate values.

[0074] FIGs. 34A-34E show optimization of PE strategies to correct ATP1A3 G947R c.2839C, related to FIGs. 25A-25N. FIG. 34A is a schematic (left) showing the pathogenic ATP1A3 G947R c.2839C mutation targeted for PE correction (c.2839C) and the two epegRNA protospacers (NGG120nt and NGG220nt) used for PE. On the right is a schematic of the G947 c.2839G corrective edit (c.2839G) with the concomitantly installed silent editing strategy 1 (SE1, TGGCT, where the G corresponds to c.2839G and the italicized nucleotides are the silent edits). FIG. 34B shows monoclonal HEK293T cells homozygous for the ATP1A3 G947R c.2839C mutation were transfected with epegRNA expression plasmids to optimize spacer selection, PBS lengths, and RTT lengths to correct G947R c.2839C with PEmax. Bar charts show HTS-quantified G947 c.2839G genotype and indels after editing with epegRNAs with different combinations of PBS and RTT lengths (in nucleotides), with protospacers and silent edit strategies listed above each chart. Two top performing epegRNAs (blue: NGG120nt SE1 PBS14 RTT17 and NGG220nt SE1 PBS11 RTT20) were selected for subsequent editing optimization experiments. FIG. 34C shows monoclonal HEK293T cells homozygous for the ATP1A3 G947R c.2839C mutation were transfected with ngRNA expression plasmids to optimize G947R c.2839C correction using PEmax and the epegRNAs NGG120nt SE1 PBS14 RTT17 and NGG220nt SE1 PBS11 RTT20. X-axis labels identify different ngRNAs by their nicking position relative to the epegRNA nick (in base pairs). The “No nick” x-axis label represents a control PE condition where an epegRNA was transfected without an ngRNA. A top performing epegRNA-ngRNA combination (blue: NGG120nt SE1 PBS14 RTT17 and ngRNA –1) was selected for subsequent editing optimization experiments. FIG. 34D shows monoclonal ATP1A3 G947R c.2839C iPSCs derived from patients with AHC were electroporated with various prime editor mRNAs to optimize G947R c.2839C correction with chemically synthesized NGG1 20nt SE1 PBS14 RTT17 pegRNA and –1 ngRNA. MLH1dn mRNA was used to suppress MMR. A top performing prime editor (blue: PE6b) was selected for subsequent optimization experiments. The “No edit” x-axis label specifies a control in which no editing reagents were transfected. FIG. 34E shows monoclonal ATP1A3 G947R c.2839C iPSCs derived from patients with AHC were electroporated with chemically synthesized dsgRNAs to optimize G947R c.2839C correction with chemically synthesized NGG120nt SE1 PBS14 RTT17 pegRNA and –1 ngRNA, and PE6b mRNA. MLH1dn mRNA was used to suppress MMR. X- B1195.70198WO00 13849370.1 (April 11, 2025)42 / 895 axis labels identify different dsgRNAs by their putative nicking position and protospacer strand relative to the pegRNA nick (in base pairs) and PAM, respectively. The “No dsgRNA” x-axis label represents a control PE condition in which a non-targeting dsgRNA was electroporated with the pegRNA, ngRNA, and prime editor. A top performing dsgRNA (blue: –49 PAM) was selected for subsequent editing experiments. The “No edit” x-axis label specifies a control in which no editing reagents were transfected. For FIGs. 34B and 34C, based on HTS analysis of unedited ATP1A3 G947R cells (see “No edit” x-axis label), monoclonal HEK293T cells were presumed homozygous for the ATP1A3 G947R c.2839C mutation. Therefore, PE sequencing data is shown as “percent specified genotype,” with all samples starting at approximately 0% wild type G947 c.2839G genotype. Data represent the mean of n = 3 independent biological replicates, with dots showing individual replicate values. For FIGs. 34D and 34E, because iPSCs derived from patients with AHC are heterozygous for pathogenic ATP1A3 mutations, sequencing data is shown as “percent specified genotype” and all samples start from approximately 50% wild type G947 c.2839C genotype. Data represent the mean of n = 3 independent biological replicates from two G947R c.2839C donors (two different monoclonal iPSC lines derived from one donor and one monoclonal iPSC line from the other donor were assayed). Dots shows individual replicate values.

[0075] FIGs. 35A-35O show optimization of PE strategies to correct Atp1a3 D801N c.2401A and Atp1a3 E815K c.2443A, related to FIGs. 27A-27K. FIGs. 35A and 35B are sequence schematics comparing the sequence flanking human and mouse targets for (FIG. 35A) D801N c.2401A and (FIG. 35B) E815K c.2443A. The pathogenic mutation (c.2401A) on the human and mouse sequences. Disagreements between human and mouse sequences are shown in the Atp1a3 schematic with text and linked to corresponding human base pairs with black lines. Encoded silent edits have been omitted from RTT annotations. Optimized sequence elements from (FIG. 35A) NGA220nt SE1 PBS12 RTT19 and (FIG. 35B) ATP1A3 E815K c.2443A epegRNA 19nt SE1 PBS13 RTT25 are annotated on the ATP1A3 sequence. FIG. 35C shows monoclonal N2a cells homozygous for the Atp1a3 D801N c.2401A mutation were transfected with epegRNA expression plasmids to optimize PBS and RTT lengths to correct D801N c.2401A with PEmax. Bar chart shows HTS-quantified D801 c.2401G genotype and indels after editing with epegRNAs with different combinations of PBS and RTT lengths (in nucleotides). One top performing epegRNAs (NGG120nt SE1 PBS9 RTT23) was selected for subsequent editing optimization experiments. Based on HTS B1195.70198WO00 13849370.1 (April 11, 2025)43 / 895 analysis of unedited Atp1a3 D801N cells (see “No edit” x-axis label), monoclonal N2a cells were presumed homozygous for the Atp1a3 D801N c.2401A mutation. Therefore, PE sequencing data is shown as “percent specified genotype,” with all samples starting at approximately 0% wild type D801 c.2401G genotype. Data represent the mean of n = 3 independent biological replicates, with dots showing individual replicate values. FIG. 35D shows monoclonal N2a cells homozygous for the Atp1a3 D801N c.2401A mutation were transfected with ngRNA expression plasmids to optimize D801N c.2401A correction using PEmax and the epegRNA NGG120nt SE1 PBS9 RTT23. X-axis labels identify different ngRNAs by their nicking position relative to the epegRNA nick (in base pairs). The “No nick” x-axis label represents a control PE condition where an epegRNA was transfected without an ngRNA. A top performing ngRNA (ngRNA +7) was selected for subsequent editing optimization experiments. Data represent the mean of n = 3 independent biological replicates, with dots showing individual replicate values. FIG. 35E shows monoclonal N2a cells homozygous for the Atp1a3 D801N c.2401A mutation were transfected with various prime editor plasmids to optimize D801N c.2401A correction with NGG120nt SE1 PBS9 RTT23 and ngRNA +7. A top performing prime editor (PE6c) was selected for subsequent optimization experiments. Data represent the mean of n = 3 independent biological replicates, with dots showing individual replicate values. FIG. 35F shows Monoclonal N2a cells homozygous for the Atp1a3 D801N c.2401A mutation were transfected with dsgRNA expression plasmids to optimize D801N c.2401A correction using NGG120nt SE1 PBS9 RTT23, ngRNA +7, and PE6c. Numeric x-axis labels identify different dsgRNAs by their putative nicking position relative to the epegRNA nick (in base pairs). The “None” x-axis label represents a control where no dsgRNA plasmid was transfected with the epegRNA, ngRNA, and prime editor plasmids. A top performing dsgRNA (–52 PAM) was selected for subsequent editing experiments. Data represent the mean of n = 3 independent biological replicates, with dots showing individual replicate values. FIG. 35G show Atp1a3 E815K c.2443A primary fibroblasts isolated from E815K mice were electroporated with various prime editor mRNAs to optimize E815K c.2443A correction with chemically synthesized NGG1 19nt SE1 PBS13 RTT25 pegRNA and +49 ngRNA. MLH1dn mRNA was not used to suppress MMR. A top performing prime editor (PE6c) was selected for subsequent optimization experiments. The “No edit” x-axis label specifies a control in which no editing RNA reagents were electroporated. Because primary fibroblasts isolated from E815K mice are heterozygous for E815K c.2443A, sequencing data is shown as “percent specified B1195.70198WO00 13849370.1 (April 11, 2025)44 / 895 genotype” and all samples start from approximately 50% wild type E815 c.2443A genotype. Data represent the mean of n = 3 independent biological replicates. Dots shows individual replicate values. FIG. 35H shows Atp1a3 E815K c.2443A primary fibroblasts isolated from E815K mice were electroporated with chemically synthesized dsgRNAs to optimize E815K c.2443A correction with chemically synthesized NGG119nt SE1 PBS13 RTT25 pegRNA and +49 ngRNA, and PE6c mRNA. MLH1dn mRNA was not used to suppress MMR. X-axis labels identify different dsgRNAs by their putative nicking position and protospacer strand relative to the pegRNA nick (in base pairs) and PAM, respectively. The “None” x-axis label represents a control PE condition where no dsgRNA was electroporated with the pegRNA, ngRNA, and prime editor. A top performing dsgRNA (–32 non-PAM) was selected for subsequent editing experiments. Because primary fibroblasts isolated from E815K mice are heterozygous for E815K c.2443A, sequencing data is shown as “percent specified genotype” and all samples start from approximately 50% wild type E815 c.2443A genotype. Data represent the mean of n = 3 independent biological replicates. Dots shows individual replicate values. FIG. 35I shows fluorescence-Activated Cell Sorting (FACS) gating strategy for brain nuclei. Nuclei were extracted from homogenized, previously frozen brain tissue and stained with Dye Cycle Ruby. Gating was performed to identify nuclei based on forward and side scatter area (panel 1), followed by selection of singlets using Dye Cycle Ruby intensity (panel 2). Sorting was then carried out based on GFP fluorescence, generating two populations: a "Bulk Nuclei" group, which included all nuclei regardless of GFP expression, and a "GFP-positive Nuclei" group, which consisted exclusively of GFP-positive nuclei (panels 3 and 4). Panel 3 shows GFP FACS data from vehicle-treated D801N mice, while panel 4 displays GFP FACS data from D801N-PE-AAV9-treated D801N mice. FIG. 35J are scatter plots showing the relationship between percent GFP-positive (GFP+) nuclei and percent pathogenic allele correction from gDNA HTS of bulk nuclei from cortical (top) or hippocampal (bottom) dissections. Each point represents a matched data pair from a single mouse. The strength and direction of the linear relationship for D801N and E815K between percent GFP-positive nuclei and percent pathogenic allele correction from gDNA HTS of bulk nuclei were assessed using Pearson's correlation coefficient (r). FIGs. 35K and 35L show hippocampal Atp1a3 western blot from WT and (FIG. 35K) D801N mice and (FIG. 35L) E815K mice treated with D801N-PE-AAV9 and E815K-PE-AAV9, respectively, (labeled “PE”) or phosphate-buffered saline (PBS, labeled “vehicle”). Gapdh was used as a loading control. Each gel lane represents a single mouse of the specified genotype and B1195.70198WO00 13849370.1 (April 11, 2025)45 / 895 treatment group. FIGs. 35M and 35N show densitometry to assess Atp1a3 signal intensity in WT and (FIG. 35M) D801N mice and (FIG. 35N) E815K mice treated with D801N-PE- AAV9 and E815K-PE-AAV9, respectively, (labeled “PE”) or PBS (labeled “vehicle”), relative to WT vehicle-treated Atp1a3 signal intensity. All data normalized to Gapdh signal intensity. Data represent the mean of at least n = 2 mice, with dots showing individual mice. FIG. 35O are scatter plots showing the relationship between percent pathogenic allele correction from gDNA (top) or cDNA (bottom) HTS of bulk hippocampal nuclei and the Atp1a3 activity ratio of D801N and E815K mice treated with D801-PE-AAV9 and E815K- PE-AAV9, respectively, and WT mice treated with PBS vehicle). Each point represents a matched data pair from a single mouse. The strength and direction of the linear relationship between percent pathogenic allele correction from gDNA HTS of bulk hippocampal nuclei and the Atp1a3 activity ratio were assessed using Pearson's correlation coefficient (r).

[0076] FIGs. 36A-36K show ATP1A3 gene therapy’s effect survival, multiple patient-relevant behavioral phenotypes, and transgene expression, related to FIGs. 28A-28J. FIGs. 36A and 36B show a Kaplan-Meier survival curve for male (FIG. 36A) and female (FIG. 36B) WT and D801N mice treated with phosphate-buffered saline (vehicle) or ATP1A3 gene therapy vector (ATP1A3-treated). Two-way ANOVA followed by Tukey's multiple comparisons test was used to determine P-values between groups. FIGs. 36C-36F are the results of hypothermia-induced paroxysmal episode assay for WT and D801N mice treated with PBS (vehicle) or ATP1A3 gene therapy vector (ATP1A3-treated) for males (M, left) and females (F, right). Paroxysmal event scores (FIGs. 36C, 36D) and number of convulsive-like events (DE) were measured upon hypothermia induction. Recovery period was monitored for latency to regain righting / balance (FIGs. 36E, 36F) and mobility (FIGs. 36F, 36G). FIG. 36G shows latency to fall off an accelerating rotarod was measured for D801N and WT mice treated with PBS (vehicle) or ATP1A3 gene therapy vector (ATP1A3-treated) for males (M, left) and females (F, right). FIGs. 36H and 36I show D801N and WT mice treated with PBS (vehicle) or ATP1A3 gene therapy vector (ATP1A3-treated) were subjected to open field testing to interrogate exploratory and / or locomotor activity in males (M, left) and females (F, right). FIGs. 36H and 36I show vertical episodes count. FIGs. 36I and 36J show total distance traveled. FIG. 36J shows RNA in situ hybridization of human ATP1A3 mRNA on parasagittal brain sections from ~30-week-old mice. WT mice were ICV injected with PBS or ATP1A3 gene therapy vector (pSyn-ATP1A3) at P0 (N = 2 per injection article). Sections were counterstained with hematoxylin and higher magnifications of individual brain regions B1195.70198WO00 13849370.1 (April 11, 2025)46 / 895 are shown. Scale bars: 1 mm (whole brain), 100 μm (cortex), 250 μm (hippocampus), 100 μm (cerebellum), 50 μm (cerebellar hemisphere, DCN) and 25 μm (cerebellar hemisphere, PML). ML, molecular cell layer; PL, Purkinje cell layer; GCL, granule cell layer; PML, paramedian lobe; and DCN, deep cerebellar nuclei. FIG. 36K shows ATP1A3 gene expression analysis of different brain regions of PBS(vehicle) and ATP1A3 gene therapy vector (ATP1A3-treated) treated D801N mice at ~7-weeks of age. WT and D801N mice were injected (P0 ICV) with PBS (vehicle) or ATP1A3 gene therapy vector (ATP1A3-treated). D801N mice were analyzed via quantitative RT-PCR (TaqMan®) using probes that detect both human and mouse transcripts. Data were normalized to the transcript levels of Tbp and the fold change in gene expression is relative to that of vehicle-treated WT mice per region of the brain.. The size of each cohort is displayed in the plot’s legend. One-way ANOVA followed by Tukey's multiple comparisons test was used to determine P-values between groups. For FIGs, 36C- 36I and 36K, each dot represents data for a single mouse. For (FIGs. 36A-36I and 36K, the size of each cohort is displayed in the plot’s legend. Two-way ANOVA followed by Tukey's multiple comparisons test was used to determine P-values between groups. ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0077] FIGs.37A-37J show PE installs of ATP1A3 mutations in HEK293T cells FIG.37A is a schematic of ATP1A3 D801N c.2401A installation strategy. The installation epegRNA protospacer sequence is annotated on the wild-type (WT) ATP1A3 sequence (top) and the installed D801N c.2401A mutation is shown on the edited ATP1A3 sequence (bottom). FIG. 37B shows the PE data for the optimization installation of D801N c.2401A into HEK293T cell ATP1A3. A panel of epegRNAs with variable primer binding site (PBS) and reverse transcriptase template (RTT) lengths (listed as nucleotides lengths on x-axis) were transfected into HEK293T cells with SpCas9(VRQR)-PEmax. MLH1dn was also used to suppress cellular mismatch repair (MMR). The epegRNA PBS12 RTT18 was selected to generate the ATP1A3 D801N c.2401A clonal cell line. Data represent the mean of n = 3 independent biological replicates (shown as dots). FIG. 37C is a schematic of ATP1A3 E815K c.2443A installation strategy. The installation epegRNA protospacer sequence is annotated on the wild-type (WT) ATP1A3 sequence (top) and the installed E815K c.2443A mutation is shown on the edited ATP1A3 sequence (bottom). FIG. 37D shows the PE data for the optimization installation of E815K c.2443A into HEK293T cell ATP1A3. A panel of epegRNAs with variable PBS and RTT lengths (listed as nucleotides lengths on x-axis) were transfected into HEK293T cells with PEmax. MLH1dn was also used to suppress MMR. The B1195.70198WO00 13849370.1 (April 11, 2025)47 / 895 epegRNA PBS13 RTT13 was selected to generate the ATP1A3 E815K c.2443A clonal cell line. Data represent the mean of n = 3 independent biological replicates (shown as dots). FIG. 37E is a schematic of ATP1A3 L839P c.2516C installation strategy. The installation epegRNA protospacer sequence is annotated on the wild-type (WT) ATP1A3 sequence (top) and the installed L839P c.2516C mutation is shown on the edited ATP1A3 sequence (bottom). FIG. 37F shows the PE data for the optimization installation of L839P c.2516C into HEK293T cell ATP1A3. A panel of epegRNAs with variable PBS and RTT lengths (listed as nucleotides lengths on x-axis) were transfected into HEK293T cells with PEmax. MLH1dn was also used to suppress MMR. The epegRNA PBS13 RTT26 was selected to generate the ATP1A3 L839P c.2516C clonal cell line. Data represent the mean of n = 3 independent biological replicates (shown as dots). FIG. 37G is a schematic of ATP1A3 G947R c.2839A installation strategy. The installation epegRNA protospacer sequence is annotated on the wild-type (WT) ATP1A3 sequence (top) and the installed G947R c.2839A mutation is shown on the edited ATP1A3 sequence (bottom). FIG. 37H shows the PE data for the optimization installation of G947R c.2839A into HEK293T cell ATP1A3. A panel of epegRNAs with variable PBS and RTT lengths (listed as nucleotides lengths on x-axis) were transfected into HEK293T cells with PEmax. MLH1dn was also used to suppress MMR. The epegRNA PBS13 RTT14 was selected to generate the ATP1A3 G947R c.2839A clonal cell line. Data represent the mean of n = 3 independent biological replicates (shown as dots). FIG. 37I is a schematic of ATP1A3 G947R c.2839C installation strategy. The installation epegRNA protospacer sequence is annotated on the wild-type (WT) ATP1A3 sequence (top) and the installed G947R c.2839C mutation is shown on the edited ATP1A3 sequence (bottom). FIG. 37J shows the PE data for the optimization installation of G947R c.2839C into HEK293T cell ATP1A3. A panel of epegRNAs with variable PBS and RTT lengths (listed as nucleotides lengths on x-axis) were transfected into HEK293T cells with PEmax. MLH1dn was also used to suppress MMR. The epegRNA PBS13 RTT14 was selected to generate the ATP1A3 G947R c.2839C clonal cell line. Data represent the mean of n = 3 independent biological replicates (shown as dots).

[0078] FIG. 38 shows the optimization of PE strategies to install Atp1a3 D801N c.2401A in N2A cells. To a identify PE strategy to install Atp1a3 D801N c.2401A in N2A cells and a translationally silent L802 c.2406A mutation present in the B6C3 D801N mice, panels of epegRNAs with variable PBS lengths and RTT lengths were transfected into N2A cells. One of the most efficient epegRNAs, PBS13 RTT20, was selected to generate a B1195.70198WO00 13849370.1 (April 11, 2025)48 / 895 monoclonal N2A cell lines with the Atp1a3 D801N c.2401A and L802 c.2406A mutations. PBS and RTT lengths are listed as nucleotides lengths. One monoclonal cell line homozygous for the Atp1a3 D801N c.2401A and L802 c.2406A mutations was isolated. MLH1dn was used to suppress cellular mismatch repair and enhance editing in these experiments. Data represent the mean of n = 3 independent biological replicates (shown as dots).

[0079] FIGs. 39A and 39B show weights of male (FIG. 39A) and female (FIG. 39B) WT and D801N mice treated with vehicle or D801N-PE-AAV9. Data points and error bars represent mean and standard deviation, respectively, for the number of surviving mice at each time point. Ordinary one-way ANOVA with repeated measurements was corrected for multiple comparisons using Tukey method. The size of each cohort is displayed in the plot’s legend. ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0080] FIGs. 40A and 40B show weights of male (FIG. 40A) and female (FIG. 40B) WT and D801N mice treated with vehicle or ATP1A3-AAV9 gene therapy vector. Data points and error bars represent mean and standard deviation, respectively, for the number of surviving mice at each time point. Ordinary one-way ANOVA with repeated measurements was corrected for multiple comparisons using Tukey method. The size of each cohort is displayed in the plot’s legend. ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. DEFINITIONS

[0081] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise. Base editing

[0082] “Base editing” refers to genome editing technology that involves the conversion of a specific nucleic acid base into another at a targeted genomic locus. In certain embodiments, this can be achieved without requiring double-stranded DNA breaks (DSB), or single stranded breaks (i.e., nicking). To date, other genome editing techniques, including B1195.70198WO00 13849370.1 (April 11, 2025)49 / 895 CRISPR-based systems, begin with the introduction of a DSB at a locus of interest. Subsequently, cellular DNA repair enzymes mend the break, commonly resulting in random insertions or deletions (indels) of bases at the site of the DSB. However, when the introduction or correction of a point mutation at a target locus is desired rather than stochastic disruption of the entire gene, these genome editing techniques are unsuitable, as correction rates are low (e.g. typically 0.1% to 5%), with the major genome editing products being indels. In order to increase the efficiency of gene correction without simultaneously introducing random indels, the present inventors previously modified the CRISPR / Cas9 system to directly convert one DNA base into another without DSB formation. See, Komor, A.C., et al., Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424 (2016), the entire contents of which is incorporated by reference herein.

[0083] The following base editor, which effects transitions (pyrimidine to pyrimidine, or purine to purine) mutations are relevant to the methods disclosed herein. Adenine base editor (or “ABE”). This type of editor converts an A:T Watson-Crick nucleobase pair to a G:C Watson-Crick nucleobase pair. Because the corresponding Watson-Crick paired bases are also interchanged as a result of the conversion, this category of base editor may also be referred to as a thymine base editor (or “TBE”). Cytidine base editors are also contemplated herein. Base editor

[0084] The term “base editor (BE)” as used herein, refers to an agent comprising a polypeptide that is capable of making a modification to a base (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the base editor is capable of deaminating a base within a nucleic acid such as a base within a DNA molecule. In the case of an adenine base editor, the base editor is capable of deaminating an adenine (A) in DNA. Such base editors may include a nucleic acid programmable DNA binding protein (napDNAbp) fused to an adenosine deaminase. Some base editors include CRISPR-mediated fusion proteins that are utilized in the base editing methods described herein. In some embodiments, the base editor comprises a nuclease-inactive Cas9 (dCas9) fused to a deaminase which binds a nucleic acid in a guide RNA-programmed manner via the formation of an R-loop, but does not cleave the nucleic acid. For example, the dCas9 domain of the base editor may include a D10A and a H840A mutation (which renders Cas9 capable of B1195.70198WO00 13849370.1 (April 11, 2025)50 / 895 cleaving only one strand of a nucleic acid duplex), as described in PCT / US2016 / 058344, which published as WO 2017 / 070632 on April 27, 2017 and is incorporated herein by reference in its entirety. The DNA cleavage domain of S. pyogenes Cas9 includes two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA (the “targeted strand”, or the strand in which editing or deamination occurs), whereas the RuvC1 subdomain cleaves the non- complementary strand containing the PAM sequence (the “non-edited strand”). The RuvC1 mutant D10A generates a nick in the targeted strand, while the HNH mutant H840A generates a nick on the non-edited strand (see Jinek et al., Science, 337:816-821(2012); Qi et al., Cell. 28;152(5):1173-83 (2013)).

[0085] The term “base editor” encompasses the CRISPR-mediated fusion proteins utilized in the multiplexed base editing methods described herein as well as any base editor known or described in the art at the time of this filing or developed in the future. Reference is made to Rees & Liu, Base editing: precision chemistry on the genome and transcriptome of living cells, Nat. Rev. Genet. 2018;19(12):770-788; as well as U.S. Patent Publication No. 2018 / 0073012, published March 15, 2018, which issued as U.S. Patent No. 10,113,163; on October 30, 2018; U.S. Patent Publication No. 2017 / 0121693, published May 4, 2017, which issued as U.S. Patent No. 10,167,457 on January 1, 2019; International Publication No. WO 2017 / 070633, published April 27, 2017; International Publication No. WO 2018 / 027078, published August 2, 2018; International Application No PCT / US2018 / 056146, filed October 16, 2018, which published as Publication No. WO 2019 / 079347 on April 25, 2019; International Application No PCT / US2019 / 033848, filed May 23, 2019, which published as Publication No. WO 2019 / 226593 on November 28, 2019; U.S. Patent Publication No. 2015 / 0166980, published June 18, 2015; U.S. Patent No. 9,840,699, issued December 12, 2017; U.S. Patent No. 10,077,453, issued September 18, 2018; International Publication No. WO 2019 / 023680, published January 31, 2019; International Publication No. WO 2018 / 0176009, published September 27, 2018; International Publication No. WO 2020 / 041751, published February 27, 2020; International Publication No. WO 2020 / 051360, published March 12, 2020; International Patent Publication No. WO 2020 / 102659, published May 22, 2020; International Publication No. WO 2020 / 086908, published April 30, 2020; International Publication No. WO 2020 / 181180, published September 10, 2020; International Publication No. WO 2020 / 214842, published October 22, 2020; International Publication No. WO 2020 / 092453, published May 7, 2020; International Patent Application No. B1195.70198WO00 13849370.1 (April 11, 2025)51 / 895 PCT / US2020 / 033873, filed May 20, 2020, published as International Publication No. WO2020 / 236982 on November 26, 2020, and International Application No. PCT / US2020 / 624628, filed November 25, 2020, each of which is incorporated herein by reference in its entirety. Cas9

[0086] 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 casn1 nuclease or a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, correct processing of pre-crRNA requires a trans- encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas9 domain. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cleaves a linear or circular dsDNA target complementary to the spacer. The 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 M1 strain of Streptococcus pyogenes.” Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., B1195.70198WO00 13849370.1 (April 11, 2025)52 / 895 White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E., Science 337:816- 821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease comprises one or more mutations that partially impair or inactivate the DNA cleavage domain.

[0087] A nuclease-inactivated Cas9 domain may interchangeably be referred to as a “dCas9” protein (for nuclease-“dead” Cas9). Methods for generating a Cas9 domain (or a fragment thereof) having an inactive DNA cleavage domain are known (see, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell. 28;152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28;152(5):1173-83 (2013)). In some embodiments, a Cas9 protein comprises one or more mutations to inactivate the nuclease activity of only one of the HNH subdomain or the RuvC1 subdomain.

[0088] In some embodiments, proteins comprising fragments of a Cas9 protein are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9, or fragments thereof, are referred to as “Cas9 B1195.70198WO00 13849370.1 (April 11, 2025)53 / 895 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.8% identical, or at least about 99.9% identical to wild type Cas9 (e.g., SpCas9). In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes compared to wild type Cas9 (e.g., SpCas9). In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9 (e.g., SpCas9). In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9 (e.g., SpCas9). Dead single guide RNA (dsgRNA)

[0089] The terms “dead guide RNA,” “dgRNA,” “dead single guide RNA,” and “dsgRNA” refer to 5′-truncated guide RNAs that enable Cas9 to bind to a target sequence but do not support DNA cleavage. DsgRNAs may have, for example, only 14-16 nucleotides of protospacer complementarity. DsgRNAs may be useful, e.g., for manipulating the accessibility of a target site to be edited by facilitating the unfolding of genomic DNA. DsgRNAs are further described in Kiani, S. et al., Cas9 gRNA engineering for genome editing, activation and repression. Nat. Methods 12, 1051–1054 (2015), which is incorporated herein by reference. Fusion protein

[0090] 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 B1195.70198WO00 13849370.1 (April 11, 2025)54 / 895 the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C- terminal) protein, thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. A protein may comprise different domains, for example, a Cas9 protein fused to a polymerase such as a reverse transcriptase (i.e., a prime editor). Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which is incorporated herein by reference. Guide RNA (“gRNA”)

[0091] As used herein, the term “guide RNA” is a particular type of guide nucleic acid which is commonly associated with a Cas protein (e.g., a Cas9 protein), directing the Cas protein to a specific sequence in a DNA molecule that includes complementarity to the protospacer sequence of the guide RNA. A gRNA, as disclosed herein, may refer to a pegRNA, ngRNA, dsgRNA (e.g., for prime editing) or a sgRNA (e.g., for base editing).For example, a gRNA may direct a Cas protein (e.g., as part of a prime editor) to a target site in the ATP1A3 gene. However, this term also embraces the equivalent guide nucleic acid molecules that associate with Cas protein equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and which otherwise program the Cas protein equivalent to localize to a specific target nucleotide sequence. The Cas protein equivalents may include other napDNAbps from any type of CRISPR system (e.g., type II, V, VI), including Cpf1 (a type-V CRISPR-Cas system), C2c1 (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system), and C2c3 (a type V CRISPR-Cas system). Further Cas-equivalents are described in Makarova et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299), which is incorporated herein by reference. Exemplary sequences and structures of guide RNAs are provided herein.

[0092] Functionally, guide RNAs associate with a Cas protein, directing (or programming) the Cas 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 B1195.70198WO00 13849370.1 (April 11, 2025)55 / 895 component of the CRISPR / Cas system. The sequence specificity of a Cas 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) Specificity Determining Sequence (SDS), or 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 Cas protein. In some embodiments, an SDS of the present disclosure has a length of 15 to 100 nucleotides, or more. For example, an SDS 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 SDS is 20 nucleotides long. For example, the SDS 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 SDS of the gRNA. For a Cas protein to successfully bind to the DNA target sequence, a region of the target sequence is complementary to the SDS of the gRNA sequence and is immediately followed by the correct protospacer adjacent motif (PAM) sequence. In some embodiments, an SDS is 100% complementary to its target sequence. In some embodiments, the SDS 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 SDS of template DNA or target DNA may differ from a complementary region of a gRNA by 1, 2, 3, 4, or 5 nucleotides.

[0093] 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 (e.g., a target sequence in ATP1A3). 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. B1195.70198WO00 13849370.1 (April 11, 2025)56 / 895 Linker

[0094] 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 napDNAbp (e.g., 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 gRNA). In other embodiments, the linker is a non-peptidic 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. MLH1dn

[0095] “MLH1dn” refers to a dominant negative variant of the DNA-mismatch repair (MMR) enzyme MLH1. MLH1dn is an inhibitor of the DNA mismatch repair pathway and can help increase the efficiency of prime editing.

[0096] In some embodiments, the dominant negative MLH1 can include, for example, MLH1 Δ754-Δ756, which has the following amino acid sequence:

[0097] MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQV IVKEGGLKLIQIQDNGTGIRKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISH VAHVTITTKTADGKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKAL KNPSEEYGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFGNAV SRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAY LPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSSRM YFTQTLLPGLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQP LSKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELPAPAEVAAKNQSLEGDTTKGTSE MSEKRGPTSSNPRKRHREDSDVEMVEDDSRKEMTAACTPRRRIINLTSVLSLQEEINE QGHEVLREMLHNHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANF GVLRLSEPAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSL EIDEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEKECFESLSKECAMFYSIRK QYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKALRSHILPPKHFTEDGNILQLANLP DLYKVF[- - -] (SEQ ID NO: 1412), or an amino acid sequence having at least 70%, at least B1195.70198WO00 13849370.1 (April 11, 2025)57 / 895 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity thereto (wherein the [- - -] indicates deleted amino acid residue(s) relative to the parent or wildtype MLH1 sequence). napDNAbp

[0098] 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., guide RNA), which localizes the napDNAbp to a DNA sequence that comprises a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid, or a portion thereof (e.g., the protospacer of a guide RNA). In other words, the guide nucleic-acid “programs” the napDNAbp (e.g., Cas9, or a variant thereof) to localize and bind to a complementary sequence.

[0099] 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. Nickase

[0100] As used herein, a “nickase” refers to a napDNAbp (e.g., 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 B1195.70198WO00 13849370.1 (April 11, 2025)58 / 895 domain of a canonical napDNAbp (e.g., 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 an aspartate-to-alanine substitution (D10A) in the RuvC1 catalytic domain of Cas9 relative to a canonical SpCas9 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 an H840A, N854A, and / or N863A mutation relative to a canonical SpCas9 sequence, or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the term “Cas9 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. In some embodiments, the nickase is a Cas protein that is not a Cas9 nickase.

[0101] In some embodiments, the napDNAbp of a prime editor or base editor is a Cas9 nickase (nCas9) that nicks only a single strand. In other embodiments, the napDNAbp can be selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Cas12f1, Cas12j (CasΦ), and Argonaute and optionally has a nickase activity such that only one strand is cut. In some embodiments, the napDNAbp is selected from Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Cas12f1, Cas12j (CasΦ), and Argonaute and optionally has a nickase activity such that one DNA strand is cut preferentially to the other DNA strand. Nicking Guide RNA (ngRNA)

[0102] In some embodiments, a guide RNA is a “nicking guide RNA.” Nicking guide RNAs may be used to nick the non-edited strand of a target nucleic acid molecule, which may facilitate incorporation of the edit by cellular DNA repair mechanisms. Nuclear localization sequence (NLS)

[0103] 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 B1195.70198WO00 13849370.1 (April 11, 2025)59 / 895 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 prime editor comprises one or more NLS as described herein. Nucleic acid molecule

[0104] The term “nucleic acid,” as used herein, (also referred to as a “polynucleotide”) refers to a polymer of nucleotides. The polymer may include natural nucleosides (i.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, O(6) methylguanine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1-methyl adenosine, 1-methyl guanosine, N6-methyl adenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'- deoxyribose, 2´-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5ʹ N phosphoramidite linkages). pegRNA

[0105] 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 the 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 nucleic acid sequence. The extended regions may comprise, but are not limited to, single-stranded RNA or DNA. Further, the extended regions may occur at the 3′ end of a traditional guide RNA. In other arrangements, the extended regions may occur at the 5′ end of a traditional guide RNA. In still other arrangements, the extended region may occur at an intramolecular region of the traditional guide RNA, for example, in the gRNA core region which associates and / or binds B1195.70198WO00 13849370.1 (April 11, 2025)60 / 895 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.

[0106] In certain embodiments, the pegRNAs have a 3ʹ extension arm, a spacer, and a gRNA core. The 3ʹ extension arm further comprises in the 5ʹ to 3ʹ 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.

[0107] In certain other embodiments, the pegRNAs have a 5ʹ extension arm, a spacer, and a gRNA core. The 5ʹ extension further comprises in the 5ʹ to 3ʹ direction a 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.

[0108] In still other embodiments, the pegRNAs have in the 5ʹ to 3ʹ direction a spacer, a gRNA core, and an extension arm. The extension arm is at the 3ʹ end of the pegRNA. The extension arm further comprises in the 5ʹ to 3ʹ direction a homology arm, an edit template, and a primer binding site. The extension arm may also comprise an optional modifier region at the 3ʹ and 5ʹ ends, which may be the same sequences or different sequences. In addition, the 3ʹ end of the pegRNA may comprise a transcriptional terminator sequence. These sequence elements of the pegRNAs are further described and defined herein.

[0109] In still other embodiments, the pegRNAs have in the 5ʹ to 3ʹ direction an extension arm, a spacer, and a gRNA core. The extension arm is at the 5ʹ end of the pegRNA. The extension arm further comprises in the 3ʹ to 5ʹ direction a primer binding site, an edit template, and a homology arm. The extension arm may also comprise an optional modifier B1195.70198WO00 13849370.1 (April 11, 2025)61 / 895 region at the 3ʹ and 5ʹ ends, which may be the same sequences or different sequences. The pegRNAs may also comprise a transcriptional terminator sequence at the 3ʹ end. These sequence elements of the pegRNAs are further described and defined herein.

[0110] 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.

[0111] In some embodiments, a pegRNA is an “engineered pegRNA” (“epegRNA”). Relative 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 3′ structured motif comprises evopreq1. In some embodiments, the 3′ structured motif in the pegRNAs disclosed herein comprises the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 281), or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence: CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 281).

[0112] pegRNAs are further described, e.g., in International Patent Application No. PCT / US2020 / 023721, filed March 19, 2020, which published as WO 2020 / 191239; International 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 B1195.70198WO00 13849370.1 (April 11, 2025)62 / 895 published as WO 2023 / 076898; and International Patent Application No. PCT / US2022 / 074628, filed August 5, 2022, which published as WO 2023 / 015309; the contents of each of which is incorporated by reference herein.

[0113] 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. PE1

[0114] As used herein, “PE1” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a wild type MMLV RT having the following structure: [NLS]- [Cas9(H840A)]-[linker]-[MMLV_RT(wt)] + a desired pegRNA, wherein the PE fusion has the following amino acid sequence: MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNT DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDS FFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEH HQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKIL TFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVP QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRD KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETR IDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPD VSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKP HIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPY NLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQ GFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTL B1195.70198WO00 13849370.1 (April 11, 2025)63 / 895 GNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLRE FLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALG LPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMV AAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTD GSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNV YTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQ KGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKR KV (SEQ ID NO: 1413) KEY: NUCLEAR LOCALIZATION SEQUENCE (NLS) CAS9(H840A) 33-AMINO ACID LINKER M-MLV reverse transcriptase PE2

[0115] As used herein, “PE2” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a variant MMLV RT having the following structure: [NLS]- [Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] + a desired pegRNA, wherein the PE fusion has the amino acid sequence of: MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNT DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDS FFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEH HQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKIL TFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVP QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRD KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETR B1195.70198WO00 13849370.1 (April 11, 2025)64 / 895 IDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPD VSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKP HIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPY NLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQ GFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTL GNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLRE FLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGL PDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVA AIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDR VQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYT DSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKG HSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 1414) KEY: NUCLEAR LOCALIZATION SEQUENCE (NLS) CAS9(H840A) 33-AMINO ACID LINKER M-MLV reverse transcriptase PE3

[0116] As used herein, “PE3” refers to a prime editing composition comprising a PE2 prime editor and further comprising a second-strand nicking guide RNA that complexes with PE2 and introduces a nick in the non-edit DNA strand in order to induce preferential replacement of the edit strand. PE3b

[0117] As used herein, “PE3b” refers to a prime editing composition comprising PE2 and further comprising a second-strand nicking guide RNA that complexes with PE2 and introduces a nick in the non-edit DNA strand, wherein the second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing the second strand nicking guide RNA with a spacer sequence that comprises complementarity to, and only hybridizes with, the edited strand after installation of the desired nucleotide edit(s), but not the endogenous target DNA sequence. Using this strategy, mismatches between the nicking guide RNA spacer and the unedited target DNA should disfavor nicking by the sgRNA until after the editing event on the PAM strand takes place. PE4 B1195.70198WO00 13849370.1 (April 11, 2025)65 / 895

[0118] As used herein, “PE4” refers to a prime editing composition comprising a PE2 and further comprising an MLH1 dominant negative protein variant (i.e., wild-type MLH1 with amino acids 754-756 truncated, which may be referred to herein as “MLH1 Δ754-756” or “MLH1dn”). The MLH1 dominant negative protein variant may be expressed in trans in some embodiments. In some embodiments, a PE4 system comprises a fusion protein comprising a PE2 protein and an MLH1 dominant negative protein joined via an optional linker. PE5 and PE5b

[0119] As used herein, “PE5” refers to a prime editing composition comprising a PE3 prime editor and further comprising an MLH1 dominant negative protein variant (i.e., wild- type MLH1 with amino acids 754-756 truncated, which may be referred to as “MLH1 Δ754- 756” or “MLH1dn”). The MLH1 dominant negative variant may be expressed in trans in some embodiments. In some embodiments, a PE5 system comprises a fusion protein comprising a PE2 protein and an MLH1 dominant negative protein joined via an optional linker. “PE5b” refers to a prime editing composition comprising a PE3 and an MLH1 dominant negative protein, wherein the second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing the second strand nicking guide RNA with a spacer sequence that comprise complementarity to, and hybridize with, only the edited strand after installation of the desired nucleotide edit(s), but not the endogenous target DNA sequence. PE6

[0120] The term “PE6” refers to a suite of prime editors (PE6a, PE6b, PE6c, PE6d, PE6e, PE6f, and PE6g) comprising improved reverse transcriptase and / or Cas9 variants. The improved reverse transcriptase and Cas9 domains of the PE6 variants can also be combined with each other to offer cumulative benefits. For example, a PE6 prime editor comprising an improved reverse transcriptase variant of PE6a and an improved Cas9 variant of PE6e is referred to herein as the prime editor “PE6a-e” (or “PE6e-a”). Any possible combination of PE6 prime editors is contemplated by the present disclosure including, for example, PE6a-e, PE6a-f, PE6a-g, PE6b-e, PE6b-f, PE6b-g, PE6c-e, PE6c-f, PE6c-g, PE6d-e, PE6d-f, and PE6d-g. B1195.70198WO00 13849370.1 (April 11, 2025)66 / 895

[0121] Any of the PE6 prime editors may also comprise the architecture of the PEmax protein as provided herein. In some embodiments, any of the PE6 prime editors provided herein may further comprise additional amino acid mutations, e.g., any of those included in PEmax as provided herein.

[0122] In some embodiments, a PE6 protein comprises a reverse transcriptase of the following amino acid sequence (the RT domain of “PE6a”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence: GRPYVTLNLNGMFMDKFKPYSKSNAPITTLEKLSKALSISVEELKAIAELSLDEKYTL KKIPKIDGSKRIVYSLHPKMRLLQSRINERIFKELVVFPSFLFGSVPSKNDVLNSNVKR DYVSCAKAHCGAKTVLKVDISNFFDNIHRDLVRSVFEEILHIKDEALDYLVDICTKDD FVVQGALTSSYIATLCLFAVEGDVVRRAQRKGLVYTRLVDDITVSSKISNYDFSQMQ SHIERMLSEHNLPINKHKTKIFHCSSEPIKVHGLIVDYDSPRLPSDKVKRIRASIHNLKL LAAKNNTKTSVAYRKEFNRCMGRVNELGRVGHEKYESFKKQLQAIKPMPSNRDVA VIDAAIKSLELSYSKGNQNKHWYKRKYDLTRYKMIILTRSESFKEKLECFKSRLASLK PL (SEQ ID NO: 1415)

[0123] In some embodiments, a PE6 protein comprises a reverse transcriptase of the following amino acid sequence (the RT domain of “PE6b”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence: ISSSKHTLSQMNKVSNIVKEPELPDIYKEFKDITADTNTEKLPKPIKGLEFEVELTQEN YRLPIRNYPLTPVKMQAMNDEINQGLKGGIIRESKAINACPVIFVPRKEGTLRMVVDY RPLNKYVKPNVYPLPLIEQLLAKIQGSTIFTKLDLKSAYHQIRVRKGDEHKLAFRCPR GVFEYLVMPYGISTAPAHFQYFINTILGEAKESHVVCYMDDILIHSKSESEHVKHVKD VLQKLKNANLIINQAKCEFHQSQVKFIGYHISEKGLTPCQENIDKVLQWKQPKNRKE LRQFLGSVNYLRKFIPKTSQLTHPLNKLLKKDVRWKWTPTQTQAIENIKQCLVSPPV LRHFDFSKKILLETDVSDVAVGAVLSQKHDDDKYYPVGYYSAKMSKAQLNYSVSD KEMLAIIKSLEHWRHYLESTIEPFKILTDHRNLIGRITNESEPENKRLARWQLFLQDFN FEINYRPGSANHIADALSRIVDETEPIPKDNEDNSINFVNQISI (SEQ ID NO: 1416)

[0124] In some embodiments, a PE6 protein comprises a reverse transcriptase of the following amino acid sequence (the RT domain of “PE6c”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence: B1195.70198WO00 13849370.1 (April 11, 2025)67 / 895 ISSSKHTLSQMNKVSNIVKEPELPDIYKEFKDITADTNTEKLPKPIKGLEFEVELTQEN YRLPIRNYPLTPVKMQAMNDEINQGLKGGIIRESKAINACPVIFVPRKEGTLRMVVDY RPLNKYVKPNVYPLPLIEQLLAKIQGSTIFTKLDLKSAYHQIRVRKGDEHKLAFRCPR GVFEYLVMPYGIKTAPAHFQYFINTILGEAKESHVVCYMDDILIHSKSESEHVKHVK DVLQKLKNANLIINQAKCEFHQSQVKFLGYHISEKGLTPCQENIDKVLQWKQPKNQ KELRQFLGQVNYLRKFIPKTSQLTHPLNKLLKKDVRWKWTPTQTQAIENIKQCLVSP PVLRHFDFSKKILLETDVSDVAVGAVLSQKHDDDKYYPVGYYSAKMSKAQLNYSVS DKEMLAIIKSLEHWRHYLESTIEPFKILTDHRNLIGRITNESEPENKRLARWQLFLQDF NFEINYRPGSANHIADALSRIVDETEPIPKDNEDNSINFVNQISI (SEQ ID NO: 37)

[0125] In some embodiments, a PE6 protein comprises a reverse transcriptase comprising the following amino acid sequence (the RT domain of “PE6d”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence: TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTP VSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLR EVNKRVEDIHPNVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWR DPEMGISGQLTWTRLPQGFKNSPTLFCEALHRDLADFRIQHPDLILLQYYDDLLLAAT SELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKE TVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKA YQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSN ARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLD (SEQ ID NO: 1417)

[0126] In some embodiments, a PE6 protein comprises a Cas9 protein of the following amino acid sequence (the Cas9 domain of “PE6e”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence: MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHE RHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ B1195.70198WO00 13849370.1 (April 11, 2025)68 / 895 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFT VYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECF DSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQRNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIARQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVL SMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVL VVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS LFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLF VEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTN LGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 1418)

[0127] In some embodiments, a PE6 protein comprises a Cas9 protein of the following amino acid sequence (the Cas9 domain of “PE6f”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence: MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFRRLEESFLVEEDKKHE RHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEKTITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFT VYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECF DSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMVEE B1195.70198WO00 13849370.1 (April 11, 2025)69 / 895 RLKTYAHLFDNKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLYEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIARQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVL SMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVL VVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS LFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLF VEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTN LGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 1419)

[0128] In some embodiments, a PE6 protein comprises a Cas9 protein of the following amino acid sequence (the Cas9 domain of “PE6g”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence: MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFRRLEESFLVEEDKKHE RHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEKTITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFT VYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECF DSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMVEE RLKTYAHLFDNKVMKQLKRCRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLYEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRS B1195.70198WO00 13849370.1 (April 11, 2025)70 / 895 DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVL SMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVL VVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS LFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLF VEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTN LGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 1420) PE7

[0129] The term “PE7” refers to the PE6 prime editors plus a second strand nicking guide RNA. For example, “PE7a” refers to the PE6a prime editor as provided herein, plus a second strand nicking guide RNA. PEmax

[0130] As used herein, “PEmax” refers to a prime editing composition comprising 1) a fusion protein comprising a Cas9 protein variant Cas9(R221K N39K H840A) and a variant MMLV RT having the following structure: [bipartite NLS]- [Cas9(R221K)(N394K)(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)]-[bipartite NLS]-[NLS] and 2) a desired PEgRNA, wherein the fusion protein (referred to as the PEmax protein) has the following amino acid sequence: MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNT DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDS FFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEH HQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKIL TFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR B1195.70198WO00 13849370.1 (April 11, 2025)71 / 895 KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVP QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRD KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETR IDLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSTLNIEDEYRLHETSK EPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARL GIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVP NPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTR LPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL REFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPAL GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRM VAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYT DGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN VYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGH QKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFESPKK KRKVGSGPAAKRVKLD (SEQ ID NO: 1421 KEY: BIPARTITE SV40 NUCLEAR LOCALIZATION SEQUENCE (NLS), CAS9(R221K N39K H840A) SGGSx2-BIPARTITE SV40NLS-SGGSx2 LINKER M-MLV reverse transcriptase(D200N T306K W313F T330P L603W) Other linker sequence B1195.70198WO00 13849370.1 (April 11, 2025)72 / 895 BIPARTITE SV40NLS Other linker sequence c-Myc NLS Prime editing

[0131] As used herein, the term “prime editing” refers to an approach for gene editing using napDNAbps, a polymerase (e.g., a reverse transcriptase), and specialized guide RNAs that include a primer binding site and 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. See also International PCT Application, PCT / US2020 / 023721, filed March 19, 2020, and published as WO 2020 / 191239, which is incorporated herein by reference.

[0132] 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 (i.e., in the form of a fusion protein or otherwise provided in trans with the napDNAbp), wherein the prime editing system is programmed with a prime editing (PE) guide RNA (“pegRNA”) that both specifies the target site and templates the synthesis of the desired edit in the form of a replacement DNA strand by way of an extension (either DNA or RNA) engineered onto a guide RNA (e.g., at the 5ʹ or 3ʹ end, or at an internal portion of a guide RNA). The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same 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, as described herein, not only search and locate the desired target site to be edited, but at the same time, encode a replacement strand containing a desired edit that is installed in place of the corresponding target site endogenous DNA strand. The prime editors of the present disclosure relate, in part, to the discovery that the mechanism of target-primed reverse B1195.70198WO00 13849370.1 (April 11, 2025)73 / 895 transcription (TPRT) or “prime editing” can be leveraged or adapted for conducting precision CRISPR / Cas-based genome editing with high efficiency and genetic flexibility. TPRT is naturally used by mobile DNA elements, such as mammalian non-LTR retrotransposons and bacterial Group II introns. Cas protein-reverse transcriptase fusions or related systems are used to target a specific DNA sequence with a guide RNA, generate a single strand nick at the target site, and use the nicked DNA as a primer for reverse transcription of an engineered DNA synthesis template that is integrated with the guide RNA. However, while the concept begins with prime editors that use reverse transcriptase as the DNA polymerase component, the prime editors described herein are not limited to reverse transcriptases but may include the use of virtually any DNA polymerase. Indeed, while the application throughout may refer to prime editors with “reverse transcriptases,” it is set forth here that reverse transcriptases are only one type of DNA polymerase that may work with prime editing. Thus, wherever the specification mentions a “reverse transcriptase,” the person having ordinary skill in the art should appreciate that any suitable DNA polymerase may be used in place of the reverse transcriptase. Thus, in one aspect, the prime editors may comprise Cas9 (or an equivalent napDNAbp), which is programmed to target a DNA sequence by associating it with a specialized guide RNA (i.e., pegRNA) containing a spacer sequence that anneals to a complementary sequence (the complementary sequence to an endogenous protospacer sequence) in the target DNA. The pegRNA also contains new genetic information in the form of an extension that encodes a replacement strand of DNA containing a desired nucleotide change which is used to replace a corresponding endogenous DNA strand at the target site. To transfer information from the pegRNA to the target DNA, the mechanism of prime editing involves nicking the target site in one strand of the DNA to expose a 3′-hydroxyl group. The exposed 3′-hydroxyl group can then be used to prime the DNA polymerization of the edit- encoding extension on pegRNA directly into the target site. In various embodiments, the extension—which provides the template for polymerization of the replacement strand containing the edit—can be formed from RNA or DNA. In the case of an RNA extension, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (such as a reverse transcriptase). In the case of a DNA extension, the polymerase of the prime editor may be a DNA-dependent DNA polymerase. The newly synthesized strand (i.e., the replacement DNA strand containing the desired nucleotide edit) that is formed by the prime editor would be homologous to the genomic target sequence (i.e., have the same sequence as), except for the inclusion of one or more desired nucleotide changes (e.g., a single B1195.70198WO00 13849370.1 (April 11, 2025)74 / 895 nucleotide substitution, a deletion, or an insertion, or a combination thereof). The newly synthesized (or replacement) strand of DNA may also be referred to as a single strand DNA flap, which would compete for hybridization with the complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. Resolution of the hybridized intermediate (also referred to as a heteroduplex, comprising the single strand DNA flap synthesized by the reverse transcriptase hybridized to the endogenous DNA strand with the exception of mismatches at positions where desired nucleotide edits are installed in the edit strand) can include removal of the resulting displaced flap of endogenous DNA (e.g., with a 5ʹ end DNA flap endonuclease, FEN1), ligation of the synthesized single strand DNA flap to the target DNA, and assimilation of the desired nucleotide changes as a result of cellular DNA repair and / or replication processes.

[0133] In various embodiments, prime editing operates by contacting a target DNA molecule (for which a change in the nucleotide sequence is desired to be introduced) with a nucleic acid programmable DNA binding protein (napDNAbp) complexed with a prime editing guide RNA (pegRNA). In various embodiments, the prime editing guide RNA (pegRNA) comprises an extension at the 3′ or 5′ end of the guide RNA, or at an intramolecular location in the guide RNA, and encodes the desired nucleotide change (e.g., single nucleotide substitution, insertion, or deletion). First, the napDNAbp / extended gRNA complex contacts the DNA molecule, and the extended gRNA guides the napDNAbp to bind to a target locus. Next, a nick in one of the strands of DNA of the target locus is introduced (e.g., by a nuclease or chemical agent), thereby creating an available 3′ end in one of the strands of the target locus. In certain embodiments, the nick is created in the strand of DNA that corresponds to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence, i.e., the “non-target strand.” The nick, however, could be introduced in either of the strands. That is, the nick could be introduced into the R-loop “target strand” (i.e., the strand hybridized to the protospacer of the extended gRNA) or the “non-target strand” (i.e., the strand forming the single-stranded portion of the R-loop and which is complementary to the target strand). In the next step, the 3′ end of the DNA strand (formed by the nick) interacts with the extended portion of the guide RNA in order to prime reverse transcription (i.e., “target-primed RT”). In certain embodiments, the 3′ end DNA strand hybridizes to a specific RT priming sequence on the extended portion of the guide RNA, i.e., the “reverse transcriptase priming sequence” or “primer binding site” on the pegRNA. In the next step, a reverse transcriptase (or other suitable DNA polymerase) is introduced that synthesizes a B1195.70198WO00 13849370.1 (April 11, 2025)75 / 895 single strand of DNA from the 3′ end of the primed site towards the 5′ end of the prime editing guide RNA. The DNA polymerase (e.g., reverse transcriptase) can be fused to the napDNAbp or alternatively can be provided in trans to the napDNAbp. This forms a single- strand DNA flap comprising the desired nucleotide change (e.g., the single base change, insertion, or deletion, or a combination thereof) and that is otherwise homologous to the endogenous DNA at or adjacent to the nick site. In the next step, the napDNAbp and guide RNA are released. The final two steps relate to the resolution of the single strand DNA flap such that the desired nucleotide change becomes incorporated into the target locus. This process can be driven towards the desired product formation by removing the corresponding 5′ endogenous DNA flap that forms once the 3′ single strand DNA flap invades and hybridizes to the endogenous DNA sequence. Without being bound by theory, the cell’s endogenous DNA repair and replication processes resolve the mismatched DNA to incorporate the nucleotide change(s) to form the desired altered product. The process can also be driven towards product formation with “second strand nicking.” This process may introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions. Prime editor

[0134] 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., Cas9 nickase, and / or any of the Cas9 variants provided herein) and a reverse transcriptase (e.g., any of the reverse transcriptase variants 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., Cas9 nickase) 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 B1195.70198WO00 13849370.1 (April 11, 2025)76 / 895 complex comprising a fusion protein (reverse transcriptase fused to a napDNAbp), a pegRNA, and a regular guide RNA capable of directing the second-site nicking step of the non-edited strand as described herein. Primer binding site

[0135] The term “primer binding site” or “the PBS” refers to the nucleotide sequence located on a pegRNA as component of the extension arm (typically at the 3ʹ end of the extension arm) and serves to bind to the primer sequence that is formed after Cas9 nicking of the target sequence by the prime editor. As detailed elsewhere, when the Cas9 nickase component of a prime editor nicks one strand of the target DNA sequence, a 3ʹ-ended ssDNA flap is formed, which serves a primer sequence that anneals to the primer binding site on the pegRNA to prime reverse transcription. Protein, peptide, and polypeptide

[0136] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein, or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the contents of which are incorporated herein by reference. B1195.70198WO00 13849370.1 (April 11, 2025)77 / 895 Protospacer

[0137] As used herein, the term “protospacer” refers to the sequence (~20 bp) in DNA adjacent to the PAM (protospacer adjacent motif) sequence. The protospacer shares the same sequence as the spacer sequence of the guide RNA. The guide RNA anneals to the complement of the protospacer sequence on the target DNA (specifically, one strand thereof, i.e., the “target strand” versus the “non-target strand” of the target DNA sequence). 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. Protospacer adjacent motif (PAM)

[0138] As used herein, the term “protospacer adjacent motif” or “PAM” refers to a DNA sequence (e.g., an approximately 2-6 nucleotide sequence) that is an important targeting component of a Cas nuclease, e.g., a Cas9. For example, in some embodiments for a Cas9 nuclease, the PAM sequence is on either strand and is downstream in the 5ʹ to 3ʹ direction of the Cas9 cut site. The canonical PAM sequence (i.e., the PAM sequence that is associated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9) is 5ʹ-NGG-3ʹ, wherein “N” is any nucleobase followed by two guanine (“G”) nucleobases. In some embodiments, SpCas9 can also recognize additional non-canonical PAMs (e.g., NAG and NGA).

[0139] Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms. In addition, any given Cas9 nuclease, e.g., SpCas9, may be modified to alter the PAM specificity of the nuclease such that the nuclease recognizes an alternative PAM sequence. Reverse transcriptase

[0140] 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 B1195.70198WO00 13849370.1 (April 11, 2025)78 / 895 cloned into a vector for further manipulation. Avian myoblastosis virus (AMV) reverse transcriptase was the first widely used RNA-dependent DNA polymerase (Verma, Biochim. Biophys. Acta 473:1 (1977)). The enzyme has 5ʹ-3ʹ RNA-directed DNA polymerase activity, 5ʹ-3ʹ DNA-directed DNA polymerase activity, and RNase H activity. RNase H is a processive 5ʹ and 3ʹ ribonuclease specific for the RNA strand for RNA-DNA hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)). Errors in transcription cannot be corrected by reverse transcriptase because known viral reverse transcriptases lack the 3ʹ-5ʹ exonuclease activity necessary for proofreading (Saunders and Saunders, Microbial Genetics Applied to Biotechnology, London: Croom Helm (1987)). A detailed study of the activity of AMV reverse transcriptase and its associated 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 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. The invention contemplates the use of any such reverse transcriptases, or variants or mutants thereof. Silent mutation

[0141] As used herein, the term “silent mutation” refers to a mutation in a nucleic acid molecule that does not have an effect on the phenotype of the nucleic acid molecule, or the protein it produces if it encodes a protein. Silent mutations can be introduced into coding regions of a nucleic acid (i.e., segments of a gene that encode for a protein), or they can be introduced in non-coding regions of a nucleic acid. A silent mutation in a nucleic acid sequence, e.g., in a target DNA sequence or in a DNA synthesis template sequence to be installed in the target sequence, may be a nucleotide alteration that does not result in expression or function of the amino acid sequence encoded by the nucleic acid sequence, or other functional features of the target nucleic acid sequence. When silent mutations are present in a coding region, they may be synonymous mutations. Synonymous mutations refer to substitutions of one base for another in a gene such that the corresponding amino acid residue of the protein produced by the gene is not modified. This is due to the redundancy of the genetic code, allowing for multiple different codons to encode for the same amino acid in a particular organism. When a silent mutation is in a noncoding region or a junction of a B1195.70198WO00 13849370.1 (April 11, 2025)79 / 895 coding region and a non-coding region (e.g., an intron / exon junction), it may be in a region that does not impact any biological properties of the nucleic acid molecule (e.g., splicing, gene regulation, RNA lifetime, etc.). In particular embodiments, a silent mutation may also be a “benign” mutation, for example, where a nucleotide substitution results in one or more alterations in the amino acid sequence encoded, but does not result in detrimental impact on the expression or function of the polypeptide. Silent mutations may be useful, for example, for increasing the length of contiguous changes in a desired nucleotide edit or the number of nucleotide edits made to a target nucleotide sequence using prime editing to evade correction of the edit by the MMR pathway as described herein. In certain embodiments, the number of silent mutations installed may be one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or more. In certain other embodiments involving at least two silent mutations, the silent mutations may be installed within one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 nucleotides from the intended edit site. Spacer sequence

[0142] As used herein, the term “spacer sequence” in connection with a guide RNA refers to the portion of the guide RNA 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. Subject

[0143] 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. B1195.70198WO00 13849370.1 (April 11, 2025)80 / 895 Target site

[0144] The term “target site” refers to a sequence within a nucleic acid molecule that is modified (e.g., edited) by a prime editor as described herein. The target site further refers to the sequence within a nucleic acid molecule (e.g., a nucleic acid molecule comprising ATP1A3) to which a complex of, for example, a prime editor and a pegRNA binds. Treatment

[0145] The terms “treatment,” “treat,” and “treating,” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder (e.g., alternating hemiplegia of childhood), or one or more symptoms thereof, as described herein. As used herein, the terms “treatment,” “treat,” and “treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder (e.g., alternating hemiplegia of childhood), or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and / or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease (e.g., alternating hemiplegia of childhood). For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence. Variant

[0146] As used herein, the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant Cas9 is a Cas9 comprising one or more changes in amino acid residues (i.e., “substitutions”) as compared to a wild type Cas9 amino acid sequence. The term “variant” encompasses homologous proteins having 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 sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence that display the same or substantially the same functional activity or activities as the reference sequence. B1195.70198WO00 13849370.1 (April 11, 2025)81 / 895 Vector

[0147] The term “vector,” as used herein, refers to a nucleic acid that can be modified to encode a gene of interest and that is able to enter 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. INCORPORATION BY REFERENCE

[0148] The present application refers to various issued patent, published patent applications, scientific journal articles, and other publications describing prime editing including: International Patent Application Number PCT / US2023 / 065947, filed on April 19, 2023, and published on October 26, 2023, with International Publication Number WO2023 / 205687; International Patent Application Number PCT / US2022 / 078655, filed on October 25, 2022, and published on May 4, 2023, with International Publication Number WO2023 / 076898; International Patent Application Number PCT / US2022 / 080836, filed on December 2, 2022, and published on June 8, 2023, with International Publication Number WO2023 / 102538; International Patent Application Number PCT / US2023 / 076282, filed on October 6, 2023; International Patent Application Number PCT / US2023 / 085586, filed on December 21, 2023; International Patent Application Number PCT / US2023 / 080226, filed on November 17, 2023; International Patent Application Number PCT / US2024 / 014998, filed on February 8, 2024; International Patent Application Number PCT / US2024 / 011892, filed on January 17, 2024; International Patent Application Number PCT / US2024 / 021103, filed on March 22, 2024; U.S. Patent Application Number 17 / 767,777, filed on April 8, 2022., U.S. Patent Application Number 18 / 064738, filed on December 12, 2022; U.S. Patent Application Number 18 / 326588, filed on May 31, 2023, and published on October 26, 2023, with publication number US-2023-0340465-A1; U.S. Patent Application Number 18 / 326634, filed on May 31, 2023, and published on October 26, 2023, with publication number US-2023- 0340466-A1; U.S. Patent Application Number 18 / 326689, filed on May 31, 2023, and published on November 30, 2023, with publication number US-2023-0383289-A1; U.S. Patent Application Number 18 / 326708, filed on May 31, 2023, and published on October 26, 2023, with publication number US-2023-0340467-A1; U.S. Patent Application Number B1195.70198WO00 13849370.1 (April 11, 2025)82 / 895 18 / 323245, filed on May 24, 2023, and published on October 19, 2023, with publication number US-2023-0332144-A1; U.S. Patent Application Number 18 / 028183, filed on March 23, 2023, and published on November 9, 2023, with publication number US-2023-0357766- A1; U.S. Patent Application Number 18 / 271656, filed on July 10, 2023; U.S. Patent Application Number 18 / 681490, filed on February 5, 2024; U.S. Patent Application Number 17 / 440682, filed on September 17, 2021, and published on March 16, 2023, with publication number US-2023-0078265-A1; U.S. Patent Application Number 18 / 619518, filed on March 28, 2024; U.S. Patent Application Number 18 / 534489, filed on December 8, 2023; U.S. Patent Application Number 17 / 300668, filed on September 17, 2021; U.S. Patent Number 11795452, filed on May 23, 2022, and granted on October 24, 2023; U.S. Patent Number 11447770, filed on March 31, 2021, and granted on September 20, 2022; U.S. Patent Number 11643652, filed on March 31, 2021, and granted on May 9, 2023; and U.S. Patent Number 11912985, filed on November 7, 2022, and granted on February 27, 2024, all of which are incorporated herein by reference.

[0149] The present application refers to various issued patent, published patent applications, scientific journal articles, and other publications describing base editing including U.S. Patent Publication No. 2018 / 0073012, published March 15, 2018, which issued as U.S. Patent No. 10,113,163; on October 30, 2018; U.S. Patent Publication No. 2017 / 0121693, published May 4, 2017, which issued as U.S. Patent No. 10,167,457 on January 1, 2019; International Publication No. WO 2017 / 070633, published April 27, 2017; International Publication No. WO 2018 / 027078, published August 2, 2018; International Application No PCT / US2018 / 056146, filed October 16, 2018, which published as Publication No. WO 2019 / 079347 on April 25, 2019; International Application No PCT / US2019 / 033848, filed May 23, 2019, which published as Publication No. WO 2019 / 226593 on November 28, 2019; U.S. Patent Publication No.2015 / 0166980, published June 18, 2015; U.S. Patent No. 9,840,699, issued December 12, 2017; U.S. Patent No. 10,077,453, issued September 18, 2018; International Publication No. WO 2019 / 023680, published January 31, 2019; International Publication No. WO 2018 / 0176009, published September 27, 2018; International Publication No. WO 2020 / 041751, published February 27, 2020; International Publication No. WO 2020 / 051360, published March 12, 2020; International Patent Publication No. WO 2020 / 102659, published May 22, 2020; International Publication No. WO 2020 / 086908, published April 30, 2020; International Publication No. WO 2020 / 181180, published September 10, 2020; International Publication B1195.70198WO00 13849370.1 (April 11, 2025)83 / 895 No. WO 2020 / 214842, published October 22, 2020; International Publication No. WO 2020 / 092453, published May 7, 2020; International Patent Application No. PCT / US2020 / 033873, filed May 20, 2020, published as International Publication No. WO2020 / 236982 on November 26, 2020, and International Application No. PCT / US2020 / 624628, filed November 25, 2020, all of which are incorporated herein by reference. DETAILED DESCRIPTION

[0150] The present disclosure provides articles, compositions and methods of editing an ATP1A3 gene using a prime editor and a pegRNA to correct a ATP1A3 D801N c.2401A mutation, ATP1A3 E815K c.2443A mutation, a ATP1A3G947R c.2839C mutations, or aATP1A3 L839P c.2516C mutation. Additionally, the present disclosure provides articles, compositions and methods of editing an ATP1A3 gene using a base editor and a sgRNA to correct a ATP1A3 G947R c.2839A mutation. The articles, compositions, and methods disclosed herein may be useful for treating alternating hemiplegia of childhood (AHC). The present disclosure also provides ngRNAs, dsgRNAs, and systems (e.g., prime editing systems and / or base editing systems) for editing the ATP1A3 gene and treating AHC. Polynucleotides, vectors, cells, and kits for editing ATP1A3 and treating AHC are also provided herein. Guide RNAs (gRNAs)

[0151] The present disclosure provides pegRNAs, ngRNAs, and dsgRNAs for prime editing a ATP1A3 gene (e.g., a ATP1A3 D801N c.2401A mutation, a ATP1A3 E815K c.2443A mutation, a ATP1A3 G947R c.2839C mutation, a ATP1A3 L839P c.2516C mutation or a ATP1A3 G947R c.2839A mutation). The pegRNAs, ngRNAs, and dsgRNAs provided herein may be useful for treating Alternating Hemiplegia of Childhood (AHC).

[0152] A nonlimiting example of a human ATP1A3 gene comprises gene sequence is provided in Gene ID: 478. A nonlimiting example of human ATP1A3 gene sequence is provided in Homo sapiens chromosome 19, GRCh38.p14 Primary Assembly: NC_000019.10: 41966582..41994230.

[0153] In some embodiments, the provided pegRNAs target a prime editor to a site in the human ATP1A3 gene. In some embodiments, the gRNAs target a prime editor to a site in the human ATP1A3 gene such that the prime editor corrects a ATP1A3 D801N c.2401A mutation, a ATP1A3 E815K c.2443A mutation, a ATP1A3 G947R c.2839C mutation, or a B1195.70198WO00 13849370.1 (April 11, 2025)84 / 895 ATP1A3 L839P c.2516C mutation in a ATP1A3 protein by inserting a ATP1A3 D801N c.2401A>G edit, a ATP1A3 E815K c.2443A>G edit, a ATP1A3 G947R c.2839C>G edit, or a ATP1A3 L839P c.2516C>T edit.

[0154] In one aspect, the present disclosure provides prime editing pegRNAs comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 77-96, 919-1066, 1229-1317, 1408-1409, 1523-1525.

[0155] In one aspect, the present disclosure provides prime editing pegRNAs comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of GGCTCACCATGTCAGTGCCC (SEQ ID NO: 79).

[0156] In one aspect, the present disclosure provides prime editing pegRNAs comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of GCCATCTCACTGGCGTACA (SEQ ID NO: 84).

[0157] In one aspect, the present disclosure provides prime editing pegRNAs comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of GGAACAAGATCCTGATCTTC (SEQ ID NO: 81).

[0158] In one aspect, the present disclosure provides prime editing pegRNAs comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of GCCCGTAGGCCATGCTGAT (SEQ ID NO: 89).

[0159] The pegRNAs provided herein also comprise a backbone scaffold sequence that facilitates binding of the pegRNA to a napDNAbp, for example, a Cas9 protein (e.g., a Cas9 protein as part of a prime editor). In some embodiments, the provided pegRNAs comprise a backbone scaffold sequence for binding to SpCas9. In some embodiments, the pegRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 97-102, 1067.

[0160] In some embodiments, the pegRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at B1195.70198WO00 13849370.1 (April 11, 2025)85 / 895 least 97%, at least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of GTTTGAGAGCTATGCTGGAAACAGCATAGCAAGTTCAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 99).

[0161] In some embodiments, the pegRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of GTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 98).

[0162] The pegRNAs provided herein also comprise a reverse transcription template. In some embodiments, the pegRNA comprises an RTT of 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, or 50 nucleotides in length. In some embodiments, the pegRNA comprises an RTT comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 103-223, 1526-1528.

[0163] In some embodiments, the pegRNA comprises an RTT comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to the nucleic acid sequence TCCTCTGCATTGACTTAGG (SEQ ID NO: 124).

[0164] In some embodiments, the pegRNA comprises an RTT comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to the nucleic acid sequence ATGATGTCGCTTTCGGCAGCTTCAT (SEQ ID NO: 152).

[0165] In some embodiments, the pegRNA comprises an RTT comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to the nucleic acid sequence TCCTCAAACAAGCCAAA (SEQ ID NO: 171).

[0166] In some embodiments, the pegRNA comprises an RTT comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to the nucleic acid sequence GGTCAATGAGAGGTTGATC (SEQ ID NO: 188). B1195.70198WO00 13849370.1 (April 11, 2025)86 / 895

[0167] The pegRNAs provided herein also comprise a primer binding site (PBS). In some embodiments, the pegRNA comprises a PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 224-280, 1529- 1531.

[0168] The pegRNAs provided herein also comprise a primer binding site (PBS). In some embodiments, the pegRNA comprises a PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequence: CACTGACATGGT (SEQ ID NO: 231).

[0169] The pegRNAs provided herein also comprise a primer binding site (PBS). In some embodiments, the pegRNA comprises a PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequence: ACGCCAGTGAGAT (SEQ ID NO: 235).

[0170] The pegRNAs provided herein also comprise a primer binding site (PBS). In some embodiments, the pegRNA comprises a PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequence: GATCAGGATCTTGT (SEQ ID NO: 260).

[0171] The pegRNAs provided herein also comprise a primer binding site (PBS). In some embodiments, the pegRNA comprises a PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequence: AGCATGGCCTAC (SEQ ID NO: 267).

[0172] In some embodiments, the pegRNAs provided herein further comprise a structured motif at the 3' end. In some embodiments, the structured motif is an RNA pseudoknot motif. In some embodiments, the structured motif comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 281). In certain embodiments, the structured motif comprises the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 281).

[0173] In some embodiments, the structured motif comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 281-282. B1195.70198WO00 13849370.1 (April 11, 2025)87 / 895

[0174] In some embodiments, the pegRNA comprises the structure 5'-[spacer]- [backbone scaffold]-[reverse transcription template]-[primer binding site]-[structured motif]- 3'. In some embodiments, the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 283-918, 1573-1578.

[0175] In some embodiments, the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequence: GGCTCACCATGTCAGTGCCCGTTTGAGAGCTATGCTGGAAACAGCATAGCAAGTT CAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTCCT CTGCATTGACTTAGGCACTGACATGGTCGCGGTTCTATCTAGTTACGCGTTAAAC CAACTAGAA. (SEQ ID NOs: 369)

[0176] In some embodiments, the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequence: GCCCGTAGGCCATGCTGATGTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTT TAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGGT CAATGAGAGGTTGATCAGCATGGCCTACCGCGGTTCTATCTAGTTACGCGTTAAA CCAACTAGAA. (SEQ ID NO: 783)

[0177] In some embodiments, the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequence: GGAACAAGATCCTGATCTTCGTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGT TTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTCC TCAAACAAGCCAAAGATCAGGATCTTGTCGCGGTTCTATCTAGTTACGCGTTAAA CCAACTAGAA. (SEQ ID NOs: 679)

[0178] In some embodiments, the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequence: GCCCGTAGGCCATGCTGATGTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTT TAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGGT CAATGAGAGGTTGATCAGCATGGCCTACCGCGGTTCTATCTAGTTACGCGTTAAA CCAACTAGAA. (SEQ ID NO: 783) B1195.70198WO00 13849370.1 (April 11, 2025)88 / 895

[0179] In some embodiments, the pegRNA comprises a chemical modification at its 3' end. In certain embodiments, the chemical modification comprises phosphonoacetate (mP).

[0180] In some embodiments, the pegRNA targets a D801N (c.2401A) mutation in ATP1A3. In some embodiments, the pegRNA that targets a D801N (c.2401A) mutation in ATP1A3 comprises a nucleotide sequence of SEQ ID NOs 355-450 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 80% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 90% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 95% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 99.5% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the pegRNA comprises the nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs 355-450.

[0181] In some embodiments, the pegRNA targets a E815K (c.2443A) mutation in ATP1A3. In some embodiments, the pegRNA that pegRNA targets a E815K (c.2443A) mutation in ATP1A3 comprises a nucleotide sequence of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNA that pegRNA targets a E815K (c.2443A) mutation in ATP1A3 comprises a nucleotide sequence of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNA that pegRNA targets a E815K (c.2443A) mutation in ATP1A3 comprises a nucleic acid sequence having at least 80%, at least 85%, at B1195.70198WO00 13849370.1 (April 11, 2025)89 / 895 least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 80% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 90% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 95% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 99.5% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNA comprises the nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576.

[0182] In some embodiments, the pegRNA targets a L839P (c.2516C) mutation in ATP1A3. In some embodiments, the pegRNA that targets a L839P (c.2516C) mutation in ATP1A3 comprises a nucleotide sequence of SEQ ID NOs 733-827, 1574, 1577 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the pegRNA that targets a L839P (c.2516C) mutation in ATP1A3 comprises a nucleotide sequence of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the pegRNA that targets a L839P (c.2516C) mutation in ATP1A3 comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least B1195.70198WO00 13849370.1 (April 11, 2025)90 / 895 80% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733- 827, 1574, 1577. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 90% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 95% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 99.5% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the pegRNA comprises the nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577.

[0183] In some embodiments, the pegRNA targets a G947R (c.2839C) mutation in ATP1A3. In some embodiments, the pegRNA that targets a G947R (c.2839C) mutation in ATP1A3 comprises a nucleotide sequence of SEQ ID NOs 639-732, 1578 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleotide sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNA that targets a G947R (c.2839C) mutation in ATP1A3 comprises a nucleotide sequence of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNA that targets a G947R (c.2839C) mutation in ATP1A3 comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleotide sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 80% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 90% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the B1195.70198WO00 13849370.1 (April 11, 2025)91 / 895 pegRNA comprises a nucleic acid sequence having at least 95% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 99.5% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNA comprises the nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs 639-732, 1578.

[0184] In another aspect, the present disclosure provides nicking guide RNAs (ngRNAs) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 80, 85, 86, 90, 919-1066, 1532-1534.

[0185] In some embodiments, the nicking guide RNAs (ngRNAs) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequence: CCTGACCAGCAATATCCCGG. (SEQ ID NOs: 929).

[0186] In some embodiments, the nicking guide RNAs (ngRNAs) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequence: TGACCAATTTGTCCGTCCGC (SEQ ID NO: 920).

[0187] In some embodiments, the nicking guide RNAs (ngRNAs) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequence: CCTCAAACAAGCCAAAGATC (SEQ ID NO: 1011).

[0188] In some embodiments, the nicking guide RNAs (ngRNAs) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequence: CAATGAGAGGTTGATCAGCA. (SEQ ID NO: 1031).

[0189] In certain embodiments, the ngRNA spacer further comprises a G at its 5' end. B1195.70198WO00 13849370.1 (April 11, 2025)92 / 895

[0190] In some embodiments, the ngRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 97, 99, 1067.

[0191] In some embodiments, the ngRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of GTTTGAGAGCTATGCTGGAAACAGCATAGCAAGTTCAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 99).

[0192] In some embodiments, the ngRNA comprises a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 1068-1228, 1579-1583.

[0193] In some embodiments, the ngRNA comprises a sequence comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of gCCTGACCAGCAATATCCCGGGTTTGAGAGCTATGCTGGAAACAGCATAGCAAGT TCAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 1080).

[0194] In some embodiments, the ngRNA comprises a sequence comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of gTGACCAATTTGTCCGTCCGCGTTTGAGAGCTATGCTGGAAACAGCATAGCAAGT TCAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 1093).

[0195] In some embodiments, the ngRNA comprises a sequence comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of gCCTCAAACAAGCCAAAGATCGTTTGAGAGCTATGCTGGAAACAGCATAGCAAG TTCAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 1171).

[0196] In some embodiments, the ngRNA comprises a sequence comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at B1195.70198WO00 13849370.1 (April 11, 2025)93 / 895 least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of gCAATGAGAGGTTGATCAGCAGTTTGAGAGCTATGCTGGAAACAGCATAGCAAG TTCAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 1192).

[0197] In another aspect, the present disclosure provides dead single guide RNAs (dsgRNAs) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 1229-1317, 1535-1550.

[0198] In some embodiments, the dead single guide RNAs (dsgRNAs) comprises a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequence: GGGGCCCAGGAGGGT (SEQ ID NO: 1234)

[0199] In some embodiments, the dsgRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 97, 99, 1067.

[0200] In some embodiments, dsgRNAs comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequence: GTTTGAGAGCTATGCTGGAAACAGCATAGCAAGTTCAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 99)

[0201] In some embodiments, the dsgRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs. 1318-1407, 1585-1601.

[0202] In some embodiments, the dsgRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or at least 100% identical to the sequence: GGGGCCCAGGAGGGTGTTTGAGAGCTATGCTGGAAACAGCATAGCAAGTTCAAA TAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 1323).

[0203] In some embodiments, the sgRNA comprises a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, B1195.70198WO00 13849370.1 (April 11, 2025)94 / 895 at least 99%, or at least 100% identical: CTTCAGGCTGTTTGAGGAGA. (SEQ ID NO: 1408).

[0204] In another aspect, the present disclosure provides sgRNAs comprising a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 97-102, 1067.

[0205] In some embodiments, the sgRNA comprises a sgRNA backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical: GTTTGAGAGCTATGCTGGAAACAGCATAGCAAGTTCAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC. (SEQ ID NO: 99).

[0206] In another aspect, the present disclosure provides sgRNAs comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 1410-1411.

[0207] In some embodiments, the sgRNA comprises a sgRNA comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical: gCTTCAGGCTGTTTGAGGAGAGTTTGAGAGCTATGCTGGAAACAGCATAGCAAGT TCAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC. (SEQ ID NO: 1410).

[0208] In another aspect, the present disclosure provides single guide RNAs for programing a base editor to edit a target gene, e.g., ATP1A3 (e.g., ATP1A3 G947R c.2839A). In some embodiments, the sgRNA comprises a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 1408-1409.

[0209] In some embodiments, the sgRNA comprises a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical: CTTCAGGCTGTTTGAGGAGA. (SEQ ID NO: 1408).

[0210] In another aspect, the present disclosure provides sgRNAs comprising a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least B1195.70198WO00 13849370.1 (April 11, 2025)95 / 895 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 99, 1067.

[0211] In some embodiments, the sgRNA comprises a sgRNA backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to: GTTTGAGAGCTATGCTGGAAACAGCATAGCAAGTTCAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC. (SEQ ID NO: 99).

[0212] In another aspect, the present disclosure provides sgRNAs comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 1410-1411

[0213] In some embodiments, the sgRNA comprises a sgRNA comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to: gCTTCAGGCTGTTTGAGGAGAGTTTGAGAGCTATGCTGGAAACAGCATAGCAAGT TCAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC. (SEQ ID NO: 1410).

[0214] Additional sequences of suitable pegRNAs, ngRNAs, and dsgRNAs for prime editing (or sgRNAs for base editing) ATP1A3 within the scope of the present disclosure will be apparent to those of skill in the art. Such suitable pegRNAs, ngRNAs, and dsgRNAs (or sgRNAs) sequences typically comprise a spacer sequence that is complementary to a nucleic sequence within 50 nucleotides (e.g., within 45, 40, 35, 30, 25, 20, 15, or 10 nucleotides) upstream or downstream of the target nucleotide to be edited (e.g., a target mutation such as ATP1A3 D801N c.2401A, ATP1A3 E815K c.2443A, ATP1A3G947R c.2839C, or ATP1A3 L839P c.2516C.).

[0215] In general, a pegRNA, ngRNA, or dsgRNA (or sgRNA) spacer is any RNA sequence having sufficient complementarity with a target polynucleotide sequence (e.g., ATP1A3) to hybridize with the target sequence and direct sequence-specific binding of a napDNAbp (e.g., Cas9, which may be part of a prime editor) to the target sequence. In some embodiments, the degree of complementarity between the spacer and its corresponding target sequence in ATP1A3, 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 (or the spacer and the corresponding target sequence comprise one, two, three, four, five, six, seven, B1195.70198WO00 13849370.1 (April 11, 2025)96 / 895 eight, nine, or ten amino acid differences). In certain embodiments, the spacer is 100% complementary to its corresponding target sequence in ATP1A3. 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).

[0216] The ability of a pegRNA, ngRNA, or dsgRNA to direct sequence-specific binding of a prime editor (or a sgRNA to direct base editor binding) to a target sequence may also be assessed by any suitable assay. For example, a prime editor and pegRNA may be provided to a host cell having the corresponding target mutation (e.g., ATP1A3 D801N c.2401A, ATP1A3 E815K c.2443AATP1A3G947R c.2839C, or ATP1A3 L839P c.2516C mutation., or a portion thereof), such as by transfection with vectors encoding the prime editor and pegRNA or by transfection of a ribonucleoprotein (RNP) complex, followed by an assessment of preferential cleavage, nicking, or editing within the target sequence. Similarly, cleavage or editing of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, prime editor, and pegRNA to be tested and a control pegRNA different from the test pegRNA, and comparing binding or rate of cleavage or editing at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will be apparent to those skilled in the art.

[0217] In some embodiments, a pegRNA, ngRNA, or dsgRNA (or sgRNA) is about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 75, about 100, or more nucleotides in length. In some embodiments, a gRNA is about 50-150, about 60-140, about 70-130, about 80-120, or about 90-110 nucleotides in length. In some embodiments, the spacer sequence of a pegRNA, ngRNA, or dsgRNA (or sgRNA) 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, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides in length.

[0218] In some embodiments, a pegRNA, ngRNA, or dsgRNA (or sgRNA) comprises an optional linker sequence. For example, the gRNAs provided herein may comprise an optional linker sequence between the spacer and the backbone scaffold sequences. In certain embodiments, the optional linker sequence is at least 3 nucleotides, at least 4 nucleotides, at B1195.70198WO00 13849370.1 (April 11, 2025)97 / 895 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, or at least 50 nucleotides in length. Methods of Prime Editing and Base Editing ATP1A3

[0219] Some aspects of the present disclosure provide methods of prime editing a ATP1A3 gene. In one aspect, the present disclosure provides methods of prime editing a ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 gene with a prime editor and any of the pegRNAs disclosed herein. In some embodiments, the methods further comprise providing any of the ngRNAs disclosed herein. In some embodiments, the methods further comprise providing any of the dsgRNAs disclosed herein.

[0220] Any of the prime editors disclosed herein, or any prime editor known in the art, can be used in the methods of the present disclosure. In some embodiments, the prime editor comprises a nucleic acid-programmable DNA-binding protein (napDNAbp) and a polymerase. In some embodiments, the prime editor comprises a napDNAbp (e.g., a Cas9 protein, such as SpCas9, or a variant thereof, such as nCas9 or dCas9) and a polymerase (e.g., a reverse transcriptase, such as an MMLV reverse transcriptase, a Tf1 reverse transcriptase, or a variant thereof). In certain embodiments, the prime editor comprises a PE6 Cas9 variant and / or a PE6 reverse transcriptase. In some embodiments, the prime editor comprises PEmax architecture as provided herein. In other embodiments, the prime editor comprises SpCas9(VRQR)-PEmax architecture as provided herein.

[0221] In some embodiments, the method further comprises providing an inhibitor of the cellular mismatch repair (MMR) pathway. In some embodiments, the inhibitor of the MMR pathway comprises a dominant negative variant of MLH1 (MLH1dn). In certain embodiments, the MLH1dn comprises a truncated version of the wild type MLH1 protein (e.g., with the C-terminal amino acids 754-756 truncated as described herein).

[0222] In some embodiments, the step of contacting corrects a ATP1A3 D801N c.2401A, ATP1A3 E815K c.2443A, ATP1A3G947R c.2839C, or ATP1A3 L839P c.2516C mutation in the ATP1A3 protein. In some embodiments, the step of contacting installs an edit that removes the mutation, e.g., ATP1A3 D801N c.2401A>G, ATP1A3 E815K c.2443A>G edit, a ATP1A3 G947R c.2839C>G edit, a ATP1A3 L839P c.2516C>T edit from the ATP1A3 B1195.70198WO00 13849370.1 (April 11, 2025)98 / 895 gene. In some embodiments, the step of contacting results in the correction of the ATP1A3 gene to a wild-type sequence. In some embodiments, the step of contacting results in at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, or at least 50% correction of the ATP1A3 gene to a wild-type sequence within a cell. In some embodiments, the step of contacting results in an increase in ATP1A3 Na+ / K+-ATPase activity to at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of wild-type levels within a cell.

[0223] In some embodiments, the step of contacting results in the installation of one or more silent edits into the ATP1A3 gene. In certain embodiments, the one or more silent edits comprise one or more PAM-disrupting edits. In certain embodiments, the one or more silent edits comprise one or more MMR pathway-inhibiting edits.

[0224] In some embodiments, the contacting step comprises delivering one or more polynucleotides encoding the pegRNA and the prime editor (and optionally a ngRNA and / or dsgRNA) to the nucleic acid sequence encoding the ATP1A3 gene. In some embodiments, the contacting step is performed in a cell. In some embodiments, the cell is an immortalized cell line. In some embodiments, the cell line is an immortalized human cell line. In some embodiments, the methods are performed in a cell line capable of achieving stable growth and / or efficient transfection, e.g., transfection with a trans-gene bearing plasmid. Such cell lines may be referred to as “workhorse cell lines.” In some embodiments, a workhorse cell line exhibits plasmid transfection efficiency of at least 70%, 80%, 85%, 90%, 95%, or 99% when tested with Calcium phosphate transfection method as described in Kingston et al. , Curr. Protoc. Mol. Biol. Chapter 9: Unit 9.1 (2003), the contents of which are hereby incorporated by reference in its entirety. In certain embodiments, the workhorse cell line comprises HEK293 cells. In some embodiments, the workhorse cell line comprises HEK293T cells (e.g., for human targets). In certain embodiments, the workhorse cell lines comprises N2A cells (e.g., for murine targets).

[0225] In some embodiments, the contacting step is performed in vitro. In some embodiments, the contacting step is performed in vivo. In certain embodiments, the contacting step is performed in a subject. A subject may have been diagnosed with a disease, or be at risk for having a disease. In some embodiments, the method is a method for treating a disease in a subject. In some embodiments, the disease is Alternating Hemiplegia of Childhood (AHC). B1195.70198WO00 13849370.1 (April 11, 2025)99 / 895

[0226] Some aspects of the present disclosure provide methods of base editing a ATP1A3 gene. In one aspect, the present disclosure provides methods of base editing a ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 gene with a base editor and any of the sgRNAs disclosed herein.

[0227] Some aspects of the present disclosure provide methods of base editing a ATP1A3 gene. In one aspect, the present disclosure provides methods of base editing a ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 gene with a base editor and any of the pegRNAs disclosed herein.

[0228] In some embodiments, method comprises a method of prime editing an ATP1A3 D801N c.2401A mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 D801N c.2401A mutation with a prime editor and a prime editing guide RNA (pegRNA). In some embodiments, the pegRNA comprises a nucleotide sequence of SEQ ID NOs 355-450. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450.

[0229] In some embodiments, the method of prime editing an ATP1A3 D801N c.2401A mutation in an ATP1A3 gene described herein further comprises providing a ngRNA. In some embodiments, the ngRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 929-939 or 1050-1065. In some embodiments, the ngRNA comprises a spacer sequence comprising a nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs: 929-939 or 1050-1065. In some embodiments, the ngRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211-1227. In some embodiments, the ngRNA comprises a sequence comprising a nucleic acid sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211-1227.

[0230] In some embodiments, the method of prime editing an ATP1A3 D801N c.2401A mutation in an ATP1A3 gene described herein further comprises providing a dsgRNA. In some embodiments, the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at B1195.70198WO00 13849370.1 (April 11, 2025)100 / 895 least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1229-1252 or 1277-1316. In some embodiments, the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 1229-1252 or 1277-1316. In some embodiments, the dsgRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406. In some embodiments, the dsgRNA comprises a sequence comprising a nucleic acid sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406.

[0231] In some embodiments, in any one of the methods of prime editing an ATP1A3 D801N c.2401A mutation in an ATP1A3 gene described herein, the prime editor is selected from the group consisting of: PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

[0232] In some embodiments, method comprises a method of prime editing an ATP1A3 L839P c.2516C mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 L839P c.2516C mutation with a prime editor and a prime editing guide RNA (pegRNA). In some embodiments, the pegRNA comprises a nucleotide sequence of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the pegRNA comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577.

[0233] In some embodiments, the method of prime editing an ATP1A3 L839P c.2516C mutation in an ATP1A3 gene described herein further comprises providing a ngRNA. In some embodiments, the ngRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 929-939 or 1050-1065. In some embodiments, the ngRNA comprises a spacer sequence comprising a nucleic acid sequence of any one of the nucleic acid sequences of B1195.70198WO00 13849370.1 (April 11, 2025)101 / 895 SEQ ID NOs: 929-939 or 1050-1065. In some embodiments, the ngRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211-1227. In some embodiments, the ngRNA comprises a sequence comprising a nucleic acid sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211-1227.

[0234] In some embodiments, the method of prime editing an ATP1A3 L839P c.2516C mutation in an ATP1A3 gene described herein further comprises providing a dsgRNA. In some embodiments, the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1229-1252 or 1277-1316. In some embodiments, the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 1229-1252 or 1277-1316. In some embodiments, the dsgRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406. In some embodiments, the dsgRNA comprises a sequence comprising a nucleic acid sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406.

[0235] In some embodiments, in any one of the methods of prime editing an ATP1A3 L839P c.2516C mutation in an ATP1A3 gene described herein, the prime editor is selected from the group consisting of: PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

[0236] In some embodiments, method comprises a method of prime editing an ATP1A3 G947R c.2839C mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 G947R c.2839C mutation with a prime editor and a prime editing guide RNA (pegRNA). In some embodiments, the pegRNA comprises a nucleotide sequence of SEQ ID NOs 639-732, 1578 or a nucleic acid sequence having at least 80%, at B1195.70198WO00 13849370.1 (April 11, 2025)102 / 895 least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleotide sequences of SEQ ID NOs 639-732, 1578.

[0237] In some embodiments, the method of prime editing an ATP1A3 G947R c.2839C mutation in an ATP1A3 gene described herein further comprises providing a ngRNA. In some embodiments, the ngRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 929-939 or 1050-1065. In some embodiments, the ngRNA comprises a spacer sequence comprising a nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs: 929-939 or 1050-1065. In some embodiments, the ngRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211-1227. In some embodiments, the ngRNA comprises a sequence comprising a nucleic acid sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211-1227.

[0238] In some embodiments, the method of prime editing an ATP1A3 G947R c.2839C mutation in an ATP1A3 gene described herein further comprises providing a dsgRNA. In some embodiments, the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1229-1252 or 1277-1316. In some embodiments, the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 1229-1252 or 1277-1316. In some embodiments, the dsgRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406. In some embodiments, the dsgRNA comprises a sequence comprising a nucleic acid sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406.

[0239] In some embodiments, in any one of the methods of prime editing an ATP1A3 G947R c.2839C mutation in an ATP1A3 gene described herein, the prime editor is selected from the group consisting of: PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: B1195.70198WO00 13849370.1 (April 11, 2025)103 / 895 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

[0240] In some embodiments, method comprises a method of prime editing an ATP1A3 E815K c.2443A mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 E815K c.2443A mutation with a prime editor and a prime editing guide RNA (pegRNA). In some embodiments, the pegRNA comprises a nucleotide sequence of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576.

[0241] In some embodiments, the method of prime editing an ATP1A3 E815K c.2443A mutation in an ATP1A3 gene described herein further comprises providing a ngRNA. In some embodiments, the ngRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 929-939 or 1050-1065. In some embodiments, the ngRNA comprises a spacer sequence comprising a nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs: 929-939 or 1050-1065. In some embodiments, the ngRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211-1227. In some embodiments, the ngRNA comprises a sequence comprising a nucleic acid sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211-1227.

[0242] In some embodiments, the method of prime editing an ATP1A3 E815K c.2443A mutation in an ATP1A3 gene described herein further comprises providing a dsgRNA. In some embodiments, the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1229-1252 or 1277-1316. In some embodiments, the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 1229-1252 or 1277-1316. In some embodiments, the dsgRNA B1195.70198WO00 13849370.1 (April 11, 2025)104 / 895 comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406. In some embodiments, the dsgRNA comprises a sequence comprising a nucleic acid sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406.

[0243] In some embodiments, in any one of the methods of prime editing an ATP1A3 E815K c.2443A mutation in an ATP1A3 gene described herein, the prime editor is selected from the group consisting of: PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

[0244] Any of the base editors disclosed herein, or any base editor known in the art, can be used in the methods of the present disclosure. In some embodiments, the base editor comprises a nucleic acid-programmable DNA-binding protein (napDNAbp) and a deaminase. In some embodiments, the base editor comprises a napDNAbp (e.g., a Cas9 protein, such as SpCas9, or a variant thereof, such as nCas9 or dCas9) and a deaminase (e.g., an evolved deaminase such as TadA-8e ). In certain embodiments, the base editor comprises a ABE8e base editor and / or variant thereof. In some embodiments, the base editor comprises TadAmax architecture as provided herein. In other embodiments, the base editor comprises SpCas9(D10A)-TadAmax architecture as provided herein.

[0245] In some embodiments, the step of contacting corrects a ATP1A3 D801N c.2401A, ATP1A3 E815K c.2443A, ATP1A3 G947R c.2839C, or ATP1A3 L839P c.2516C mutation in the ATP1A3 protein. In some embodiments, the step of contacting installs an edit that removes the mutation, e.g., ATP1A3 D801N c.2401A>G, ATP1A3 E815K c.2443A>G edit, a ATP1A3 G947R c.2839C>G edit, a ATP1A3 L839P c.2516C>T edit from the ATP1A3 gene. In some embodiments, the step of contacting results in the correction of the ATP1A3 gene to a wild-type sequence. In some embodiments, the step of contacting results in at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, or at least 50% correction of the ATP1A3 gene to a wild-type sequence within a cell. In some embodiments, the step of contacting results in an increase in ATP1A3 Na+ / K+-ATPase activity to at least 20%, at least B1195.70198WO00 13849370.1 (April 11, 2025)105 / 895 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of wild-type levels within a cell.

[0246] In some embodiments, the contacting step comprises delivering one or more polynucleotides encoding the sgRNA and the base editor to the nucleic acid sequence encoding the ATP1A3 gene. In some embodiments, the contacting step is performed in a cell. In some embodiments, the cell is an immortalized cell line. In some embodiments, the cell line is an immortalized human cell line. In some embodiments, the methods are performed in a cell line capable of achieving stable growth and / or efficient transfection, e.g., transfection with a trans-gene bearing plasmid. Such cell lines may be referred to as “workhorse cell lines.” In some embodiments, a workhorse cell line exhibits plasmid transfection efficiency of at least 70%, 80%, 85%, 90%, 95%, or 99% when tested with Calcium phosphate transfection method as described in Kingston et al., Curr. Protoc. Mol. Biol. Chapter 9: Unit 9.1 (2003), the contents of which are hereby incorporated by reference in its entirety. In certain embodiments, the workhorse cell line comprises HEK293 cells. In some embodiments, the workhorse cell line comprises HEK293T cells (e.g., for human targets). In certain embodiments, the workhorse cell lines comprises N2A cells (e.g., for murine targets).

[0247] In some embodiments, the contacting step is performed in vitro. In some embodiments, the contacting step is performed in vivo. In certain embodiments, the contacting step is performed in a subject. A subject may have been diagnosed with a disease, or be at risk for having a disease. In some embodiments, the method is a method for treating a disease in a subject. In some embodiments, the disease is Alternating Hemiplegia of Childhood (AHC).

[0248] In some aspects, the present disclosure contemplates use of any of the ngRNAs, dsgRNAs, prime editors, sgRNAs, base editors, compositions, polynucleotides, vectors, pharmaceutical compositions, and / or cells disclosed herein in the manufacture of a medicament for the treatment of a disease or disorder (e.g., AHC). In some aspects, any of the pegRNAs, ngRNAs, dsgRNAs, prime editors, base editors, sgRNAs compositions (e.g., PE systems and / or BE systems), polynucleotides, vectors, pharmaceutical compositions, and / or cells disclosed herein are for use in medicine. In some embodiments, the present disclosure provides for veterinary uses (e.g., in non-human animals) of any of the pegRNAs, ngRNAs, dsgRNAs, prime editors, sgRNAs, base editors, compositions, polynucleotides, vectors, pharmaceutical compositions, cells, and / or methods provided herein. B1195.70198WO00 13849370.1 (April 11, 2025)106 / 895 Pharmaceutical compositions

[0249] Other aspects of the present disclosure relate to pharmaceutical compositions comprising any of the pegRNAs, ngRNAs, dsgRNAs, prime editors, compositions, systems, polynucleotides, vectors, and / or cells described herein. 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).

[0250] 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 corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, 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, B1195.70198WO00 13849370.1 (April 11, 2025)107 / 895 release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, and antioxidants can also be present in the formulation. Terms such as “excipient,” “carrier,” “pharmaceutically acceptable carrier,” “pharmaceutically acceptable excipient,” or the like are used interchangeably herein.

[0251] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject for gene editing (e.g., prime editing).

[0252] 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.

[0253] 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.

[0254] In some embodiments, the pharmaceutical composition comprises any one of the pegRNAs described herein and pharmaceutically acceptable excipient. In some embodiments, the pegRNAs target a D801N (c.2401A) mutation, a E815K (c.2443A) mutation, a L839P (c.2516C) mutation, or a G947R (c.2839C) mutation in ATP1A3.

[0255] In some embodiments, the pharmaceutical composition comprises any one of the pegRNAs that target a D801N (c.2401A) mutation in ATP1A3 and pharmaceutically acceptable excipient. In some embodiments, the pegRNAs comprise a nucleotide sequence of B1195.70198WO00 13849370.1 (April 11, 2025)108 / 895 SEQ ID NOs 355-450 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 80% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 90% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 95% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 99.5% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450. In some embodiments, the pegRNAs comprise the nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs 355-450.

[0256] In some embodiments, the pharmaceutical composition comprises any one of the pegRNAs that target a E815K (c.2443A) mutation in ATP1A3 and pharmaceutically acceptable excipient. In some embodiments, the pegRNAs comprise a nucleotide sequence of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 80% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 90% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNAs B1195.70198WO00 13849370.1 (April 11, 2025)109 / 895 comprise a nucleic acid sequence having at least 95% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 99.5% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576. In some embodiments, the pegRNAs comprise the nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576.

[0257] In some embodiments, the pharmaceutical composition comprises any one of the pegRNAs that target a L839P (c.2516C) mutation in ATP1A3 and pharmaceutically acceptable excipient. In some embodiments, the pegRNAs comprise a nucleotide sequence of SEQ ID NOs 733-827, 1574, 1577 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 80% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733- 827, 1574, 1577. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 90% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 95% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 99.5% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577. In some embodiments, the B1195.70198WO00 13849370.1 (April 11, 2025)110 / 895 pegRNAs comprise the nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577.

[0258] In some embodiments, the pharmaceutical composition comprises any one of the pegRNAs that target a G947R (c.2839C) mutation in ATP1A3 and pharmaceutically acceptable excipient. In some embodiments, the pegRNAs comprise a nucleotide sequence of SEQ ID NOs 639-732, 1578 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleotide sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 80% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 90% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 95% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNAs comprise a nucleic acid sequence having at least 99.5% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 639-732, 1578. In some embodiments, the pegRNAs comprise the nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs 639-732, 1578.

[0259] In some embodiments, the pharmaceutical composition comprises any one of the ngRNAs described herein and pharmaceutically acceptable excipient. In some embodiments, the ngRNAs comprise a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 80, 85- 86, 90, or 919-1066. In some embodiments, the ngRNAs comprise a spacer sequence comprising a nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs: 80, 85-86, 90, or 919-1066. In some embodiments, the ngRNAs further comprise a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least B1195.70198WO00 13849370.1 (April 11, 2025)111 / 895 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1068- 1228. In some embodiments, the ngRNAs further comprise a sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 1068-1228.

[0260] In some embodiments, the pharmaceutical composition comprises any one of the dsgRNAs described herein and pharmaceutically acceptable excipient. In some embodiments, the dsgRNAs comprise a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1229- 1317. In some embodiments, the dsgRNAs comprise a spacer sequence comprising a nucleic acid sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 1229-1317. In some embodiments, the dsgRNAs further comprise a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1318-1407. In some embodiments, the dsgRNAs further comprise a sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 1318-1407.

[0261] In some embodiments, the pharmaceutical composition comprises any one of the sgRNAs described herein and pharmaceutically acceptable excipient. In some embodiments, the sgRNAs comprise a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to the nucleic acid sequences of SEQ ID NOs: 1408 or 1409. In some embodiments, the sgRNAs comprise a spacer sequence comprising a nucleic acid sequence comprising the nucleic acid sequences of SEQ ID NOs: 1408 or 1409. In some embodiments, the sgRNAs further comprise a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to the nucleic acid sequences of SEQ ID NOs: 1410 or 1411. In some embodiments, the sgRNAs further comprise a sequence comprising the nucleic acid sequences of SEQ ID NOs: 1410 or 1411.

[0262] In some embodiments, the pharmaceutical composition comprises any one of the complexes described herein and pharmaceutically acceptable excipient. In some embodiments, the complexes comprise a prime editor and any one of the pegRNAs described herein. In some embodiments, the pegRNAs target a D801N (c.2401A) mutation, a E815K (c.2443A) mutation, a L839P (c.2516C) mutation, or a G947R (c.2839C) mutation in ATP1A3. In some embodiments, the prime editor is a fusion protein comprising a napDNAbp B1195.70198WO00 13849370.1 (April 11, 2025)112 / 895 domain and a reverse transcriptase (RT) domain. In some embodiments, the prime editor is selected from the group consisting of PE2, PE3, PE3b, PE4, PE5, PE5b, PEmax, SpCas9(VRQR)-PEmax, PE3max, PE3bmax, PE4max, PE4max, PE5bmax, PE6, PE6b, PE6c, PE6d, SpCas9(VRQR)-PE6b, SpCas9(VRQR)-PE6c, and SpCas9(VRQR)-PE6d. In some embodiments, the prime editor is selected from the group consisting of: PEmax, PE6b, PE6c, PE6d, SpCas9(VRQR)-PEmax, SpCas9(VRQR)-PE6b, SpCas9(VRQR)-PE6c, and SpCas9(VRQR)-PE6d. In some embodiments, the prime editor is selected from the group consisting of: PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

[0263] In some embodiments, the pharmaceutical composition comprises any one of the compositions described herein and pharmaceutically acceptable excipient. In some embodiments, the compositions comprise any one of the pegRNAs described herein, any one of the ngRNAs described herein, and / or any one of the dsgRNAs described herein. In some embodiments, the complexes comprise a prime editor and any one of the pegRNAs described herein. In some embodiments, the compositions further comprise a prime editor. In some embodiments, the prime editor selected from the group consisting of: PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

[0264] In some embodiments, the pharmaceutical composition comprises any one of the systems described herein and pharmaceutically acceptable excipient. In some embodiments, the systems comprise any one of the pegRNA described herein and a prime edi...

Claims

861 / 895 CLAIMS What is claimed is:

1. A prime editing guide RNA (pegRNA) comprising a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 77-96, 919-1066, 1229-1317, 1408-1409, 1523-1525, or a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to: a) any one of the following nucleic acid sequences that target a D801N (c.2401A) mutation: gCACCATGTCAGTGCCCAGAT (SEQ ID NO: 78) (“NGA120nt”) or GGCTCACCATGTCAGTGCCC (SEQ ID NO: 79) (“NGA220nt”); b) any one of the following nucleic acid sequences that target a E815K (c.2443A) mutation: gTGCCATCTCACTGGCGTACA (SEQ ID NO: 83) (“NGG120nt”); GCCATCTCACTGGCGTACA (SEQ ID NO: 84) (“NGG119nt”); gCTAGGTCCCTGCCATCTCAC (SEQ ID NO: 85) (“NGG220nt”); or gTCTCTTCATGATGTCGCTTT (SEQ ID NO: 86) (“NGG320nt”); c) any one of the following nucleic acid sequences that target a L839P (c.2516T) mutation: gTGCCCGTAGGCCATGCTGAT (SEQ ID NO: 88) (“NGG120nt”); GCCCGTAGGCCATGCTGAT (SEQ ID NO: 89) (“NGG119nt”); or gCTGCCCGTAGGCCATGCTGA (SEQ ID NO: 90) (“NGG220nt”); d) any one of the following nucleic acid sequences that target a G947R (c.2839C) mutation: GGAACAAGATCCTGATCTTC (SEQ ID NO: 81) (“NGG120nt”) or GGGCCGTCTCCTCAAACAGC (SEQ ID NO: 87) (“NGG220nt”).

2. The pegRNA of claim 1, further comprising a reverse transcriptase template (RTT) sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 103- 223 and 1526-1528, or a reverse transcriptase template (RTT) comprising a nucleic acid B1195.70198WO00 13849370.1 (April 11, 2025)862 / 895 sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to: a) any one of the following RTT sequences that edits a D801N (c.2401A) mutation: TCCTCTGCATTGACT (SEQ ID NO: 140) ACCATCCTCTGCATTGACT (SEQ ID NO: 141) TCACCATCCTCTGCATTGACT (SEQ ID NO: 142) ACCATCACCATCCTCTGCATTGACT (SEQ ID NO: 143) GCACCATCACCATCCTCTGCATTGACT (SEQ ID NO: 144) TGGGCACCATCACCATCCTCTGCATTGACT (SEQ ID NO: 145) TGCATTGACTTAGG (SEQ ID NO: 122) TCTGCATTGACTTAGG (SEQ ID NO: 123) TCCTCTGCATTGACTTAGG (SEQ ID NO: 124) ACCATCCTCTGCATTGACTTAGG (SEQ ID NO: 125) ATCACCATCCTCTGCATTGACTTAGG (SEQ ID NO: 126) ACCATCACCATCCTCTGCATTGACTTAGG (SEQ ID NO: 127) TGCATCGACTTGGG (SEQ ID NO: 128) TCTGCATCGACTTGGG (SEQ ID NO: 129) TCCTCTGCATCGACTTGGG (SEQ ID NO: 130) ACCATCCTCTGCATCGACTTGGG (SEQ ID NO: 131) ATCACCATCCTCTGCATCGACTTGGG (SEQ ID NO: 132) ACCATCACCATCCTCTGCATCGACTTGGG (SEQ ID NO: 133) TGCATTGACTTGGG (SEQ ID NO: 134) TCTGCATTGACTTGGG (SEQ ID NO: 135) TCCTCTGCATTGACTTGGG (SEQ ID NO: 136) ACCATCCTCTGCATTGACTTGGG (SEQ ID NO: 137) ATCACCATCCTCTGCATTGACTTGGG (SEQ ID NO: 138) ACCATCACCATCCTCTGCATTGACTTGGG (SEQ ID NO: 139) b) any one of the following RTT sequences that edits a E815K (c.2443A) mutation: TCGGCAGCTTCAT (SEQ ID NO: 146) TTTCGGCAGCTTCAT (SEQ ID NO: 147) GCTTTCGGCAGCTTCAT (SEQ ID NO: 148) TCGCTTTCGGCAGCTTCAT (SEQ ID NO: 149) TGTCGCTTTCGGCAGCTTCAT (SEQ ID NO: 150) GATGTCGCTTTCGGCAGCTTCAT (SEQ ID NO: 151) ATGATGTCGCTTTCGGCAGCTTCAT (SEQ ID NO: 152) TCATGATGTCGCTTTCGGCAGCTTCAT (SEQ ID NO: 153) TCGGCAGCTTCAT (SEQ ID NO: 146) TTTCGGCAGCTTCAT (SEQ ID NO: 147) GCTTTCGGCAGCTTCAT (SEQ ID NO: 148) TCGCTTTCGGCAGCTTCAT (SEQ ID NO: 149) TGTCGCTTTCGGCAGCTTCAT (SEQ ID NO: 150) GATGTCGCTTTCGGCAGCTTCAT (SEQ ID NO: 151) ATGATGTCGCTTTCGGCAGCTTCAT (SEQ ID NO: 152) B1195.70198WO00 13849370.1 (April 11, 2025)863 / 895 TCATGATGTCGCTTTCGGCAGCTTCAT (SEQ ID NO: 153) TCGGCAGCTTCATACGCCAGTG (SEQ ID NO: 154) TTTCGGCAGCTTCATACGCCAGTG (SEQ ID NO: 155) GCTTTCGGCAGCTTCATACGCCAGTG (SEQ ID NO: 156) TCGCTTTCGGCAGCTTCATACGCCAGTG (SEQ ID NO: 157) TGTCGCTTTCGGCAGCTTCATACGCCAGTG (SEQ ID NO: 161) GATGTCGCTTTCGGCAGCTTCATACGCCAGTG (SEQ ID NO: 158) ATGATGTCGCTTTCGGCAGCTTCATACGCCAGTG (SEQ ID NO: 159) TCATGATGTCGCTTTCGGCAGCTTCATACGCCAGTG (SEQ ID NO: 160) ACTGGCGTATGAAGCTGCCGAAA (SEQ ID NO: 162) TCACTGGCGTATGAAGCTGCCGAAA (SEQ ID NO: 163) TCTCACTGGCGTATGAAGCTGCCGAAA (SEQ ID NO: 164) ATCTCACTGGCGTATGAAGCTGCCGAAA (SEQ ID NO: 165) GCCATCTCACTGGCGTATGAAGCTGCCGAAA (SEQ ID NO: 166) TGCCATCTCACTGGCGTATGAAGCTGCCGAAA (SEQ ID NO: 167) TCCCTGCCATCTCACTGGCGTATGAAGCTGCCGAAA (SEQ ID NO: 168) c) GTCCCTGCCATCTCACTGGCGTATGAAGCTGCCGAAA (SEQ ID NO: 169)any one of the following RTT sequences that edits a L839P (c.2516T) mutation: AATGAGAGGTTGATC (SEQ ID NO: 186) TCAATGAGAGGTTGATC (SEQ ID NO: 187) GGTCAATGAGAGGTTGATC (SEQ ID NO: 188) TTGGTCAATGAGAGGTTGATC (SEQ ID NO: 189) AATTGGTCAATGAGAGGTTGATC (SEQ ID NO: 190) ACAAATTGGTCAATGAGAGGTTGATC (SEQ ID NO: 191) GGACAAATTGGTCAATGAGAGGTTGATC (SEQ ID NO: 192) ACGGACAAATTGGTCAATGAGAGGTTGATC (SEQ ID NO: 193) AATGAGAGGTTGATC (SEQ ID NO: 186) TCAATGAGAGGTTGATC (SEQ ID NO: 187) GGTCAATGAGAGGTTGATC (SEQ ID NO: 188) TTGGTCAATGAGAGGTTGATC (SEQ ID NO: 189) AATTGGTCAATGAGAGGTTGATC (SEQ ID NO: 190) ACAAATTGGTCAATGAGAGGTTGATC (SEQ ID NO: 191) GGACAAATTGGTCAATGAGAGGTTGATC (SEQ ID NO: 192) ACGGACAAATTGGTCAATGAGAGGTTGATC (SEQ ID NO: 193) AATGAGAGGTTGATCA (SEQ ID NO: 194) TCAATGAGAGGTTGATCA (SEQ ID NO: 195) GGTCAATGAGAGGTTGATCA (SEQ ID NO: 196) TTGGTCAATGAGAGGTTGATCA (SEQ ID NO: 197) AATTGGTCAATGAGAGGTTGATCA (SEQ ID NO: 198) ACAAATTGGTCAATGAGAGGTTGATCA (SEQ ID NO: 199) GGACAAATTGGTCAATGAGAGGTTGATCA (SEQ ID NO: 200) B1195.70198WO00 13849370.1 (April 11, 2025)864 / 895 d) ACGGACAAATTGGTCAATGAGAGGTTGATCA (SEQ ID NO: 201)any one of the following RTT sequences that edits a G947R (c.2839C) mutation: TCAAACAAGCCAAA (SEQ ID NO: 170) TCCTCAAACAAGCCAAA (SEQ ID NO: 171) TCTCCTCAAACAAGCCAAA (SEQ ID NO: 177) GTCTCCTCAAACAAGCCAAA (SEQ ID NO: 172) GCCGTCTCCTCAAACAAGCCAAA (SEQ ID NO: 173) GGGCCGTCTCCTCAAACAAGCCAAA (SEQ ID NO: 174) GCCAGGGCCGTCTCCTCAAACAAGCCAAA (SEQ ID NO: 175) GCAGCCAGGGCCGTCTCCTCAAACAAGCCAAA (SEQ ID NO: 176) TGATCTTTGGCTT (SEQ ID NO: 178) TCCTGATCTTTGGCTT (SEQ ID NO: 179) GATCCTGATCTTTGGCTT (SEQ ID NO: 180) AAGATCCTGATCTTTGGCTT (SEQ ID NO: 181) ACAAGATCCTGATCTTTGGCTT (SEQ ID NO: 182) GAACAAGATCCTGATCTTTGGCTT (SEQ ID NO: 183) AGGAACAAGATCCTGATCTTTGGCTT (SEQ ID NO: 184) TCCAGGAACAAGATCCTGATCTTTGGCTT (SEQ ID NO: 185).

3. The pegRNA of claim 1 or 2, further comprising a primer binding site (PBS) sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 224-280, 1529-1531.

4. The pegRNA of any one of claims 1-3, wherein the PBS site is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of sequences: ACGCCAGTG, AGATGGCAG, AGCATGGCC ,CACTGACAT, GATCAGGAT, GCATGGCCT, GCGACATCA, GTTTGAGGA, TACCGACAT, TGACCAATT, TGGGCACTG, or UACCGACAU.

5. The pegRNA of any one of claims 1-4, further comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 283-918, 1573-1578.

6. A nicking guide RNA (ngRNA) comprising a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at B1195.70198WO00 13849370.1 (April 11, 2025)865 / 895 least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 80, 85- 86, 90, 919-1066, or 1532-1534.

7. The ngRNA of claim 6, further comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1068-1228 and 1579-1583.

8. A dead single guide RNA (dsgRNA) comprising a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1229- 1317 and 1535-1550.

9. The dsgRNA of claim 8, further comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1318-1407 and 1585-1601.

10. A single guide RNA (sgRNA) comprising a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the nucleic acid sequences of SEQ ID NOs: 1408 or 1409.

11. The sgRNA of claim 10, further comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the nucleic acid sequences of SEQ ID NOs: 1410 or 1411.

12. A complex comprising a prime editor and a pegRNA comprising a spacer sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 77-96, 919-1066, 1229-1317, 1408-1409, and 1523-1525. B1195.70198WO00 13849370.1 (April 11, 2025)866 / 895 13. The complex of claim 12, wherein the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 283-918 and 1573-1578.

14. The complex of claim 12 or 13, wherein the prime editor is a fusion protein comprising a napDNAbp domain and a reverse transcriptase (RT) domain.

15. The complex of any one of claims 12-14, wherein the prime editor is selected from the group consisting of: PE2, PE3, PE3b, PE4, PE5, PE5b, PEmax, SpCas9(VRQR)-PEmax, PE3max, PE3bmax, PE4max, PE4max, PE5bmax, PE6, PE6b, PE6c, and PE6d.

16. The complex of any one of claim 12-15, wherein the prime editor is selected from the group consisting of: SpCas9(VRQR)-PEmax, PEmax, PE2, PE6b, PE6c, and PE6d.

17. The complex of any one of claims 12-16, wherein the prime editor has the following architecture (in the N- to C- direction): [bipartite NLS]- [Cas9:(R221K+N394K+H840A)+(D1135V+G1218R+R1335Q+T1337R)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)]-[bipartite NLS]-[NLS] or [bipartite NLS]- [Cas9(R221K+N394K+H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)]-[bipartite NLS]-[NLS].

18. The complex of any one of claims 12-17, wherein the prime editor is encoded by a nucleic acid comprising a sequence with at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to the nucleic acid sequence of SEQ ID NOs: 5 (Sp Cas9 N-terminal fragment, PEmax architecture), 23 (Sp Cas9 C-terminal fragment, PEmax architecture), 1515 (SpCas9 – VRQR variant – PEmax), 1517 (SpCas9 – VRQR variant – PE6b), 1519 (pCas9 – VRQR variant – PE6c), 1521 (SpCas9 – VRQR variant – PE6d). B1195.70198WO00 13849370.1 (April 11, 2025)867 / 895 19. A complex comprising a base editor and a sgRNA comprising a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to the nucleic acid sequences of SEQ ID NOs: 1408 or 1409.

20. The complex of claim 19, wherein the sgRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to the nucleic acid sequences of SEQ ID NOs: 1410 or 1411.

21. The complex of claim 19 or 20, wherein the base editor comprises a fusion protein comprising a napDNAbp and a deaminase.

22. The complex of claim 21, wherein the deaminase is an adenosine deaminase.

23. The complex of claim 22, wherein the adenosine deaminase comprises an evolved TadA8e deaminase (SEQ ID NO: 48) or a variant thereof.

24. The complex of any one of claims 19-23, wherein the base editor comprises ABE8e.

25. A composition comprising the pegRNA of any one of claims 1-5, the ngRNA of claim 6 or 7, and / or the dsgRNA of claim 8 or 9.

26. The composition of claim 25, further comprising a prime editor.

27. The composition of claim 26, wherein the prime editor comprises a nucleic acid- programmable DNA-binding protein (napDNAbp) and a polymerase.

28. The composition of claim 27, wherein the napDNAbp comprises a Cas9 protein. B1195.70198WO00 13849370.1 (April 11, 2025)868 / 895 29. The composition of claim 27 or 28, wherein the napDNAbp comprises a Cas9 nickase (nCas9).

30. The composition of claim 28, wherein the Cas9 protein comprises a Streptococcus pyogenes Cas9 protein, or a variant thereof.

31. The composition of any one of claims 27-30, wherein the polymerase is a reverse transcriptase.

32. The composition of claim 27-30, wherein the polymerase is an MMLV reverse transcriptase, a Tf1 reverse transcriptase, or a variant thereof.

33. The composition of any one of claims 27-30, wherein the polymerase comprises a reverse transcriptase of a PE6 prime editor.

34. The composition of any one of claims 26-33, wherein the prime editor comprises PEmax or a SpCas9(VRQR)-PEmax architecture.

35. The composition of any one of claims 26-34, wherein the prime editor is encoded by a nucleic acid comprising a sequence with at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to the nucleic acid sequence of SEQ ID NOs: 5 (Sp Cas9 N-terminal fragment, PEmax architecture), 23 (Sp Cas9 C-terminal fragment, PEmax architecture), 1515 (SpCas9 – VRQR variant – PEmax), 1517 (SpCas9 – VRQR variant – PE6b), 1519 (pCas9 – VRQR variant – PE6c), 1521 (SpCas9 – VRQR variant – PE6d).

36. A system comprising the pegRNA of any one of claims 1-5, the ngRNA of claim 6 or 7, and / or the dsgRNA of claim 8 or 9.

37. The system of claim 36, further comprising a prime editor. B1195.70198WO00 13849370.1 (April 11, 2025)869 / 895 38. The system of claim 37, wherein the prime editor comprises a nucleic acid-programmable DNA-binding protein (napDNAbp) and a polymerase.

39. The system of claim 38, wherein the napDNAbp comprises a Cas9 protein.

40. The system of claim 38 or 39, wherein the napDNAbp comprises a Cas9 nickase (nCas9).

41. The system of claim 38 or 39, wherein the Cas9 protein comprises a Streptococcus pyogenes Cas9 protein, or a variant thereof.

42. The system of any one of claims 38-41, wherein the polymerase is a reverse transcriptase.

43. The system of any one of claims 38-41, wherein the polymerase is an MMLV reverse transcriptase, a Tf1 reverse transcriptase, or a variant thereof.

44. The system of any one of claims 38-43, wherein the polymerase comprises a reverse transcriptase of a PE6 prime editor.

45. The system of any one of claims 38-44, wherein the prime editor comprises PEmax or a SpCas9(VRQR)-PEmax architecture.

46. The system of any one of claims 36-45, wherein the prime editor is encoded by a nucleic acid comprising a sequence with at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to the nucleic acid sequence of SEQ ID NOs: 5 (Sp Cas9 N-terminal fragment, PEmax architecture), 23 (Sp Cas9 C-terminal fragment, PEmax architecture), 1515 (SpCas9 – VRQR variant – PEmax), 1517 (SpCas9 – VRQR variant – PE6b), 1519 (pCas9 – VRQR variant – PE6c), 1521 (SpCas9 – VRQR variant – PE6d). B1195.70198WO00 13849370.1 (April 11, 2025)870 / 895 47. A recombinant adeno associated viral (rAAV) vector comprising a first nucleotide sequence encoding a N-terminal portion of the prime editor fused at its C-terminus to an intein- N, wherein the N-terminal portion of the prime editor comprises an amino acid sequence with at least 80% identity to SEQ ID NO:

15.

48. The rAAV vector of claim 47, further comprising a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the prime editor, wherein the C-terminal portion of the prime editor comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 33, 1432.

49. A recombinant adeno associated viral (rAAV) vector comprising a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the prime editor, wherein the C-terminal portion of the prime editor comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 33 or 1432.

50. The rAAV vector of claim 49, further comprising a first nucleotide sequence encoding a N-terminal portion of the prime editor fused at its C-terminus to an intein-N, wherein the N- terminal portion of the prime editor comprises an amino acid sequence with at least 80% identity to SEQ ID NO:

15.

51. A recombinant adeno associated viral (rAAV) vector comprising: (i) a first nucleotide sequence encoding a N-terminal portion of the prime editor fused at its C-terminus to an intein-N, wherein the N-terminal portion of the prime editor comprises an amino acid sequence least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to SEQ ID NO: 15, and (ii) a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the prime editor, wherein the C-terminal portion of the prime editor comprises an amino acid sequence least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to SEQ ID NO:

33. B1195.70198WO00 13849370.1 (April 11, 2025)871 / 895 52. A recombinant adeno associated viral (rAAV) vector comprising: (i) a first nucleotide sequence encoding a N-terminal portion of the prime editor fused at its C- terminus to an intein-N, wherein the N-terminal portion of the prime editor comprises an amino acid sequence least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to SEQ ID NO: 47, and (ii)a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the prime editor, wherein the C-terminal portion of the prime editor comprises an amino acid sequence least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to SEQ ID NO:

56.

53. A recombinant adeno associated viral (rAAV) vector comprising: (i)a first nucleotide sequence encoding a N-terminal portion of the prime editor fused at its C-terminus to an intein-N, wherein the N-terminal portion of the prime editor comprises an amino acid sequence least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to SEQ ID NO: 15, and (ii)a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C- terminal portion of the prime editor, wherein the C-terminal portion of the prime editor comprises an amino acid sequence least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to SEQ ID NO:

33.

54. A recombinant adeno associated viral (rAAV) vector comprising: (i) a first nucleotide sequence encoding a N-terminal portion of the prime editor fused at its C- terminus to an intein-N, wherein the N-terminal portion of the prime editor comprises an amino acid sequence least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to SEQ ID NO: 15, and (ii)a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the prime editor, wherein the C-terminal portion of the prime editor comprises an amino acid sequence least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to SEQ ID NO: 1432. B1195.70198WO00 13849370.1 (April 11, 2025)872 / 895 55. A recombinant adeno associated viral (rAAV) vector comprising: (i) a first nucleotide sequence encoding a N-terminal portion of the prime editor fused at its C- terminus to an intein-N, wherein the N-terminal portion of the prime editor comprises an amino acid sequence least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to SEQ ID NO: 15, and (ii) a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the prime editor, wherein the C-terminal portion of the prime editor comprises an amino acid sequence least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to SEQ ID NO:

33.

56. A recombinant adeno associated viral (rAAV) vector comprising: (i) a first nucleotide sequence encoding a N-terminal portion of the prime editor fused at its C- terminus to an intein-N, wherein the N-terminal portion of the prime editor comprises an amino acid sequence least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to SEQ ID NO: 15, and (ii)a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the prime editor, wherein the C-terminal portion of the prime editor comprises an amino acid sequence least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to SEQ ID NO:

33.

57. A recombinant adeno associated viral (rAAV) vector comprising: (i) a first nucleotide sequence encoding a N-terminal portion of a base editor fused at its C- terminus to an intein-N, wherein the N-terminal portion of the base editor comprises an amino acid sequence least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to SEQ ID NO: 47, and (ii) a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the base editor, wherein the C-terminal portion of the base editor comprises an amino acid sequence least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to SEQ ID NO:

56. B1195.70198WO00 13849370.1 (April 11, 2025)873 / 895 58. A polynucleotide encoding the pegRNA of any one of claims 1-5, the ngRNA of claim 6 or 7, and / or the dsgRNA of claim 8 or 9.

59. One or more polynucleotides encoding the composition of any one of claims 25-35 or the system of any one of claims 36-46.

60. A vector comprising the polynucleotide of claim 58.

61. One or more vectors comprising one or more polynucleotides of claim 59.

62. A pharmaceutical composition comprising the pegRNA of any one of claims 1-5, the ngRNA of claims 6 or 7, the dsgRNA of claim 8 or 9, the sgRNA of claims 10 or 11, the complex of any one of claims 12-24, the composition of any one of claims 25-35, the system of any one of claims 36-46, the rAAV vector of any one of claims 47-57, the polynucleotide of claim 58, the one or more polynucleotides of claim 59, the vector of claim 60, or the one or more vectors of claim 61.

63. A cell comprising the pegRNA of any one of claims 1-5, the ngRNA of claims 6 or 7, the dsgRNA of claim 8 or 9, the sgRNA of claims 10 or 11, the complex of any one of claims 12-24, the composition of any one of claims 25-35, the system of any one of claims 36-46, the rAAV vector of any one of claims 47-57, the polynucleotide of claim 58, the one or more polynucleotides of claim 59, the vector of claim 60, the one or more vectors of claim 61, or the pharmaceutical composition of claim 62.

64. A kit the pegRNA of any one of claims 1-5, the ngRNA of claims 6 or 7, the dsgRNA of claim 8 or 9, the sgRNA of claims 10 or 11, the complex of any one of claims 12-24, the composition of any one of claims 25-35, the system of any one of claims 36-46, the rAAV vector of any one of claims 47-57, the polynucleotide of claim 58, the one or more polynucleotides of B1195.70198WO00 13849370.1 (April 11, 2025)874 / 895 claim 59, the vector of claim 60, the one or more vectors of claim 61, the pharmaceutical composition of claim 62, or cells of claim 63.

65. A method of prime editing an ATP1A3 D801N c.2401A mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 gene comprising the mutation with a prime editor and a prime editing guide RNA (pegRNA), wherein the pegRNA comprises a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 78-79 or 94-95.

66. The method of claim 65, wherein the pegRNA further comprises a RTT comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 97-99 or 101-102 or 122-144.

67. The method of claim 65 or 66, wherein the pegRNA further comprises a PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 228-229 or 231-232 or 243-244 or 274-276.

68. The method of any one of claims 65-67, wherein the PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to CACTGACAT, TACCGACAT, TGGGCACTG, UACCGACAU.

69. The method of any one of claims 65-68, wherein the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 355- 450 or 868-917. B1195.70198WO00 13849370.1 (April 11, 2025)875 / 895 70. The method of any one of claims 65-69, further comprising providing a ngRNA.

71. The method of claim 70, wherein the ngRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 929-939 or 1050-1065.

72. The method of claim 70 or 71, wherein the ngRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211-1227.

73. The method of any one of claims 65-72, further comprising providing a dsgRNA.

74. The method of claim 73, wherein the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1229-1252 or 1277-1316 75. The method of claim 72 or 73, wherein the dsgRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406.

76. The method of claim 65-75, wherein the prime editor comprises a nucleic acid- programmable DNA-binding protein (napDNAbp) and a polymerase.

77. The method of claim 76, wherein the napDNAbp comprises a Cas9 protein. B1195.70198WO00 13849370.1 (April 11, 2025)876 / 895 78. The method of claim 76 or 77, wherein the napDNAbp comprises a Cas9 nickase (nCas9).

79. The method of claim 76 or 77, wherein the Cas9 protein comprises a Streptococcus pyogenes Cas9 protein, or a variant thereof.

80. The method of any one of claims 76-79, wherein the polymerase is a reverse transcriptase.

81. The method of claim 76-80, wherein the polymerase is an MMLV reverse transcriptase, a Tf1 reverse transcriptase, or a variant thereof.

82. The method of any one of claims 76-81, wherein the polymerase comprises a reverse transcriptase of a PE6 prime editor.

83. The method of any one of claims 65-82, wherein the prime editor comprises PEmax (SEQ ID NO: 1421) or a SpCas9(VRQR)-PEmax architecture (SEQ ID NO: 67).

84. The method of any one of claims 65-83, wherein the prime editor is encoded by a nucleic acid comprising a sequence with at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to the nucleic acid sequence of SEQ ID NOs: 67 or PEmax (SEQ ID NO: 1421).

85. A method of prime editing an ATP1A3 L839P c.2516C mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 gene comprising the mutation with a prime editor and a prime editing guide RNA (pegRNA), wherein the pegRNA comprises a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 88-90. B1195.70198WO00 13849370.1 (April 11, 2025)877 / 895 86. The method of claim 85, wherein the pegRNA further comprises a RTT comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 186-201.

87. The method of claim 85 or 86, wherein the pegRNA further comprises a PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 266-271.

88. The method of any one of claims 85-87, wherein the PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to AGCATGGCC or GCATGGCCT.

89. The method of any one of claims 85-88, wherein the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 733- 827.

90. The method of any one of claims 85-89, further comprising providing a ngRNA.

91. The method of claim 90, wherein the ngRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 80, 85, 975-986, 1030-1047.

92. The method of claim 90 or 91, wherein the ngRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least B1195.70198WO00 13849370.1 (April 11, 2025)878 / 895 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1130-1134, 1138-1142, 1190-1208.

93. The method of any one of claims 85-92, wherein the prime editor comprises a nucleic acid-programmable DNA-binding protein (napDNAbp) and a polymerase.

94. The method of claim 93, wherein the napDNAbp comprises a Cas9 protein.

95. The method of claim 93 or 94, wherein the napDNAbp comprises a Cas9 nickase (nCas9).

96. The method of claim 94, wherein the Cas9 protein comprises a Streptococcus pyogenes Cas9 protein, or a variant thereof.

97. The method of any one of claims 93-96, wherein the polymerase is a reverse transcriptase.

98. The method of any one of claims 93-97, wherein the polymerase is an MMLV reverse transcriptase, a Tf1 reverse transcriptase, or a variant thereof.

99. The method of any one of claims 93-98, wherein the polymerase comprises a reverse transcriptase of a PE6 prime editor.

100. The method of any one of claims 85-99, wherein the prime editor comprises PEmax or a SpCas9(VRQR)-PEmax architecture.

101. The method of any one of claims 82-100, wherein the prime editor is encoded by a nucleic acid comprising a sequence with at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, B1195.70198WO00 13849370.1 (April 11, 2025)879 / 895 or 100% identity to the nucleic acid sequence of SEQ ID NOs: 67 or PEmax (SEQ ID NO: 1421).

102. A method of prime editing an ATP1A3 G947R c.2839C mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 gene comprising the mutation with a prime editor and a prime editing guide RNA (pegRNA), wherein the pegRNA comprises a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 81 or 87.

103. The method of claim 102, wherein the pegRNA further comprises a RTT comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 170-185.

104. The method of claim 102 or 103, wherein the pegRNA further comprises a PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 237-238 or 258-265.

105. The method of any one of claims 102-104, wherein the PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to GTTTGAGGA or GATCAGGAT.

106. The method of any one of claims 102-105, wherein the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 639- 732.

107. The method of any one of claims 102-106, further comprising providing a ngRNA. B1195.70198WO00 13849370.1 (April 11, 2025)880 / 895 108. The method of claim 107, wherein the ngRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 922-925 or 988-1029.

109. The method of claim 107 or 108, wherein the ngRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1144-1189.

110. The method of any one of claims 102-109, wherein the prime editor comprises a nucleic acid-programmable DNA-binding protein (napDNAbp) and a polymerase.

111. The method of claim 110, wherein the napDNAbp comprises a Cas9 protein.

112. The method of claim 110 or 111, wherein the napDNAbp comprises a Cas9 nickase (nCas9).

113. The method of claim 111, wherein the Cas9 protein comprises a Streptococcus pyogenes Cas9 protein, or a variant thereof.

114. The method of any one of claims 110-113, wherein the polymerase is a reverse transcriptase.

115. The method of claim 110-114, wherein the polymerase is an MMLV reverse transcriptase, a Tf1 reverse transcriptase, or a variant thereof.

116. The method of any one of claims 110-115, wherein the polymerase comprises a reverse transcriptase of a PE6 prime editor. B1195.70198WO00 13849370.1 (April 11, 2025)881 / 895 117. The method of any one of claims 102-116, wherein the prime editor comprises PEmax or a SpCas9(VRQR)-PEmax architecture.

118. The method of any one of claims 101-117, wherein the prime editor is encoded by a nucleic acid comprising a sequence with at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to the nucleic acid sequence of SEQ ID NOs: 5, 23, or 67.

119. A method of prime editing an ATP1A3 E815K c.2443A mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 gene comprising the mutation with a prime editor and a prime editing guide RNA (pegRNA), wherein the pegRNA comprises a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 83-86, 91-92 or 96.

120. The method of claim 119, wherein the pegRNA further comprises a RTT comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 99-100 or 146-169.

121. The method of claim 119 or 120, wherein the pegRNA further comprises a PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 99, 234-257, or 272-273.

122. The method of any one of claims 119-121, wherein the PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to ACGCCAGTG, AGATGGCAG, or GCGACATCA. B1195.70198WO00 13849370.1 (April 11, 2025)882 / 895 123. The method of any one of claims 119-122, wherein the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 451- 638, 828-829, or 918.

124. The method of any one of claims 119-123, further comprising providing a ngRNA.

125. The method of claim 124, wherein the ngRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 85-86, 919-921, 940-987, 1048-1049, or 1066.

126. The method of claim 124 or 125, wherein the ngRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1091-1143, 1209-1210, or 1228.

127. The method of any one of claims 119-126, further comprising providing a dsgRNA.

128. The method of claim 127, wherein the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1254-1276 or 1317.

129. The method of claim 127 or 128, wherein the dsgRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1343-1365 or 1407. B1195.70198WO00 13849370.1 (April 11, 2025)883 / 895 130. The method of claim 119-129, wherein the prime editor comprises a nucleic acid- programmable DNA-binding protein (napDNAbp) and a polymerase.

131. The method of claim 130, wherein the napDNAbp comprises a Cas9 protein.

132. The method of claim 130 or 131, wherein the napDNAbp comprises a Cas9 nickase (nCas9).

133. The method of claim 131, wherein the Cas9 protein comprises a Streptococcus pyogenes Cas9 protein, or a variant thereof.

134. The method of any one of claims 130-134, wherein the polymerase is a reverse transcriptase.

135. The method of any one of claims 130-134, wherein the polymerase is an MMLV reverse transcriptase, a Tf1 reverse transcriptase, or a variant thereof.

136. The method of any one of claims 130-135, wherein the polymerase comprises a reverse transcriptase of a PE6 prime editor.

137. The method of any one of claims 119-136, wherein the prime editor comprises PEmax or a SpCas9(VRQR)-PEmax architecture.

138. The method of any one of claims 119-137, wherein the prime editor is encoded by a nucleic acid comprising a sequence with at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to the nucleic acid sequence of SEQ ID NOs: 5 (Sp Cas9 N-terminal fragment, PEmax architecture), 23 (Sp Cas9 C-terminal fragment, PEmax architecture), 1515 (SpCas9 – VRQR variant – PEmax), 1517 (SpCas9 – VRQR variant – PE6b), 1519 (pCas9 – VRQR variant – PE6c), 1521 (SpCas9 – VRQR variant – PE6d). B1195.70198WO00 13849370.1 (April 11, 2025)884 / 895 139. A method of base editing an ATP1A3 G947R c.2839A mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 gene comprising the mutation with a base editor and a single guide RNA (sgRNA), wherein the sgRNA comprises a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to a nucleic acid sequence of any one of SEQ ID NOs:1408 or 1409.

140. The method of any one of claims 139, wherein the sgRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1410 or 1411.

141. The method of claim 139 or 140, wherein the base editor comprises a fusion protein comprising a napDNAbp and a deaminase.

142. The method of claim 141, wherein the deaminase is an adenosine deaminase.

143. The method of claim 142, wherein the adenosine deaminase comprises an evolved TadA8e deaminase (SEQ ID NO: 48) or a variant thereof.

144. The method of any one of claims 139-143, wherein the base editor comprises ABE8e (SEQ ID NO: 47).

145. Use of the pegRNA of any one of claims 1-5, the ngRNA of claims 6 or 7, the dsgRNA of claim 8 or 9, the sgRNA of claims 10 or 11, the complex of any one of claims 12-24, the composition of any one of claims 25-35, the system of any one of claims 36-46, the rAAV vector of any one of claims 47-57, the polynucleotide of claim 58, the one or more polynucleotides of claim 59, the vector of claim 60, the one or more vectors of claim 61, the pharmaceutical composition of claim 62, or cells of claim 63 in the manufacture of a medicament for the treatment of AHC. B1195.70198WO00 13849370.1 (April 11, 2025)885 / 895 146. The pegRNA of any one of claims 1-5, the ngRNA of claims 6 or 7, the dsgRNA of claim 8 or 9, the sgRNA of claims 10 or 11, the complex of any one of claims 12-24, the composition of any one of claims 25-35, the system of any one of claims 36-46, the rAAV vector of any one of claims 47-57, the polynucleotide of claim 58, the one or more polynucleotides of claim 59, the vector of claim 60, the one or more vectors of claim 61, the pharmaceutical composition of claim 62, or cells of claim 63, for use in medicine.

147. A prime editing guide RNA (pegRNA) for targeting a D801N (c.2401A) mutation in ATP1A3, comprising a nucleotide sequence of SEQ ID NOs 355-450 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 355-450.

148. A prime editing guide RNA (pegRNA) for targeting a E815K (c.2443A) mutation in ATP1A3, comprising a nucleotide sequence of SEQ ID NOs 451-638, 831-834, 843-846, 849- 852, 1576 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 451-638, 831-834, 843-846, 849-852, 1576.

149. A prime editing guide RNA (pegRNA) for targeting a L839P (c.2516C) mutation in ATP1A3, comprising a nucleotide sequence of SEQ ID NOs 733-827, 1574, 1577 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs 733-827, 1574, 1577.

150. A prime editing guide RNA (pegRNA) for targeting a G947R (c.2839C) mutation in ATP1A3, comprising a nucleotide sequence of SEQ ID NOs 639-732, 1578 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity to any one of the nucleotide sequences of SEQ ID NOs 639-732, 1578. B1195.70198WO00 13849370.1 (April 11, 2025)886 / 895 151. A complex comprising a prime editor and a pegRNA of any one of claims 147-150.

152. The complex of claim 151, wherein the prime editor is a fusion protein comprising a napDNAbp domain and a reverse transcriptase (RT) domain.

153. The complex of any one of claims 151 or 152, wherein the prime editor is selected from the group consisting of PE2, PE3, PE3b, PE4, PE5, PE5b, PEmax, SpCas9(VRQR)-PEmax, PE3max, PE3bmax, PE4max, PE4max, PE5bmax, PE6, PE6b, PE6c, PE6d, SpCas9(VRQR)- PE6b, SpCas9(VRQR)-PE6c, and SpCas9(VRQR)-PE6d.

154. The complex of any one of claim 151-153, wherein the prime editor is selected from the group consisting of PEmax, PE6b, PE6c, PE6d, SpCas9(VRQR)-PEmax, SpCas9(VRQR)-PE6b, SpCas9(VRQR)-PE6c, and SpCas9(VRQR)-PE6d.

155. The complex of any one of claim 151-153, wherein the prime editor is selected from the group consisting of: PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

156. A composition comprising the pegRNA of any one of claims 147-150, the ngRNA of claims 6 or 7, and / or the dsgRNA of claim 8 or 9.

157. The composition of claim 156, further comprising a prime editor.

158. The composition of claim 157, wherein the prime editor selected from the group consisting of: (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least B1195.70198WO00 13849370.1 (April 11, 2025)887 / 895 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

159. A system comprising the pegRNA of any one of claims 147-150 and a prime editor.

160. The system of claim 159, wherein the prime editor selected from the group consisting of: PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

161. The system of any one of claims 159 or 160, further comprising a dsgRNA.

162. The system of any one of claim 159-161, further comprising a ngRNA.

163. The system of claim 161, wherein the dsgRNA is the dsgRNA of claim 8 or 9.

164. The system of claim 162, wherein the ngRNA is the ngRNA of claim 6 or 7.

165. A recombinant adeno-associated viral (rAAV) vector comprising one or more nucleotide sequences encoding (a) a pegRNA of any one of claims 147-150, and (b) a prime editor.

166. The rAAV of claim 165, wherein the prime editor is selected from the group consisting of: PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522. B1195.70198WO00 13849370.1 (April 11, 2025)888 / 895 167. The rAAV of claim 165 or 166, wherein the prime editor is a split prime editor encoded by a first and a second nucleotide sequence.

168. The rAAV of claim 167, wherein the first nucleotide sequence encodes a N-terminal portion of the prime editor fused at its C-terminus to an intein-N.

169. The rAAV of claim 165, wherein the prime editor is provided as a split variant of PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), or SpCas9(VRQR)-PE6d (SEQ ID NO: 1522).

170. The rAAV of claim 167, wherein the first nucleotide sequence encodes an intein-C fused to the N-terminus of a C-terminal portion of the prime editor.

171. The rAAV of claim 165, wherein the prime editor is provided as a split variant of PEmax (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), or SpCas9(VRQR)-PE6d (SEQ ID NO: 1522).

172. A polynucleotide encoding the pegRNA of any one of claims 147-150.

173. One or more polynucleotides encoding the pegRNA of any one of claims 147-150, the complex of any one of claims 151-155, the composition of any one of claims 156-158, or the system of any one of claims 159-164.

174. A vector comprising the polynucleotide of claim 58.

175. A vector comprising the one or more polynucleotides of claim 173.

176. A pharmaceutical composition comprising the pegRNA of any one of claims 147-150, the ngRNA of claims 6 or 7, the dsgRNA of claim 8 or 9, the sgRNA of claims 10 or 11, the complex of any one of claims 151-155, the composition of any one of claims 156-158, the B1195.70198WO00 13849370.1 (April 11, 2025)889 / 895 system of any one of claims 159-164, the rAAV vector of any one of claims 165-171, the polynucleotides of claim 172-173, and / or the vectors of claim 174 or 175, and a pharmaceutically acceptable excipient.

177. A cell comprising the pegRNA of any one of claims 147-150, the ngRNA of claims 6 or 7, the dsgRNA of claim 8 or 9, the sgRNA of claims 10 or 11, the complex of any one of claims 151-155, the composition of any one of claims 156-158, the system of any one of claims 159- 164, the rAAV vector of any one of claims 165-171, the polynucleotides of claim 172-173, and / or the vectors of claim 174 or 175.

178. A kit comprising the pegRNA of any one of claims 147-150, the ngRNA of claims 6 or 7, the dsgRNA of claim 8 or 9, the sgRNA of claims 10 or 11, the complex of any one of claims 151-155, the composition of any one of claims 156-158, the system of any one of claims 159- 164, the rAAV vector of any one of claims 165-171, the polynucleotides of claim 172-173, and / or the vectors of claim 174 or 175, and optionally one or more containers, delivery devices, and / or set of instructions.

179. A method of prime editing an ATP1A3 D801N c.2401A mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 D801N c.2401A mutation with a prime editor and a prime editing guide RNA (pegRNA) of claim 147.

180. The method of claim 179, further comprising providing a ngRNA.

181. The method of claim 180, wherein the ngRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 929-939 or 1050-1065.

182. The method of claim 180 or 181, wherein the ngRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211-1227. B1195.70198WO00 13849370.1 (April 11, 2025)890 / 895 183. The method of any one of claims 179-182, further comprising providing a dsgRNA.

184. The method of claim 183, wherein the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1229-1252 or 1277-1316.

185. The method of claim 183 or 184, wherein the dsgRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406.

186. The method of any one of claims 179-185, wherein the prime editor is selected from the group consisting of: (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

187. A method of prime editing an ATP1A3 L839P c.2516C mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 D801N c.2401A mutation with a prime editor and a prime editing guide RNA (pegRNA) of claim 149.

188. The method of claim 187, further comprising providing a ngRNA.

189. The method of claim 188, wherein the ngRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 929-939 or 1050-1065. B1195.70198WO00 13849370.1 (April 11, 2025)891 / 895 190. The method of claim 188 or 189, wherein the ngRNA comprises a sequence comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211-1227.

191. The method of any one of claims 187-190, further comprising providing a dsgRNA.

192. The method of claim 192, wherein the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity with any one of the nucleic acid sequences of SEQ ID NOs: 1229-1252 or 1277-1316.

193. The method of claim 191 or 192, wherein the dsgRNA comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% sequence identity with any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406.

194. The method of any one of claims 187-193, wherein the prime editor is selected from the group consisting of: (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522.

195. A method of prime editing an ATP1A3 G947R c.2839C mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 G947R c.2839C mutation with a prime editor and a prime editing guide RNA (pegRNA) of claim 150.

196. The method of claim 195, further comprising providing a ngRNA. B1195.70198WO00 13849370.1 (April 11, 2025)892 / 895 197. The method of claim 196, wherein the ngRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 929-939 or 1050-1065.

198. The method of claim 196 or 197, wherein the ngRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211-1227.

199. The method of any one of claims 195-198, further comprising providing a dsgRNA.

200. The method of claim 199, wherein the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1229-1252 or 1277-1316.

201. The method of claim 199 or 200, wherein the dsgRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406.

202. The method of any one of claims 195-201, wherein the prime editor is selected from the group consisting of: (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522. B1195.70198WO00 13849370.1 (April 11, 2025)893 / 895 203. A method of prime editing an ATP1A3 E815K c.2443A mutation in an ATP1A3 gene comprising contacting a nucleic acid sequence encoding the ATP1A3 E815K c.2443A mutation with a prime editor and a prime editing guide RNA (pegRNA) of claim 148.

204. The method of claim 203, further comprising providing a ngRNA.

205. The method of claim 204, wherein the ngRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 929-939 or 1050-1065.

206. The method of claim 203 or 204, wherein the ngRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1080-1090 or 1211-1227.

207. The method of any one of claims 203-206, further comprising providing a dsgRNA.

208. The method of claim 207, wherein the dsgRNA comprises a spacer sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1229-1252 or 1277-1316.

209. The method of claim 207 or 208, wherein the dsgRNA comprises a sequence comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 1318-1342 or 1366-1406.

210. The method of any one of claims 203-209, wherein the prime editor is selected from the group consisting of: (SEQ ID NO: 1421), PE6b (SEQ ID NO: 1418), PE6c (SEQ ID NO: 1419), PE6d (SEQ ID NO: 1420), SpCas9(VRQR)-PEmax (SEQ ID NO: 1516), SpCas9(VRQR)-PE6b (SEQ ID NO: 1518), SpCas9(VRQR)-PE6c (SEQ ID NO: 1520), SpCas9(VRQR)-PE6d (SEQ ID NO: 1522), and an amino acid sequence having at least 80% identity, at least 85% identity, at B1195.70198WO00 13849370.1 (April 11, 2025)894 / 895 least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity to any one of SEQ ID NOs: 1421, 1418, 1419, 1420, 1516, 1518, 1520, or 1522. B1195.70198WO00 13849370.1 (April 11, 2025)

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