Use of prime editing in correcting mutations in CDKL5

Abstract

The present disclosure provides methods of using a prime editor in combination with prime editing guide RNAs (pegRNAs) and, optionally, nicking gRNAs to carry out prime editing to directly correct mutations (i.e., c,1412delA) in the CDKL5 gene that cause CDKL5 deficiency disorder. The present disclosure also provides compositions, complexes, and systems comprising the pegRNAs, nicking gRNAs, and / or prime editors disclosed herein, as well as polynucleotides and vectors encoding the same, and cells, kits, and pharmaceutical compositions comprising the same.

Description

USE OF PRIME EDITING IN CORRECTING MUTATIONS IN CDKL5 RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application, U.S.S.N.63 / 596,037, filed November 3, 2023, the contents of which are incorporated herein by reference. GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant numbers U01 AI142756, RM1 HG009490, R01 EB022376, and R35 GM118062, awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0003] CDKL5 Deficiency Disorder (CDD) is a developmental encephalopathy that is caused by dominant negative mutations in the cyclin-dependent kinase-like 5 (CDKL5) gene, such as the single “A” nucleotide deletion in Asp471 (c.1412delA), as well as other deletions, truncations, splice variants, and missense mutations, which result in pathogenic variants of the CDKL5 protein. See Olson et al., “Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder: clinical review,” Pediatr Neurol, 2019, 97:18-25. The CDKL5 protein is widely expressed in the brain, predominantly in neurons, with roles in cell proliferation, neuronal migration, axonal outgrowth, dendritic morphogenesis, and synapse development. CDD is a rare disease which occurs in about 1 in 42,000 live births. Symptoms include seizures, intellectual disability, developmental delay, feeding difficulties, sleeping difficulties, and low muscle tone. Typically, symptoms begin within a few months after birth. Treatment for CDD is limited to symptom management. Symptom management options include broad-range anti- seizure medications (ASMs) such as levetiracetam, the only FDA-approved ASM specifically for CDD called ganaxolone, ketogenic diet, and non-FDA approved cannabidiol derivatives198,199.

[0004] As a precise and versatile genome editing technology, prime editing (PE) enables all base-to-base changes, insertions, deletions, and any combination thereof in living cells. As such, PE has the potential to correct the majority of disease-causing mutations, including CDD-causing pathogenic mutations, such as the c.1412delA mutation. PE requires a prime editing guide RNA (pegRNA) and a prime editor protein, which comprises a programmable 1 / 189 B1195.70194WO00 13324217.2nickase and a reverse transcriptase (RT). In contrast to nucleases, PEs do not require the formation of a double stranded break (DSB) intermediates and thus offer a higher ratio of the desired to undesired editing products (i.e., the intended edit versus unwanted insertions and deletions or indels). However, standard prime editors (e.g., PEmax) are large and, as a result, often require a triple-AAV delivery strategy for in vivo studies. Recently evolved and engineered PE6a, PE6b, PE6c, PE6d prime editors (as reported in Doman et al., “Phage- assisted evolution and protein engineering yield compact, efficient prime editors,” Cell, 2023 Aug 31; 186(18): 3983-4002, and International PCT Application No. PCT / US2024 / 030786, filed May 23, 2024, each of which is incorporated herein by reference), a series of prime editors that are sufficiently compact for dual-AAV delivery, address this issue while retaining or exceeding the high editing efficiency afforded by the previous state-of-the-art prime editor, PEmax. Since CDD is caused by dominant negative mutations in the CDKL5 gene (i.e., mutations which result in a pathogenic variant of CDKL5 protein that interferes with the normal function of wild-type CDKL5), prime editing strategies that can specifically target and correct pathogenic mutations are needed. SUMMARY OF THE INVENTION

[0005] The present disclosure describes prime editing compositions, systems, and components thereof, constructs encoding the prime editing systems and / or components thereof, delivery vehicles (e.g., AAV delivery vectors or LNP delivery systems) encoding the prime editing systems and / or components thereof, amino acid and nucleotide sequences of the prime editing systems and / or components thereof, cells expressing or containing the prime editing systems and / or components thereof, and methods of gene editing using the prime editing systems and / or components, including ex vivo and in vivo methods of editing. In certain embodiments, the prime editing systems are capable of editing a cyclin-dependent kinase-like 5 (CDKL5) gene in order to correct and / or repair a CDD-associated pathogenic mutation, including, but not limited to, the single “A” nucleotide deletion in Asp471 (i.e., the c.1412delA mutation) known to be causative of CDD, as well as the c.826-1 G-to-A point mutation in CDKL5 and clusters of mutations in exon 8 of CDKL5.

[0006] In embodiments, the prime editing systems described herein comprise prime editing guide RNAs (pegRNAs) and prime editors, including pegRNAs and modified prime editors based on PE6, herein referred to as “PE6b-NRCH,” “PE6c-NRCH,” and “PE6d-NRCH”, which are capable of correcting pathogenic mutations in the CDKL5 gene, including, but not limited to, the c.1412delA mutation. 2 / 189 B1195.70194WO00 13324217.2

[0007] In certain embodiments, the prime editing systems described herein have been shown to achieve over 40% correction of the c.1412delA mutation in a heterozygous mouse N2a cell line with less than 2% indel byproducts.

[0008] In one aspect, the present disclosure provides pegRNAs for correcting a pathogenic mutation in the CDKL5 gene, including, but not limited to, the c.1412delA mutation, and therefore treating CDD. In some embodiments, the provided pegRNAs comprise reverse transcription templates (RTTs) that further introduce one or more silent edits into the CDKL5 gene to increase the efficiency at which the mutation is corrected (e.g., by impairing a cell’s DNA mismatch repair mechanisms).

[0009] In some embodiments, the present disclosure provides pegRNAs for targeting the c.1412delA mutation comprising a spacer sequence of GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), or GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5), or a spacer 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 GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), or GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), or GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5).

[0010] In some embodiments, the pegRNA comprises the sequence of any of the pegRNAs provided in Tables 1, 2, and 4 below, 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 any of the pegRNAs provided in Tables 1 and 2 below.

[0011] In another aspect, the present disclosure provides compositions comprising any of the pegRNAs provided herein, including pegRNAs comprising the sequence of any of the pegRNAs provided in Tables 1, 2, and 4 below, 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 any of the pegRNAs provided in Tables 1, 2, and 4 below. 3 / 189 B1195.70194WO00 13324217.2

[0012] In some embodiments, the composition further comprises a nicking guide RNA (ngRNA). In some embodiments, the ngRNA comprises a spacer sequence of GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13), or a spacer 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 GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13).

[0013] In certain embodiments, the ngRNA comprises the sequence of any one of the ngRNAs provided in Table 3 below, 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 any one of the ngRNAs provided in Table 3 below.

[0014] In another embodiment, the present disclosure provides compositions comprising any of the nicking guides (ngRNA) provided in Table 3 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 any of the ngRNAs provided in Table 3 below.

[0015] In some embodiments, the composition further comprises a prime editor. In certain embodiments, the prime editor is PE4max-NRCH, or a variant thereof. In some embodiments, the prime editor is a PE6 prime editor, or a variant thereof. In certain embodiments, the prime editor is PE6b-NRCH, PE6c-NRCH, PE6d-NRCH, or a variant thereof.

[0016] In another aspect, the present disclosure provides systems comprising a pegRNA, prime editor, and ngRNA described herein.

[0017] In another aspect, the present disclosure provides one or more polynucleotides encoding any of the pegRNAs and / or ngRNAs provided herein. In some embodiments, the one or more polynucleotides encode any of the prime editors provided herein. 4 / 189 B1195.70194WO00 13324217.2

[0018] In another aspect, the present disclosure provides one or more vectors comprising the one or more polynucleotides provided herein. In some embodiments, the vectors are AAV vectors.

[0019] In another aspect, the present disclosure provides virus-like particles (VLPs) or engineered virus-like particles (eVLPs) comprising any of the pegRNAs, ngRNAs, and / or prime editors disclosed herein.

[0020] In another aspect, the present disclosure provides compositions comprising any of the polynucleotides and / or vectors provided herein.

[0021] In another aspect, the present disclosure provides one or more AAV particles comprising any of the pegRNAs, compositions, polynucleotides, and / or vectors provided herein.

[0022] In another aspect, the present disclosure provides virus-like particles (VLPs) comprising any of the pegRNAs, compositions, polynucleotides and / or vectors provided herein.

[0023] In another aspect, the present disclosure provides pharmaceutical compositions comprising any of the pegRNAs, compositions, polynucleotides, vectors, AAV particles, or VLPs provided herein, and a pharmaceutically acceptable excipient.

[0024] In another aspect, the present disclosure provides cells comprising any of the pegRNAs, compositions, polynucleotides, vectors, AAV particles, or VLPs provided herein.

[0025] In another aspect, the present disclosure provides kits comprising any of the pegRNAs, compositions, polynucleotides, vectors, AAV particles, or VLPs provided herein.

[0026] In another aspect, the present disclosure provides methods of using a prime editor in combination with a pegRNA (and optionally an ngRNA) to carry out prime editing to directly correct mutations in the CDKL5 gene that cause CDKL5 deficiency disorder. In one aspect, the present disclosure provides methods of correcting a 1412delA mutation in a CDKL5 gene by prime editing comprising contacting a target DNA sequence comprising the CDKL5 gene with a prime editor and a prime editing guide RNA (pegRNA), wherein the pegRNA comprises a spacer sequence of GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), or GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5), or a spacer 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 GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), 5 / 189 B1195.70194WO00 13324217.2GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), or GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), or GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5).

[0027] In some embodiments, the contacting further comprises using a nicking guide RNA (ngRNA). In some embodiments, the ngRNA comprises a spacer sequence of GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13), or a spacer 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 GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13).

[0028] In certain embodiments, the prime editor is PE4max-NRCH, or a variant thereof. In some embodiments, the prime editor is a PE6 prime editor, or a variant thereof. In certain embodiments, the prime editor is PE6b-NRCH, PE6c-NRCH, PE6d-NRCH, or a variant thereof.

[0029] In some embodiments, the method is performed in a subject. In some embodiments, the method is a method of treating CDKL5 deficiency disorder in the subject.

[0030] In another aspect, the present disclosure provides for the use of any of the pegRNAs, compositions, polynucleotides, vectors, AAV particles, VLPs, or pharmaceutical compositions provided herein in the treatment of CDKL5 deficiency disorder.

[0031] In another aspect, the present disclosure provides for the use of any of the PE editing systems, prime editors, pegRNAs, ngRNAs, compositions, expression constructs, 6 / 189 B1195.70194WO00 13324217.2polynucleotides, vectors, AAV particles, VLPs, or pharmaceutical compositions provided herein in the manufacture of a medicament for the treatment of CDKL5 deficiency disorder.

[0032] The foregoing concepts, and additional concepts discussed below, may be arranged in any suitable combination, as the present disclosure is not limited in this respect. Further, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0034] FIGs.1A-1B show further developments of an improved prime editor-NRCH strategy to specifically target the c.1412delA variant allele in CDKL5 and limit potential indel formation in the wildtype allele. A PE4max-NRCH strategy that specifically targets the pathogenic allele was developed. The NRCH PAM is disrupted when the pathogenic deletion is reverted. The strategy also disrupts the protospacer with a seed edit. Additional strategies incorporating PE6 prime editors and variants, nicking guide RNA (ngRNAs), use of a flip + extension (F+E) scaffold, and a shortened PBS were also tested.

[0035] FIG.2 shows screening for the best epegRNA. PE6b-NRCH and PE6c-NRCH perform as well as PE4-max NRCH with nicking guide 1 (NG1) as provided in the first row of Table 3 below.

[0036] FIG.3 shows that the F+E scaffold improved editing with epegRNA primer binding site (PBS) 11 and reverse transcriptase template (RTT) 15 as shown in Tables 1 and 2 below. Shortening the PBS to 9 or 10 nucleotides improved editing even further. PBS10 and RTT15 were used in subsequent screens in N2a cells.

[0037] FIG.4 shows that screening PBS10 and RTT15 in a similar manner as the previous best epegRNA again revealed that PE6b-NRCH and PE6c-NRCH are nearly as active as PE5max-NRCH with NG1.

[0038] FIG.5 shows that the desired edit-to-indel ratio with PE6b-NRCH is better than with PE6c-NRCH. 7 / 189 B1195.70194WO00 13324217.2

[0039] FIG.6 shows establishment of homozygous and 2 / 3 heterozygous HEK293T cell lines used for epegRNA PE-NRCH screening.

[0040] FIG.7 shows screening of epegRNAs with PE4max-NRCH in HEK293T cells.

[0041] FIG.8 shows screening of PE4max-SpRY (recognizing an NRN PAM). Highest editing efficiencies of 2.4% of all alleles and 4.8% of available alleles was observed.

[0042] FIG.9 shows screening of epegRNAs with a PE6 prime editor comprising an evolved Cas9 variant. Highest editing efficiencies of 6.6% of all alleles and 13.2% of available alleles was observed.

[0043] FIG.10 shows screening of epegRNAs with a PE6 prime editor comprising an evolved reverse transcriptase variant. Highest editing efficiencies of 6.2% of all alleles and 12.4% of available alleles was observed.

[0044] FIGs.11A-11D show prime editing optimization for the correction of c.1412delA in murine Cdkl5. Bars reflect mean ± s.d. of n = 2-4 independent biological replicates from different thaws of a monoclonal heterozygous N2a cell line harboring the Cdkl5 equivalent of c.1412delA in 50% of alleles, with replicate values shown as individual dots. Editing percentages represent desired prime editing outcomes with no indels in the 50% of targetable alleles with the pathogenic mutation. Indel frequencies reflect indel formation across all sequencing reads. FIG.11A shows that PE-NRCH can revert the c.1412delA mutation (underlined) back to the wildtype allele. The pathogenic allele makes an NRCH PAM which allows for allele-specific targeting in addition to eliminating potential re-targeting of the corrected allele as the PAM is disrupted when the allele is corrected. The addition of a silent seed edit (+2 G > T, double underline) may also help the prime editing intermediate evade MMR86. FIG.11B shows editing efficiencies for corrective epegRNAs of various PBS and RTT lengths in a PE4max-NRCH screen. FIG.11C shows editing efficiencies of epegRNAs with PBS lengths of 9-11 nt and an RTT length of 15 nt with either the standard sgRNA scaffold or the “flip and extension” (F+E) scaffold203. FIG.11D shows editing efficiencies for various PE systems when using an epegRNA with a standard scaffold, a PBS of 10 nt, and an RTT of 15 nt, and with the optional inclusion of a nicking sgRNA that nicks at 87 nt (NG1), 63 nt (NG2), or 47 nt (NG3) 3' of the epegRNA nick site.

[0045] FIGs.12A-12E show optimizing prime editing outcomes for c.1412delA in human CDKL5. Bars reflect mean ± s.d. of n = 2-3 independent biological replicates from monoclonal homozygous HEK293T cell lines harboring the c.1412delA in CDKL5, with replicate values shown as individual dots. FIG.12A shows that initial efforts to optimize an 8 / 189 B1195.70194WO00 13324217.2SpCas9-NRCH-based prime editing strategy for the correction of the pathogenic c.1412delA mutation (underline) required the inclusion of a non- silent seed edit . A corrective prime editing strategy was previously described with PEmax and a pegRNA which includes a silent seed edit (double underline)86. FIG.12B shows editing efficiencies for an epegRNA screen for the PE-NRCH strategy. All epegRNAs encoded a non-silent I470L mutation. FIG.12C shows editing efficiencies from a PE-NRCH variant screen utilizing the most efficient epegRNA from FIG.12B. Each prime editor variant was screened with either no nicking sgRNA, a nicking sgRNA designed not to nick until after the desired edit has already been made in attempt to minimize indels3(NG4), or a nick-to-nick distance of 22 nt (NG5) or 76 nt (NG6). FIG.12D shows editing outcomes after screening PE variants with the epegRNA version of the corrective pegRNA86 either with or without an 8 nt linker designed to minimize interactions between the structured 3' motif of the epegRNA and the RTT89. All conditions utilized the nicking sgRNA86.

[0046] FIG.13 shows prime editing of patient primary fibroblasts. Quantification of editing frequencies from PE3max, PE6c, and untreated patient primary fibroblasts heterozygous for c.1412delA. Bars represent average desired editing frequency of the 50% of editable alleles with the c.1412delA mutation. Dots represent n = 2 independent replicates and error bars represent ± s.d. Indel frequencies represent indels observed from all reads.2.5 × 105fibroblasts isolated from a patient heterozygous for the c.1412delA mutation were electroporated and cultured for 6 days. At day 3, the cells underwent media exchange, and genomic DNA was extracted at day 6.

[0047] FIGs.14A-14B show off-target editing in prime-edited primary patient fibroblasts. Bars represent average ± s.d. of n = 2 independent biological replicates. FIG.14A shows HTS quantification of the first epegRNA-encoded mismatch at Cas-OFFinder-nominated off- target loci in patient primary fibroblasts heterozygous for the c.1412delA mutation. FIG.14B shows HTS quantification of indels at epegRNA- and nicking sgRNA-dependent off-target loci nominated by Cas-OFFinder in patient primary fibroblasts heterozygous for the c.1412delA mutation. Nick OT3 was not analyzed due to its location on the Y chromosome and the fibroblasts being isolated from a female patient.

[0048] FIGs.15A-15C shows screening of prime editing strategies for the correction of c.826-1 G > A. Development of a corrective prime editing strategy for c.826-1 G > A. For bar graphs, bars reflect mean ± s.d. of n = 2 independent biological replicates from heterozygous HEK293T cell lines harboring the c.826- 1G > A mutation in approximately 30% of CDKL5 9 / 189 B1195.70194WO00 13324217.2alleles, with replicate values shown as individual dots. FIG.15A shows design of the corrective prime editing strategy for c.826-1 G > A (underline). The epegRNA (protospacer in pink, PAM in light blue) was designed to also encode silent edits (double underline) which may help the prime editing intermediate evade MMR86. FIG.15B shows editing efficiencies from an epegRNA screen to determine the epegRNAs resulting in the highest percentage of desired c.826-1 G > A correction. FIG.15C shows a screen for various prime editors and for multiple nicking sgRNAs, including an edit-specific nicking sgRNA (NG3) as this leads to fewer indels, using the epegRNA with a PBS length of 11 nt and an RTT length of 33 nt from FIG.15B.

[0049] FIGs.16A-16B shows installation of pathogenic CDD mutations in exon 8 of the CDKL5 catalytic domain. FIG.16A shows a location of various pathogenic CDKL5 mutations in exon 8 and related seed edits that could be introduced with a single epegRNA. FIG.16B shows editing efficiencies of the installation of pathogenic CDKL5 mutations or related seed edits in exon 8 from two epegRNAs that have not been completely optimized. Bars reflect mean ± s.d. of n = 3 independent biological replicates from wildtype HEK293T cell lines, with replicate values shown as individual dots.

[0050] FIG.17 shows CDKL5 function is important for brain function and development.

[0051] FIG.18 shows that pathogenic mutations causing CDKL5 deficiency disorder (CDD) are typically spontaneous and result in loss of CDKL5 function.

[0052] FIGs.19A-19B show that pathogenic mutations causing CDKL5 deficiency disorder (CDD) are typically spontaneous and result in loss of CDKL5 function.

[0053] FIGs.20A-20C show that CDD is clinically distinct from other neurological disorders like Rett syndrome, West syndrome, and X-linked dominant infantile spasm syndrome. Clinical features highly suggestive of CDD include: Seizures within the first year of life (90% by 3 months); Global developmental delay; Severely impaired gross motor function; Sleep disturbances; Abnormal muscle tone; Bruxism (grinding or gnashing of teeth); and Gastrointestinal issues.

[0054] FIG.21 shows that treatments for CDD are limited to symptom management. None of the therapies shown are disease-modifying, nor do they address the root genetic cause of CDD.

[0055] FIGs.22A-22B show that treatments for CDD are limited to symptom management. With gene therapy, endogenous transcriptional control is lost, consequences of 10 / 189 B1195.70194WO00 13324217.2overexpression of CDKL5 are unknown, patients with dominant negative mutations would be ineligible, and the genetic cause of CDD would still not be addressed.

[0056] FIGs.23A-23C show that genome editing could enable disease-modifying therapeutic development for CDD by directly correcting pathogenic mutations. Given their versatility, prime editors are uniquely suited to address pathogenic CDKL5 mutations.

[0057] FIGs.24A-24B show efficiency of PE3 strategies for installation of c.1412delA in mCDKL5.

[0058] FIGs.25A-25B show that use of a PBS + RTT length combination that showed initial success in hCDKL5 did not translate to efficient editing in mCDKL5.

[0059] FIGs.26A-26B show that development of epegRNAs and MLH1dn benefited c.1412delA installation in mCDKL5.

[0060] FIG.27 shows that PE3max and epegRNAs with a silent seed edit can enable c.1412delA installation at mCDKL5 in a knock-in HEK293T cell line.

[0061] FIG.28 shows that allele-specific targeting with an SpCas9-NRCH DNA targeting domain may reduce indels.

[0062] FIG.29 shows that PE-NRCH can target the c.1412delA allele specifically, reducing indel formation in a knock-in HEK293T cell line.

[0063] FIGs.30A-30B show that female MEFs harboring c.1412delA could not be efficiently electroporated with either prime editing strategy.

[0064] FIG.31 shows PE3max-NRCH editing in c.1412delA mice.

[0065] FIG.32 shows a protocol for PE3max-NRCH editing in c.1412delA mice.

[0066] FIGs.33A-33D show expression of full-length CDKL5 in male mice 4 weeks post- injection. Western blot analysis of forebrain CDKL5 expression in male Cdkl5-FS and WT littermate mice, 4 weeks post-administration of a single dose. Mice were injected at P0 days with either AAV9-epegRNA+nicking guide (GFP), AAV9-PE3max-NRCH (TX), or saline (SAL).

[0067] FIG.34 shows behavioral responses of treated and untreated female mice 8 weeks post-injection. FWT, female WT; FFS, female FS.

[0068] FIG.35 shows allele-specific PE3max-NRCH editing in a triple AAV system.

[0069] FIG.36 shows that efficient prime editing for the generation of an N2a cell line with c.1412delA in 50% of alleles enabled extensive screening.

[0070] FIGs.37A-37D show screening of epegRNAs with the indicated prime editor. 11 / 189 B1195.70194WO00 13324217.2

[0071] FIG.38 shows that PE3max and PE6c enable 25-27% c.1412delA correction in primary patient fibroblasts.

[0072] FIGs.39A-39B show that off-target editing is minimal in prime-edited primary patient fibroblasts. FIG.39A shows Cas-OFFinder pegRNA-nominated off-targets. FIG. 39B shows Cas-OFFinder nicking sgRNA-nominated off-targets. Both preliminary “off- targets” are in intragenic regions.

[0073] FIGs.40A-40C show that precise editing is required for c.826-1 G>A correction to avoid changes to bystander adenines.

[0074] FIG.41 shows that missense pathogenic variants cluster in the catalytic domain.

[0075] FIG.42 shows screening of optiprime epegRNAs with PEmax-NRCH. DEFINITIONS

[0076] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise. Cas9

[0077] 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 12 / 189 B1195.70194WO00 13324217.2(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., 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.

[0078] 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. 13 / 189 B1195.70194WO00 13324217.2337:816-821(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, 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 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. 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. 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. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9.

[0079] In some embodiments, a Cas9 protein used in a prime editor is Cas9-NRCH. 14 / 189 B1195.70194WO00 13324217.2CDKL5 Deficiency Disorder (CDD)

[0080] “CDKL5 deficiency disorder (CDD)” is a rare genetic disorder caused by pathogenic variants in the CDKL5 gene (e.g., c.1412delA as described herein), which encodes for cyclin- dependent kinase-like 5. Symptoms of CDD include epilepsy, hypotonia, developmental, intellectual, and motor disabilities, and cortical visual impairment. CDD occurs in approximately 1 in 42,000 live births, and each case of CDD is caused by a variety of mutations in the CDKL5 gene. Guide RNA (“gRNA”)

[0081] As used herein, the term “guide RNA” is a particular type of guide nucleic acid which is mostly commonly associated with a Cas protein of a CRISPR-Cas9 and which associates with Cas9, directing the Cas9 protein to a specific sequence in a DNA molecule that includes complementarity to protospacer sequence of the guide RNA. However, this term also embraces the equivalent guide nucleic acid molecules that associate with Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and which otherwise program the Cas9 equivalent to localize to a specific target nucleotide sequence. The Cas9 equivalents may include other napDNAbp from any type of CRISPR system (e.g., type II, V, VI), including Cpf1 (a type-V CRISPR-Cas systems), C2c1 (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system) and C2c3 (a type V CRISPR-Cas system). Further Cas-equivalents are described in Makarova et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299), the contents of which are incorporated herein by reference. Exemplary sequences are and structures of guide RNAs are provided herein. In addition, methods for designing appropriate guide RNA sequences are provided herein. As used herein, the “guide RNA” may also be referred to as a “traditional guide RNA” to contrast it with the modified forms of guide RNA termed “prime editing guide RNAs” (or “pegRNAs”) which have been invented for the prime editing methods and composition disclosed herein.

[0082] Guide RNAs or pegRNAs may comprise various structural elements that include, but are not limited to:

[0083] Spacer sequence – the sequence in the guide RNA or pegRNA (having about 20 nts in length) which binds to the protospacer in the target DNA. 15 / 189 B1195.70194WO00 13324217.2

[0084] gRNA core (or gRNA scaffold or backbone sequence) - refers to the sequence within the gRNA that is responsible for Cas9 binding, it does not include the 20 bp spacer / targeting sequence that is used to guide Cas9 to target DNA.

[0085] Extension arm – a single strand extension at the 3ʹ end or the 5ʹ end of the pegRNA which comprises a primer binding site and a DNA synthesis template sequence that encodes via a polymerase (e.g., a reverse transcriptase) a single stranded DNA flap containing the genetic change of interest, which then integrates into the endogenous DNA by replacing the corresponding endogenous strand, thereby installing the desired genetic change.

[0086] Transcription terminator – the guide RNA or pegRNA may comprise a transcriptional termination sequence at the 3ʹ of the molecule. napDNAbp

[0087] As used herein, the term “nucleic acid programmable DNA binding protein” or “napDNAbp,” of which Cas9 is an example, 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 equivalent) to localize and bind to a complementary sequence.

[0088] 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. Exemplary napDNAbp with different nuclease activities include “Cas9 nickase” (“nCas9”) and a deactivated Cas9 having no nuclease activities (“dead Cas9” or “dCas9”). Exemplary sequences for these and other napDNAbp are provided herein. In 16 / 189 B1195.70194WO00 13324217.2some embodiments, a napDNAbp has nickase activity in a RuvC domain and / or an HNH domain. In some embodiments, a napDNAbp has nickase activity in a RuvC domain or an HNH domain. Nickase

[0089] As used herein, a “nickase” refers to a napDNAbp (e.g., a Cas protein) which is capable of cleaving only one of the two complementary strands of a double-stranded target DNA sequence, thereby generating a nick in that strand. In some embodiments, the nickase cleaves a non-target strand of a double stranded target DNA sequence. In some embodiments, the nickase comprises an amino acid sequence with one or more mutations in a catalytic domain of a canonical napDNAbp (e.g., a Cas 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 RuvC I 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.

[0090] In some embodiments, the napDNAbp of the prime editing complex comprises an endonuclease having nucleic acid programmable DNA binding ability. In some embodiments, the napDNAbp comprises an active endonuclease capable of cleaving both strands of a double stranded target DNA. In some embodiments, the napDNAbp is a nuclease active endonuclease, e.g., a nuclease active Cas protein, that can cleave both strands of a double stranded target DNA by generating a nick on each strand. For example, a nuclease active Cas protein can generate a cleavage (a nick) on each strand of a double stranded target DNA. In some embodiments, the two nicks on both strands are staggered nicks, for example, generated by a napDNAbp comprising a Cas12a or Cas12b1. In some embodiments, the two nicks on 17 / 189 B1195.70194WO00 13324217.2both strands are at the same genomic position, for example, generated by a napDNAbp comprising a nuclease active Cas9. In some embodiments, the napDNAbp comprises an endonuclease that is a nickase. For example, in some embodiments, the napDNAbp comprises an endonuclease comprising one or more mutations that reduce nuclease activity of the endonuclease, rendering it a nickase. In some embodiments, the napDNAbp comprises an inactive endonuclease, for example, in some embodiments, the napDNAbp comprises an endonuclease comprising one or more mutations that abolish the nuclease activity. In various embodiments, the napDNAbp is a Cas9 protein or variant thereof. The napDNAbp can also be a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9). In a preferred embodiment, the napDNAbp is 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)

[0091] 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)

[0092] The term “nuclear localization sequence” or “NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., international PCT application, PCT / EP2000 / 011690, filed November 23, 2000, published as WO 2001 / 038547 on May 31, 2001, the contents of which are incorporated herein by reference for its disclosure of exemplary nuclear localization sequences. In some embodiments, an NLS is included in a fusion protein (e.g., in a prime editor as described herein). In certain embodiments, an NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 21), MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 22), 18 / 189 B1195.70194WO00 13324217.2KRTADGSEFESPKKKRKV (SEQ ID NO: 23), KRTADGSEFEPKKKRKV (SEQ ID NO: 24), NLSKRPAAIKKAGQAKKKK (SEQ ID NO: 25), PAAKRVKLD (SEQ ID NO: 26), RQRRNELKRSF (SEQ ID NO: 27), or NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 28). pegRNA

[0093] 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 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.

[0094] 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. 19 / 189 B1195.70194WO00 13324217.2

[0095] 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.

[0096] 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.

[0097] 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 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.

[0098] 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.

[0099] In some embodiments, a pegRNA is an “engineered pegRNA” (“epegRNA”). Relative to a pegRNA, an epegRNA comprises an additional structured motif, for example, 20 / 189 B1195.70194WO00 13324217.2attached 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: 14), 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: 14).

[0100] 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 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. Prime editing

[0101] 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.

[0102] 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 21 / 189 B1195.70194WO00 13324217.2napDNAbp), 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 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 22 / 189 B1195.70194WO00 13324217.2sequence) 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 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.

[0103] 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 23 / 189 B1195.70194WO00 13324217.2the 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 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

[0104] As used herein, the term “prime editor” in some embodiments refers to the polypeptide or polypeptide components (e.g., a nucleic acid programmable DNA binding 24 / 189 B1195.70194WO00 13324217.2protein (“napDNAbp”) working in association with a polymerase, such as a reverse transcriptase, which are in the form of a fusion protein or otherwise provided in trans) involved in prime editing as described herein, including PE1, PE2, PE3 (including PE3b), PE4, PE5 (including PE5b), PE6 (including PE6a, PE6b, PE6c, PE6d, PE6e, PE6f, PE6a-e, PE6a-f, PE6a-g, PE6b-e, PE6b-f, PE6b-g, PE6c-e, PE6c-f, PE6c-g, PE6d-e, PE6d-f, PE6d-g, PE6b-NRCH, PE6c-NRCH, and PE6d-NRCH), PE7, and PEmax, or to one or more nucleotide sequence encoding any of PE1, PE2, PE3 (including PE3b), PE4, PE5 (including PE5b), PE6 (including PE6a, PE6b, PE6c, PE6d, PE6e, PE6f, PE6a-e, PE6a-f, PE6a-g, PE6b- e, PE6b-f, PE6b-g, PE6c-e, PE6c-f, PE6c-g, PE6d-e, PE6d-f, PE6d-g, PE6b-NRCH, PE6c- NRCH, and PE6d-NRCH), PE7, and PEmax). The prime editor 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).

[0105] In some embodiments, a prime editor comprises 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). These components may be provided as a fusion protein (or fusion construct) in some embodiments. In other embodiments, these components may be provided in trans.

[0106] 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.

[0107] In some embodiments, a prime editor composition, system, or complex provided herein comprises a fusion protein or a fusion protein complexed with a pegRNA, and / or further complexed with a second-strand nicking sgRNA. In some embodiments, the prime editor system may also refer to the complex comprising a fusion protein (reverse transcriptase fused to a napDNAbp), a pegRNA, and a regular guide RNA capable of directing the second- site nicking step of the non-edited strand as described herein. 25 / 189 B1195.70194WO00 13324217.2Prime editor / editing system

[0108] As used here, the expression “prime editor system” (or equivalent term “prime editing system”) refers to a composition or otherwise a set of components needed to conduct prime editing (e.g., in a cell). In embodiments, a prime editing system minimally includes a prime editor (or a nucleotide sequence encoding a prime editor) and a pegRNA (or a nucleotide sequence encoding the pegRNA). In embodiments, the prime editor and the pegRNA form a complex which is capable of binding to a target DNA site that has complementarity to the pegRNA (i.e., the spacer of the pegRNA). In other embodiments, the prime editing system can include a second-strand nicking guide RNA (ngRNA). In still other embodiments, the prime editing system can include a delivery vector (e.g., a lipid nanoparticle (LNP), virus-like particle (VLP) or engineered virus-like particle (eVLP), adeno-associated virus (AAV) vector, or a lentivirus vector (LV). In still other embodiments, the prime editing system can be formulated as a pharmaceutical composition which may include one or more delivery vectors comprising the prime editing system (e.g., protein components, pegRNAs, or nucleotide sequence encoding same, or a mixture of said components) and one or more pharmaceutical excipients. In further embodiments, the prime editing system may also include a device or applicator for administering same. Primer binding site

[0109] 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. Protospacer

[0110] As used herein, the term “protospacer” refers to the sequence (e.g., of ~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 (except that a protospacer contains Thymine and the spacer sequence contains Uracil). The guide RNA anneals to the complement of the protospacer sequence on the target DNA (specifically, one strand thereof, i.e., the “target strand” versus the “non-target strand” of the target DNA sequence). In some embodiments, in order for a Cas nickase component of a prime editor to function, it also 26 / 189 B1195.70194WO00 13324217.2requires a specific protospacer adjacent motif (PAM) that varies depending on the Cas protein component itself, e.g., the type of Cas protein and the bacterial species from which it is derived. The most commonly used Cas9 nuclease, derived from S. pyogenes, recognizes a PAM sequence of NGG that is directly downstream of the protospacer sequence in the genomic DNA, on the non-target strand. Protospacer adjacent motif (PAM)

[0111] 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).

[0112] 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

[0113] The term “reverse transcriptase” describes a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Historically, reverse transcriptase has been used primarily to transcribe mRNA into cDNA, which can then be cloned into a vector for further manipulation. Avian myoblastosis virus (AMV) reverse transcriptase was the first widely used RNA-dependent DNA polymerase (Verma, Biochim. Biophys. Acta 473:1 (1977)). The enzyme has 5ʹ-3ʹ RNA-directed DNA polymerase activity, 5ʹ-3ʹ DNA-directed DNA polymerase activity, and RNase H activity. RNase H is a processive 5ʹ and 3ʹ ribonuclease specific for the RNA strand for RNA-DNA hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)). Errors in transcription cannot be corrected by reverse transcriptase because known viral reverse transcriptases lack the 3ʹ-5ʹ exonuclease activity necessary for proofreading (Saunders and Saunders, Microbial Genetics Applied to Biotechnology, London: Croom Helm (1987)). A detailed study of the activity of AMV reverse transcriptase and its associated RNaseH activity has been presented by Berger 27 / 189 B1195.70194WO00 13324217.2et 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. Spacer sequence

[0114] As used herein, the term “spacer sequence” in connection with a guide RNA or a pegRNA refers to the portion of the guide RNA or pegRNA of about 20 nucleotides which contains a nucleotide sequence that corresponds to the protospacer sequence in the target DNA sequence. The spacer sequence anneals to the target strand (which is complementary to the protospacer strand) to form a ssRNA / ssDNA hybrid structure at the target site and a corresponding R loop ssDNA structure of the endogenous DNA strand. Subject

[0115] 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. Target site

[0116] The term “target site” refers to a sequence within a nucleic acid molecule that is edited by a prime editor (PE) disclosed herein. The target site further refers to the sequence within a nucleic acid molecule to which a complex of the prime editor (PE) and gRNA binds. Treatment

[0117] 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, 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 28 / 189 B1195.70194WO00 13324217.2inhibit the progress of a disease or disorder, 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. 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

[0118] As used herein the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant Cas9 is a Cas9 comprising one or more changes in amino acid residues as compared to a wild type Cas9 amino acid sequence. The term “variant” encompasses homologous proteins having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% percent identity with a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence, and which display the same or substantially the same functional activity or activities as the reference sequence. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0119] This disclosure provides prime editing systems, prime editors, pegRNAs, ngRNAs, pharmaceutical compositions, and methods for correcting one or more pathogenic mutations (e.g., c.1412delA, the c.826-1 G-to-A point mutation in CDKL5, and clusters of mutations in exon 8 of CDKL5) in a cyclin-dependent kinase-like 5 (CDKL5) gene for treating CDD (CDKL5 deficiency disorder).

[0120] In a first aspect, the disclosure provides prime editing systems and components thereof. In a second aspect, the disclosure provides constructs encoding the prime editing systems and / or components thereof. In a third aspect, the disclosure provides delivery vehicles (e.g., AAV delivery vectors or LNP delivery systems) comprising and / or encoding the prime editing systems and / or components thereof. In a fourth aspect, the disclosure provides amino acid and nucleotide sequences of the prime editing systems and / or components thereof. In a fifth aspect, the disclosure provides cells expressing or containing 29 / 189 B1195.70194WO00 13324217.2the prime editing systems and / or components thereof. In a sixth aspect, the disclosure provides methods of gene editing using the prime editing systems and / or components, including ex vivo and in vivo methods of editing. In some embodiments, the prime editing systems are capable of gene editing a cyclin-dependent kinase-like 5 (CDKL5) gene in order to correct and / or repair a CDD-associated pathogenic mutation, including, but not limited to, the single “A” nucleotide deletion in Asp471 (i.e., the c.1412delA mutation) known to be causative of CDD.

[0121] In certain embodiments, the prime editing systems described herein comprise prime editing guide RNAs (pegRNAs) and prime editors, including pegRNAs and modified prime editors based on PE6, herein referred to as “PE6b-NRCH,” “PE6c-NRCH,” and “PE6d- NRCH”, which are capable of correcting pathogenic mutations in the CDKL5 gene, including, but not limited to the c.1412delA mutation.

[0122] In some embodiments, the prime editing systems described herein have been demonstrated to achieve over 40% correction of the c.1412delA mutation in a heterozygous mouse N2a cell line with less than 2% indel byproducts.

[0123] In certain embodiments, the present disclosure provides methods of using a prime editor in combination with prime editing guide RNAs (pegRNAs and, optionally, nicking gRNAs) to carry out prime editing to directly correct mutations (e.g., c.1412delA) in the CDKL5 gene that cause CDKL5 deficiency disorder.

[0124] The present disclosure also provides compositions, systems, and complexes comprising pegRNAs, nicking gRNAs, and / or prime editors provided herein, as well as polynucleotides and vectors encoding the same, and cells, kits, and pharmaceutical compositions comprising the same. pegRNAs and Compositions Thereof

[0125] In one aspect, the present disclosure provides pegRNAs for correcting a pathogenic mutation in the CDKL5 gene, including, but not limited to, the c.1412delA mutation, and therefore treating CDKL5 deficiency disorder.

[0126] In one embodiment, the present disclosure provides pegRNAs for correcting a c.1412delA mutation in CDKL5 and treating CDKL5 deficiency disorder.

[0127] In one aspect, the present disclosure provides pegRNAs for correcting a c.1412delA mutation in CDKL5 gene comprising a spacer sequence of GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG 30 / 189 B1195.70194WO00 13324217.2(SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC(SEQ ID NO: 4), GCAGAGTCGGCATAGCTATAT(SEQ ID NO: 5), or TAGCTATGCCGACT (SEQ ID NO: 6), or a spacer 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 GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5), or TAGCTATGCCGACT (SEQ ID NO: 6), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5), or TAGCTATGCCGACT (SEQ ID NO: 6). In some embodiments, the pegRNA comprises the sequence of any of the pegRNAs provided in Tables 1, 2, and 4 below, 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 any of the pegRNAs provided in Tables 1, 2, and 4 below.

[0128] In some embodiments, the pegRNAs of the present disclosure comprise a gRNA scaffold sequence of GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 576) or GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 577), 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 GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 576) or GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 577), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 576) or 31 / 189 B1195.70194WO00 13324217.2GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 577).

[0129] In some embodiments, the pegRNAs of the present disclosure comprise a reverse transcription sequence of CTATATTGACACAA (SEQ ID NO: 404), GCTATATTGACACAA (SEQ ID NO: 405), AGCTATATTGACACAA (SEQ ID NO: 406), CAGCTATATTGACACAA (SEQ ID NO: 407), ACAGCTATATTGACACAA (SEQ ID NO: 408), CACAGCTATATTGACACAA (SEQ ID NO: 409), GCACAGCTATATTGACACAA (SEQ ID NO: 410), GGCACAGCTATATTGACACAA (SEQ ID NO: 411), GAATTGTGTCAAGA (SEQ ID NO: 412), GGAATTGTGTCAAGA (SEQ ID NO: 413), GGGAATTGTGTCAAGA (SEQ ID NO: 414), GGGGAATTGTGTCAAGA (SEQ ID NO: 415), TGGGGAATTGTGTCAAGA (SEQ ID NO: 416), CTGGGGAATTGTGTCAAGA (SEQ ID NO: 417), ACTGGGGAATTGTGTCAAGA (SEQ ID NO: 418), GACTGGGGAATTGTGTCAAGA (SEQ ID NO: 419), GACTGGGGAATTGTGTCGATG (SEQ ID NO: 420), GACTGGGGAATTGTGTCAATG (SEQ ID NO: 421), GACTGGGGAATTGTGTCGATA (SEQ ID NO: 422), GACTGGGGAATTGTGTCTATG (SEQ ID NO: 423), GACTGGGGAATTGTGTCAATA (SEQ ID NO: 424), GACTGGGGAATTGTGTCTATA (SEQ ID NO: 425), GGACTGGGGAATTGTATCGATG (SEQ ID NO: 426), GGACTGGGGAATTGTATCAATG (SEQ ID NO: 427), GGACTGGGGAATTGTATCTATG (SEQ ID NO: 428), TAGAGGACTGGGGAATCGTGTCAATA (SEQ ID NO: 429), GGACTGGGGAATTGTATCGATA (SEQ ID NO: 430), TAGAGGACTGGGGAATCGTGTCGATA (SEQ ID NO: 431), GGACTGGGGAATTGTATCTATA (SEQ ID NO: 432), GACTCCTAGAGGACTGGGGGATCGTGTCAATA (SEQ ID NO: 433), TCCTAGAGGACTGGGGAATCGTATCAATA (SEQ ID NO: 434), GGACTGGGGAATTGTATCAATA (SEQ ID NO: 435), TAGAGGACTGGGGAATAGTGTCAATA (SEQ ID NO: 436), TAGAGGACTGGGGAATCGTGTCGATG (SEQ ID NO: 437), TAGAGGACTGGGGAATCGTGTCAATG (SEQ ID NO: 438), TAGAGGACTGGGGAATCGTGTCTATA (SEQ ID NO: 439), TAGAGGACTGGGGAATAGTATCAATA (SEQ ID NO: 440), TAGAGGACTGGGGAATGGTGTCAATA (SEQ ID NO: 441), 32 / 189 B1195.70194WO00 13324217.2CTCCTAGAGGACTGGGGGATCGTATCAATA (SEQ ID NO: 442), CTCCTAGAGGACTGGGGGATGGTATCAATA (SEQ ID NO: 443), CTCCTAGAGGACTGGGGGATGGTATCAATG (SEQ ID NO: 444), GACTCCTAGAGGACTGGGGGATAGTGTCAATG (SEQ ID NO: 445), CTCCTAGAGGACTGGGGGATCGTATCGATA (SEQ ID NO: 446), TCCTAGAGGACTGGGGAATCGTATCGATA (SEQ ID NO: 447), GACTCCTAGAGGACTGGGGGATGGTGTCAATG (SEQ ID NO: 448), TCCTAGAGGACTGGGGAATCGTATCAATG (SEQ ID NO: 449), TAGAGGACTGGGGAATGGTATCAATA (SEQ ID NO: 450), TAGAGGACTGGGGAATGGTGTCGATA (SEQ ID NO: 451), TAGAGGACTGGGGAATAGTGTCGATG (SEQ ID NO: 452), TAGAGGACTGGGGAATGGTGTCAATG (SEQ ID NO: 453), TAGAGGACTGGGGAATAGTATCGATA (SEQ ID NO: 454), TAGAGGACTGGGGAATAGTGTCAATG (SEQ ID NO: 455), TAGAGGACTGGGGAATAGTATCAATG (SEQ ID NO: 456), CCTAGAGGACTGGGGGATCGTGTCGATA (SEQ ID NO: 457), TAGAGGACTGGGGAATGGTGTCGATG (SEQ ID NO: 458), TCCTAGAGGACTGGGGTATCGTGTCAATA (SEQ ID NO: 459), TAGAGGACTGGGGAATAGTATCGATG (SEQ ID NO: 460), TAGAGGACTGGGGAATAGTGTCGATA (SEQ ID NO: 461), CTCCTAGAGGACTGGGGGATAGTATCAATG (SEQ ID NO: 462), TAGAGGACTGGGGAATGGTATCGATA (SEQ ID NO: 463), CCTAGAGGACTGGGGGATCGTGTCGATG (SEQ ID NO: 464), GACTCCTAGAGGACTGGGGGATGGTGTCAATA (SEQ ID NO: 465), CTCCTAGAGGACTGGGGGATAGTATCAATA (SEQ ID NO: 466), TAGAGGACTGGGGAATGGTATCAATG (SEQ ID NO: 467), TCCTAGAGGACTGGGGAATCGTATCGATG (SEQ ID NO: 468), TAGAGGACTGGGGAATAGTATCTATA (SEQ ID NO: 469), GACTCCTAGAGGACTGGGGGATCGTGTCAATG (SEQ ID NO: 470), TAGAGGACTGGGGAATCGTGTCTATG (SEQ ID NO: 471), TAGAGGACTGGGGAATGGTATCGATG (SEQ ID NO: 472), GACTCCTAGAGGACTGGGGGATAGTGTCAATA (SEQ ID NO: 473), TAGAGGACTGGGGAATGGTGTCTATA (SEQ ID NO: 474), 33 / 189 B1195.70194WO00 13324217.2TAGAGGACTGGGGAATGGTATCTATA (SEQ ID NO: 475), TAGAGGACTGGGGAATAGTGTCTATG (SEQ ID NO: 476), CCTAGAGGACTGGGGTATCGTATCAATA (SEQ ID NO: 477), TAGAGGACTGGGGAATAGTGTCTATA (SEQ ID NO: 478), TCCTAGAGGACTGGGGGATGGTGTCGATA (SEQ ID NO: 479), TAGAGGACTGGGGAATCGTATCTATA (SEQ ID NO: 480), CTCCTAGAGGACTGGGGGATCGTATCGATG (SEQ ID NO: 481), TCCTAGAGGACTGGGGGATGGTGTCGATG (SEQ ID NO: 482), TAGAGGACTGGGGAATAGTATCTATG (SEQ ID NO: 483), TAGAGGACTGGGGAATGGTGTCTATG (SEQ ID NO: 484), CTCCTAGAGGACTGGGGGATTGTATCAATA (SEQ ID NO: 485), GACTCCTAGAGGACTGGGGGATAGTGTCGATG (SEQ ID NO: 486), TCCTAGAGGACTGGGGTATCGTATCGATA (SEQ ID NO: 487), CTCCTAGAGGACTGGGGGATAGTATCGATA (SEQ ID NO: 488), CTCCTAGAGGACTGGGGGATCGTATCAATG (SEQ ID NO: 489), CTCCTAGAGGACTGGGGGATTGTATCAATG (SEQ ID NO: 490), CTCCTAGAGGACTGGGGGATGGTATCGATA (SEQ ID NO: 491), GACTCCTAGAGGACTGGGGGATAGTGTCGATA (SEQ ID NO: 492), CTCCTAGAGGACTGGGGGATGGTATCGATG (SEQ ID NO: 493), CTCCTAGAGGACTGGGGGATAGTATCGATG (SEQ ID NO: 494), TAGAGGACTGGGGAATGGTATCTATG (SEQ ID NO: 495), TCCTAGAGGACTGGGGTATCGTGTCGATA (SEQ ID NO: 496), CTCCTAGAGGACTGGGGGATTGTATCGATA (SEQ ID NO: 497), CTCCTAGAGGACTGGGGGATCGTATCTATA (SEQ ID NO: 498), CTCCTAGAGGACTGGGGGATTGTATCGATG (SEQ ID NO: 499), TAGAGGACTGGGGAATCGTATCTATG (SEQ ID NO: 500), CCTAGAGGACTGGGGGATTGTGTCAATG (SEQ ID NO: 501), CCTAGAGGACTGGGGTATGGTATCGATA (SEQ ID NO: 502), CCTAGAGGACTGGGGTATTGTGTCAATA (SEQ ID NO: 503), CTCCTAGAGGACTGGGGGATGGTATCTATA (SEQ ID NO: 504), TCCTAGAGGACTGGGGTATCGTGTCGATG (SEQ ID NO: 505), CTCCTAGAGGACTGGGGTATGGTATCAATA (SEQ ID NO: 506), GACTCCTAGAGGACTGGGGGATCGTGTCTATA (SEQ ID NO: 507), 34 / 189 B1195.70194WO00 13324217.2CCTAGAGGACTGGGGGATTGTGTCAATA (SEQ ID NO: 508), CCTAGAGGACTGGGGGATTGTGTCGATG (SEQ ID NO: 509), GACTCCTAGAGGACTGGGGGATCGTGTCTATG (SEQ ID NO: 510), CTCCTAGAGGACTGGGGGATGGTATCTATG (SEQ ID NO: 511), CTCCTAGAGGACTGGGGGATAGTATCTATA (SEQ ID NO: 512), CCTAGAGGACTGGGGTATTGTGTCAATG (SEQ ID NO: 513), TCCTAGAGGACTGGGGTATAGTGTCAATA (SEQ ID NO: 514), CCTAGAGGACTGGGGGATTGTGTCGATA (SEQ ID NO: 515), GACTCCTAGAGGACTGGGGTATAGTATCAATA (SEQ ID NO: 516), TCCTAGAGGACTGGGGTATCGTATCGATG (SEQ ID NO: 517), CCTAGAGGACTGGGGTATGGTATCGATG (SEQ ID NO: 518), GACTCCTAGAGGACTGGGGGATGGTGTCTATG (SEQ ID NO: 519), CTCCTAGAGGACTGGGGGATCGTATCTATG (SEQ ID NO: 520), GACTCCTAGAGGACTGGGGGATGGTGTCTATA (SEQ ID NO: 521), CTCCTAGAGGACTGGGGGATAGTATCTATG (SEQ ID NO: 522), TCCTAGAGGACTGGGGTATAGTGTCAATG (SEQ ID NO: 523), GACTCCTAGAGGACTGGGGGATAGTGTCTATG (SEQ ID NO: 524), GACTCCTAGAGGACTGGGGTATAGTATCAATG (SEQ ID NO: 525), TCCTAGAGGACTGGGGTATCGTGTCAATG (SEQ ID NO: 526), GACTCCTAGAGGACTGGGGGATAGTGTCTATA (SEQ ID NO: 527), CCTAGAGGACTGGGGTATCGTATCAATG (SEQ ID NO: 528), CTCCTAGAGGACTGGGGTATGGTATCAATG (SEQ ID NO: 529), CTCCTAGAGGACTGGGGGATTGTATCTATA (SEQ ID NO: 530), GACTCCTAGAGGACTGGGGTATAGTATCGATA (SEQ ID NO: 531), TCCTAGAGGACTGGGGTATTGTGTCGATG (SEQ ID NO: 532), CCTAGAGGACTGGGGTATTGTATCAATA (SEQ ID NO: 533), TCCTAGAGGACTGGGGTATTGTGTCGATA (SEQ ID NO: 534), CTCCTAGAGGACTGGGGGATTGTATCTATG (SEQ ID NO: 535), GACTCCTAGAGGACTGGGGTATGGTGTCAATA (SEQ ID NO: 536), TCCTAGAGGACTGGGGTATAGTGTCGATA (SEQ ID NO: 537), CCTAGAGGACTGGGGGATTGTGTCTATG (SEQ ID NO: 538), GACTCCTAGAGGACTGGGGTATAGTATCGATG (SEQ ID NO: 539), CCTAGAGGACTGGGGTATTGTATCGATA (SEQ ID NO: 540), 35 / 189 B1195.70194WO00 13324217.2CCTAGAGGACTGGGGGATTGTGTCTATA (SEQ ID NO: 541), CCTAGAGGACTGGGGTATTGTATCAATG (SEQ ID NO: 542), TCCTAGAGGACTGGGGTATAGTGTCGATG (SEQ ID NO: 543), GACTCCTAGAGGACTGGGGTATGGTGTCAATG (SEQ ID NO: 544), CTCCTAGAGGACTGGGGTATGGTGTCGATA (SEQ ID NO: 545), CCTAGAGGACTGGGGTATTGTATCGATG (SEQ ID NO: 546), TCCTAGAGGACTGGGGTATCGTGTCTATA (SEQ ID NO: 547), TCCTAGAGGACTGGGGTATCGTATCTATA (SEQ ID NO: 548), CTCCTAGAGGACTGGGGTATGGTGTCGATG (SEQ ID NO: 549), TCCTAGAGGACTGGGGTATAGTGTCTATA (SEQ ID NO: 550), CCTAGAGGACTGGGGTATGGTATCTATA (SEQ ID NO: 551), TCCTAGAGGACTGGGGTATAGTGTCTATG (SEQ ID NO: 552), TCCTAGAGGACTGGGGTATCGTGTCTATG (SEQ ID NO: 553), CCTAGAGGACTGGGGTATTGTGTCTATG (SEQ ID NO: 554), CCTAGAGGACTGGGGTATTGTATCTATA (SEQ ID NO: 555), CCTAGAGGACTGGGGTATTGTGTCTATA (SEQ ID NO: 556), CTCCTAGAGGACTGGGGTATAGTATCTATA (SEQ ID NO: 557), CCTAGAGGACTGGGGTATGGTGTCTATA (SEQ ID NO: 558), TCCTAGAGGACTGGGGTATGGTATCTATG (SEQ ID NO: 559), CCTAGAGGACTGGGGTATTGTATCTATG (SEQ ID NO: 560), CCTAGAGGACTGGGGTATGGTGTCTATG (SEQ ID NO: 561), CTCCTAGAGGACTGGGGTATAGTATCTATG (SEQ ID NO: 562), or TCCTAGAGGACTGGGGTATCGTATCTATG (SEQ ID NO: 563), 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 any one of SEQ ID NOs: 404-563, or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to any one of SEQ ID NOs: 404-563.

[0130] In some embodiments, the pegRNAs of the present disclosure comprise a primer binding site (PBS) of the sequence TTCCCCAGT, TTCCCCAGTC (SEQ ID NO: 29), TTCCCCAGTCC (SEQ ID NO: 30), TTCCCCAGTCCT (SEQ ID NO: 31), TTCCCCAGTCCTC (SEQ ID NO: 32), TTCCCCAGTCCTCC (SEQ ID NO: 33), TTCCCCAGTCCTCCC (SEQ ID NO: 34), TAGCTATGC, TAGCTATGCC (SEQ ID NO: 35), TAGCTATGCCG (SEQ ID NO: 36), TAGCTATGCCGA (SEQ ID NO: 37), 36 / 189 B1195.70194WO00 13324217.2TAGCTATGCCGAC (SEQ ID NO: 38), or TAGCTATGCCGACT (SEQ ID NO: 6),, 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 TTCCCCAGT, TTCCCCAGTC (SEQ ID NO: 29), TTCCCCAGTCC (SEQ ID NO: 30), TTCCCCAGTCCT (SEQ ID NO: 31), TTCCCCAGTCCTC (SEQ ID NO: 32), TTCCCCAGTCCTCC (SEQ ID NO: 33), TTCCCCAGTCCTCCC (SEQ ID NO: 34), TAGCTATGC, TAGCTATGCC (SEQ ID NO: 35), TAGCTATGCCG (SEQ ID NO: 36), TAGCTATGCCGA (SEQ ID NO: 37), TAGCTATGCCGAC (SEQ ID NO: 38), or TAGCTATGCCGACT (SEQ ID NO: 6), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to TTCCCCAGT, TTCCCCAGTC (SEQ ID NO: 29), TTCCCCAGTCC (SEQ ID NO: 30), TTCCCCAGTCCT (SEQ ID NO: 31), TTCCCCAGTCCTC (SEQ ID NO: 32), TTCCCCAGTCCTCC (SEQ ID NO: 33), TTCCCCAGTCCTCCC (SEQ ID NO: 34), TAGCTATGC, TAGCTATGCC (SEQ ID NO: 35), TAGCTATGCCG (SEQ ID NO: 36), TAGCTATGCCGA (SEQ ID NO: 37), TAGCTATGCCGAC (SEQ ID NO: 38), or TAGCTATGCCGACT (SEQ ID NO: 6).

[0131] In some embodiments, the pegRNAs of the present disclosure comprise a 3' structured motif of the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 14), 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 CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 14), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 14).

[0132] In some embodiments, the pegRNAs of the present disclosure comprise a terminator sequence of TTTTTT, 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 TTTTTT, or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to TTTTTT. In some embodiments, the pegRNAs comprise 0, 1, 2, 3, 4, 5, or 6 T’s of the terminator sequence.

[0133] In some embodiments, the pegRNAs of the present disclosure comprise the structure 5'-[spacer]-[gRNA scaffold]-[RTT]-[PBS]-[3' motif]-[terminator]- 3'. In some embodiments, the pegRNAs of the present disclosure comprise the structure 5'-[spacer]-[gRNA scaffold]- [RTT]-[PBS]-[3' motif]- 3'. 37 / 189 B1195.70194WO00 13324217.2

[0134] In another aspect, the present disclosure provides compositions comprising any of the pegRNAs provided herein. In some embodiments, the composition further comprises a nicking guide RNA (ngRNA). In some embodiments, the ngRNA comprises a spacer sequence of GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13), or a spacer 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 GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13). In certain embodiments, the ngRNA comprises the sequence of any one of the ngRNAs provided in Table 3 below, 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 any one of the ngRNAs provided in Table 3 below.

[0135] In some embodiments, the ngRNA comprises a gRNA scaffold sequence of GTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 578), 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 GTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 578), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 578).

[0136] In some embodiments, the ngRNA comprises a terminator sequence of TTTTTT, 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 TTTTTT, or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to TTTTTT.

[0137] In some embodiments, the ngRNA comprises the structure 5'-[spacer]-[gRNA scaffold]-[terminator]- 3'. 38 / 189 B1195.70194WO00 13324217.2

[0138] In another aspect, the present disclosure provides pegRNAs for correcting a c.826-1 G-to-A point mutation in a CDKL5 gene by prime editing. In some embodiments, the pegRNA comprises a spacer sequence of GCTGTCAAGTATCTGTCAGCT (SEQ ID NO: 7), or a spacer 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 GCTGTCAAGTATCTGTCAGCT (SEQ ID NO: 7), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCTGTCAAGTATCTGTCAGCT (SEQ ID NO: 7). In certain embodiments, the pegRNA comprises the sequence GCTGTCAAGTATCTGTCAGCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATTTCCTAAGAAC CTGTTGAAGTTGGACCCAGCTGACAGATACTCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 39), 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 GCTGTCAAGTATCTGTCAGCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATTTCCTAAGAAC CTGTTGAAGTTGGACCCAGCTGACAGATACTCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 39), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCTGTCAAGTATCTGTCAGCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATTTCCTAAGAAC CTGTTGAAGTTGGACCCAGCTGACAGATACTCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 39).

[0139] In another aspect, the present disclosure provides nicking guide RNAs for use in correcting a c.826-1 G-to-A point mutation in a CDKL5 gene by prime editing. In some embodiments, the nicking guide RNA comprises the sequence GATTTCAAACCCAGAGACTTC (SEQ ID NO: 15), GTTCTGGATCGTTCTCCTTCA (SEQ ID NO: 16), or GTACTGAAGTTGGACCCAGCT (SEQ ID NO: 17), 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 GATTTCAAACCCAGAGACTTC (SEQ ID NO: 15), GTTCTGGATCGTTCTCCTTCA (SEQ ID NO: 16), or GTACTGAAGTTGGACCCAGCT (SEQ ID NO: 17), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GATTTCAAACCCAGAGACTTC (SEQ ID NO: 15), 39 / 189 B1195.70194WO00 13324217.2GTTCTGGATCGTTCTCCTTCA (SEQ ID NO: 16), or GTACTGAAGTTGGACCCAGCT (SEQ ID NO: 17).

[0140] In another aspect, the present disclosure provides pegRNAs for correcting a cluster of mutations in exon 8 of the CDKL5 gene by prime editing. In some embodiments, the pegRNA comprises a spacer sequence of CAGAGTACGTTGCCACCAGA (SEQ ID NO: 8), or a spacer 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 CAGAGTACGTTGCCACCAGA (SEQ ID NO: 8), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to CAGAGTACGTTGCCACCAGA (SEQ ID NO: 8). In certain embodiments, the pegRNA comprises the sequence CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGTAAGGGCTCT AGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTTAAAC CAACTAGAATTTTTTT (SEQ ID NO: 40) or CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCACCCAAGTAAGG GCTCTAGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 41), 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 CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGTAAGGGCTCT AGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTTAAAC CAACTAGAATTTTTTT (SEQ ID NO: 40) or CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCACCCAAGTAAGG GCTCTAGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 41), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGTAAGGGCTCT AGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTTAAAC CAACTAGAATTTTTTT (SEQ ID NO: 40) or 40 / 189 B1195.70194WO00 13324217.2CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCACCCAAGTAAGG GCTCTAGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 41).

[0141] In another aspect, the present disclosure provides nicking guide RNAs for correcting a cluster of mutations in exon 8 of the CDKL5 gene by prime editing. In some embodiments, the nicking guide RNA comprises the sequence GTGGAAATGTCATTCTATTTT (SEQ ID NO: 18), 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 GTGGAAATGTCATTCTATTTT (SEQ ID NO: 18), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GTGGAAATGTCATTCTATTTT (SEQ ID NO: 18).

[0142] In another aspect, the present disclosure provides systems comprising a pegRNA, prime editor, and ngRNA described herein.

[0143] In another aspect, the present disclosure provides one or more polynucleotides encoding any of the pegRNAs and / or ngRNAs provided herein. In some embodiments, the one or more polynucleotides encode any of the prime editors provided herein.

[0144] In another aspect, the present disclosure provides one or more vectors comprising the one or more polynucleotides provided herein. In some embodiments, the vectors are AAV vectors. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9. In certain embodiments, the AAV is AAV9.

[0145] In another aspect, the present disclosure provides compositions comprising any of the polynucleotides and / or vectors provided herein.

[0146] In another aspect, the present disclosure provides one or more AAV particles comprising any of the pegRNAs, compositions, polynucleotides, and / or vectors provided herein. The use of AAVs for delivering gene editing agents, including prime editors and pegRNAs, is described, for example, in Davis et al., Efficient Prime Editing in Mouse Brain, Liver and Heart with Dual AAVs. Biotechnol.42, 253-264 (2024), which is incorporated herein by reference.

[0147] In another aspect, the present disclosure provides virus-like particles (VLPs) comprising any of the pegRNAs, compositions, polynucleotides and / or vectors provided herein. The use of VLPs for the delivery of gene editing agents, including prime editors and pegRNAs, is described, for example, in International Patent Application Publication No. WO 2023 / 102537, published June 8, 2023; International Patent Application Publication No. WO 41 / 189 B1195.70194WO00 13324217.22023 / 102538, published June 8, 2023; International Patent Application Publication No. WO 2023 / 102550, published June 8, 2023; International Patent Application Publication No. 2024 / 215652, published October 17, 2024; International Patent Application No. PCT / US2024 / 032867, filed June 6, 2024; Banskota et al., Engineered Virus-Like Particles for Efficient In Vivo Delivery of Therapeutic Proteins. Cell 185, 250-265, (2022); and An et al., Engineered Virus-Like Particles for Transient Delivery of Prime Editor Ribonucleoprotein Complexes In Vivo. Nat. Biotechnol.42(10): 1526–1537 (2024); the contents of which are incorporated by reference herein.

[0148] In another aspect, the present disclosure provides pharmaceutical compositions comprising any of the pegRNAs, compositions, polynucleotides, vectors, AAV particles, or VLPs provided herein, and a pharmaceutically acceptable excipient.

[0149] In another aspect, the present disclosure provides cells comprising any of the pegRNAs, compositions, polynucleotides, vectors, AAV particles, or VLPs provided herein. In another aspect, the present disclosure provides kits comprising any of the pegRNAs, compositions, polynucleotides, vectors, AAV particles, or VLPs provided herein. Prime Editors

[0150] Any of the compositions, systems, or prime editing complexes provided herein may comprise a prime editor. In some embodiments, a composition comprises a prime editor. In some embodiments, a system comprises a prime editor. In some embodiments, the prime editor comprises (i) a napDNAbp domain, and (ii) a polymerase domain. In certain embodiments, the napDNAbp domain has a nickase activity. In some embodiments, the napDNAbp domain is a Cas9 protein or a variant thereof (e.g., Cas9-NRCH, or a variant thereof). In certain embodiments, the napDNAbp domain is a Cas9 nickase (nCas9). In certain embodiments, the napDNAbp domain comprises a sequence of any one of the napDNAbps provided herein, 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 any one of the napDNAbps provided herein. In some embodiments, the polymerase domain is a reverse transcriptase domain. In certain embodiments, the polymerase domain comprises a sequence of any one of the polymerases provided herein, 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 any one of the polymerases provided herein. In some embodiments, the Cas9 domain (e.g. a Cas9 nickase) and the polymerase domain (e.g., a reverse transcriptase) are a translationally fused, i.e., form a fusion protein. In other embodiments, the Cas9 domain 42 / 189 B1195.70194WO00 13324217.2(e.g., a Cas9 nickase) and the polymerase domain (e.g., a reverse transcriptase) are not translationally fused, i.e., are provided in trans. In such embodiments, the different components may be provided in trans by introduction of separate expression constructs whereby each component is independently expressed. In some embodiments, the independently expressed components each may further comprise a localization or assembly domain so that the components as assembled to form a functional complex through the coupling of the localization or assembly domains.

[0151] In some embodiments, any of the following prime editor may be used in the present disclosure: PE1

[0152] As used herein, “PE1” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) nickase 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: MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGN TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAK VDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFD LAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEI TKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYV TEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVE DRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNF MQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLV SDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYD VRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIV WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDW DPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDF LEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF LYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV LSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDAT LIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTL NIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQY PMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVED IHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLT 43 / 189 B1195.70194WO00 13324217.2WTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALL QTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREF LGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTK PFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAG KLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTET EVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSE GKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTS TLLIENSSPSGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 347) KEY: NUCLEAR LOCALIZATION SEQUENCE (NLS) CAS9(H840A) 33-AMINO ACID LINKER M-MLV reverse transcriptase PE2

[0153] As used herein, “PE2” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) nickase 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: MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGN TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAK VDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFD LAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEI TKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYV TEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVE DRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNF MQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLV SDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYD VRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIV WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDW DPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDF LEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF LYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV LSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDAT 44 / 189 B1195.70194WO00 13324217.2LIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTL NIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQY PMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVED IHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLT WTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALL QTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREF LGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTK PFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAG KLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTET EVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTS EGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDT STLLIENSSPSGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 348) KEY: NUCLEAR LOCALIZATION SEQUENCE (NLS) CAS9(H840A) 33-AMINO ACID LINKER

[0154] M-MLV reverse transcriptase PE3

[0155] As used herein, “PE3” refers to PE2 plus a second-strand nicking guide RNA (ngRNA) that complexes with the PE2 and introduces a nick in the non-edited DNA strand in order to induce preferential replacement of the edited strand. PE3b

[0156] As used herein, “PE3b” refers to PE3 but wherein the second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing a gRNA with a spacer sequence that matches only the edited strand, but not the original allele. Using this strategy, referred to hereafter as PE3b, mismatches between the protospacer and the unedited allele should disfavor nicking by the sgRNA until after the editing event on the PAM strand takes place. PE4

[0157] 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 45 / 189 B1195.70194WO00 13324217.2comprising a PE2 protein and an MLH1 dominant negative protein joined via an optional linker.

[0158] The amino acid sequence of MLH1dn is:MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLK LIQIQDNGTGIRKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITT KTADGKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKALKNPSEEY GKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFGNAVSRELIEIG CEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHP FLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSSRMYFTQTLL PGLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLSS QPQAIVTEDKTDISSGRARQQDEEMLELPAPAEVAAKNQSLEGDTTKGTSEMSEKRG PTSSNPRKRHREDSDVEMVEDDSRKEMTAACTPRRRIINLTSVLSLQEEINEQGHEVL REMLHNHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFGVLRLSE PAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGN LIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISEES TLSGQQSEVPGSIPNSWKWTVEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVF (SEQ ID NO: 351) PE5 and PE5b

[0159] 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

[0160] The term “PE6” refers to a suite of next-generation prime editors described herein (PE6a, PE6b, PE6c, PE6d, PE6e, PE6f, and PE6g) comprising improved reverse transcriptase 46 / 189 B1195.70194WO00 13324217.2and / or Cas9 variants. PE6a, PE6b, PE6c, PE6d, PE6e, PE6f, and PE6g were described in Doman et al., “Phage-assisted evolution and protein engineering yield compact, efficient prime editors,” Cell, 2023 Aug 31; 186(18): 3983-4002, and International PCT Application PCT / US2024 / 030786, each of which is incorporated herein by reference. The improved reverse transcriptase and Cas9 domains of the PE6 variants can also be combined with each other to offer cumulative benefits. For example, a PE6 prime editor comprising an improved reverse transcriptase variant of PE6a and an improved Cas9 variant of PE6e is referred to herein as the prime editor “PE6a-e” (or “PE6e-a”). Any possible combination of PE6 prime editors is contemplated by the present disclosure including, for example, PE6a-e, PE6a-f, PE6a-g, PE6b-e, PE6b-f, PE6b-g, PE6c-e, PE6c-f, PE6c-g, PE6d-e, PE6d-f, and PE6d-g.

[0161] Any of the PE6 prime editors provided herein may also comprise the architecture of the PEmax protein as provided below. 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 below.

[0162] 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: 572).

[0163] 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 47 / 189 B1195.70194WO00 13324217.2GVFEYLVMPYGISTAPAHFQYFINTILGEAKESHVVCYMDDILIHSKSESEHVKHVKD VLQKLKNANLIINQAKCEFHQSQVKFIGYHISEKGLTPCQENIDKVLQWKQPKNRKE LRQFLGSVNYLRKFIPKTSQLTHPLNKLLKKDVRWKWTPTQTQAIENIKQCLVSPPVL RHFDFSKKILLETDVSDVAVGAVLSQKHDDDKYYPVGYYSAKMSKAQLNYSVSDK EMLAIIKSLEHWRHYLESTIEPFKILTDHRNLIGRITNESEPENKRLARWQLFLQDFNF EINYRPGSANHIADALSRIVDETEPIPKDNEDNSINFVNQISI (SEQ ID NO: 573).

[0164] 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: ISSSKHTLSQMNKVSNIVKEPELPDIYKEFKDITADTNTEKLPKPIKGLEFEVELTQEN YRLPIRNYPLTPVKMQAMNDEINQGLKGGIIRESKAINACPVIFVPRKEGTLRMVVDY RPLNKYVKPNVYPLPLIEQLLAKIQGSTIFTKLDLKSAYHQIRVRKGDEHKLAFRCPR GVFEYLVMPYGIKTAPAHFQYFINTILGEAKESHVVCYMDDILIHSKSESEHVKHVKD VLQKLKNANLIINQAKCEFHQSQVKFLGYHISEKGLTPCQENIDKVLQWKQPKNQKE LRQFLGQVNYLRKFIPKTSQLTHPLNKLLKKDVRWKWTPTQTQAIENIKQCLVSPPV LRHFDFSKKILLETDVSDVAVGAVLSQKHDDDKYYPVGYYSAKMSKAQLNYSVSD KEMLAIIKSLEHWRHYLESTIEPFKILTDHRNLIGRITNESEPENKRLARWQLFLQDFN FEINYRPGSANHIADALSRIVDETEPIPKDNEDNSINFVNQISI (SEQ ID NO: 574).

[0165] 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 48 / 189 B1195.70194WO00 13324217.2DPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSN ARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLD (SEQ ID NO: 575).

[0166] 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 KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR TFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFA WMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK VMGRHKPENIVIEMARENQTTQKGQRNSRERMKRIEEGIKELGSQILKEHPVENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIA RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 352).

[0167] 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 49 / 189 B1195.70194WO00 13324217.285%, 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 KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR TFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFA WMTRKSEKTITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMVEER LKTYAHLFDNKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR NFMQLIHDDSLTFKEDIQKAQVSGQGDSLYEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQ LQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSD KNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFI ARQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYK VREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLV VAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 353).

[0168] 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 50 / 189 B1195.70194WO00 13324217.2RHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR TFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFA WMTRKSEKTITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMVEER LKTYAHLFDNKVMKQLKRCRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR NFMQLIHDDSLTFKEDIQKAQVSGQGDSLYEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQ LQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSD KNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFI KRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYK VREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLV VAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 354).

[0169] In some embodiments, a PE6 protein comprises a reverse transcriptase variant disclosed herein and a Cas9 protein that recognizes a non-canonical PAM sequence, e.g., Cas9-NRCH of the following amino acid sequence, or 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 DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRKLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA 51 / 189 B1195.70194WO00 13324217.2DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLRKQR TFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFA WMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK VMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESILPKGNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVV AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE LENGRKRMLASAGVLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTINRKQYNTTKEVLDATLIRQSITGLYETRIDLSQLGGD (SEQ ID NO: 349).

[0170] For example, the prime editor “PE6b-NRCH” comprises the reverse transcriptase of PE6b and the NRCH-Cas9 protein. PE6b-NRCH comprises the amino acid sequence: MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNT DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDS FFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDE HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD GTEELLVKLKREDLLRKQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKIEKI LTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI 52 / 189 B1195.70194WO00 13324217.2LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRD KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG SPAIKKGILQTVKVVDELVKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVP QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDKLIARKKDWDP KKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFKYFDTTINRKQYNTTKEVLDATLIRQSITGLYETRI DLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSISSSKHTLSQMNKVS NIVKEPELPDIYKEFKDITADTNTEKLPKPIKGLEFEVELTQENYRLPIRNYPLTPVKM QAMNDEINQGLKGGIIRESKAINACPVIFVPRKEGTLRMVVDYRPLNKYVKPNVYPL PLIEQLLAKIQGSTIFTKLDLKSAYHQIRVRKGDEHKLAFRCPRGVFEYLVMPYGISTA PAHFQYFINTILGEAKESHVVCYMDDILIHSKSESEHVKHVKDVLQKLKNANLIINQA KCEFHQSQVKFIGYHISEKGLTPCQENIDKVLQWKQPKNRKELRQFLGSVNYLRKFIP KTSQLTHPLNKLLKKDVRWKWTPTQTQAIENIKQCLVSPPVLRHFDFSKKILLETDVS DVAVGAVLSQKHDDDKYYPVGYYSAKMSKAQLNYSVSDKEMLAIIKSLEHWRHYL ESTIEPFKILTDHRNLIGRITNESEPENKRLARWQLFLQDFNFEINYRPGSANHIADALS RIVDETEPIPKDNEDNSINFVNQISIKRTADGSEFESPKKKRKVPAAKRVKLD (SEQ ID NO: 355).

[0171] The prime editor “PE6c-NRCH” comprises the reverse transcriptase of PE6c and the NRCH-Cas9 protein. The NRCH-Cas9 protein comprises the amino acid sequence: MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHE RHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRKLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDEHHQDLTLLKALVRQQLPE 53 / 189 B1195.70194WO00 13324217.2KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLRKQR TFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFA WMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK VMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESILPKGNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVV AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE LENGRKRMLASAGVLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTINRKQYNTTKEVLDATLIRQSITGLYETRIDLSQLGGD (SEQ ID NO: 349) PE6c-NRCH comprises the amino acid sequence: MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNT DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDS FFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDE HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD GTEELLVKLKREDLLRKQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKIEKI LTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRD KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG 54 / 189 B1195.70194WO00 13324217.2SPAIKKGILQTVKVVDELVKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVP QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDKLIARKKDWDP KKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFKYFDTTINRKQYNTTKEVLDATLIRQSITGLYETRI DLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSISSSKHTLSQMNKVS NIVKEPELPDIYKEFKDITADTNTEKLPKPIKGLEFEVELTQENYRLPIRNYPLTPVKM QAMNDEINQGLKGGIIRESKAINACPVIFVPRKEGTLRMVVDYRPLNKYVKPNVYPL PLIEQLLAKIQGSTIFTKLDLKSAYHQIRVRKGDEHKLAFRCPRGVFEYLVMPYGIKT APAHFQYFINTILGEAKESHVVCYMDDILIHSKSESEHVKHVKDVLQKLKNANLIINQ AKCEFHQSQVKFLGYHISEKGLTPCQENIDKVLQWKQPKNQKELRQFLGQVNYLRK FIPKTSQLTHPLNKLLKKDVRWKWTPTQTQAIENIKQCLVSPPVLRHFDFSKKILLET DVSDVAVGAVLSQKHDDDKYYPVGYYSAKMSKAQLNYSVSDKEMLAIIKSLEHWR HYLESTIEPFKILTDHRNLIGRITNESEPENKRLARWQLFLQDFNFEINYRPGSANHIAD ALSRIVDETEPIPKDNEDNSINFVNQISIKRTADGSEFESPKKKRKVPAAKRVKLD (SEQ ID NO: 356).

[0172] The prime editor “PE6d-NRCH” comprises the reverse transcriptase of PE6d (or the non-truncated version of the MMLV reverse transcriptase variant of PE6d) and the NRCH- Cas9 protein. PE6d-NRCH comprises the amino acid sequence: MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNT DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDS FFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDE HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD GTEELLVKLKREDLLRKQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKIEKI 55 / 189 B1195.70194WO00 13324217.2LTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRD KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG SPAIKKGILQTVKVVDELVKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVP QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDKLIARKKDWDP KKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFKYFDTTINRKQYNTTKEVLDATLIRQSITGLYETRI DLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSTLNIEDEYRLHETSKE PDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGI KPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPNVPN PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRL PQGFKNSPTLFCEALHRDLADFRIQHPDLILLQYYDDLLLAATSELDCQQGTRALLQT LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLR EFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALG LPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMV AAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDSGGSKRTADGSEFESPKKKRKVPAAKRV KLD (SEQ ID NO: 357); or the amino acid sequence (which comprises a C-terminally truncated reverse transcriptase domain): MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNT DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDS FFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT 56 / 189 B1195.70194WO00 13324217.2YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDE HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD GTEELLVKLKREDLLRKQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKIEKI LTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRD KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG SPAIKKGILQTVKVVDELVKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVP QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDKLIARKKDWDP KKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFKYFDTTINRKQYNTTKEVLDATLIRQSITGLYETRI DLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSTLNIEDEYRLHETSKE PDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGI KPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPFVPNP YNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLP QGFKNSPTLFNEALHRDLADFRIQHPDLILLQYMDDLLLAATSELDCQQGTRALLQT LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLR EFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALG LPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMV AAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDG SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVY TDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQK GHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFESPKKKR KVPAAKRVKLD (SEQ ID NO: 358). 57 / 189 B1195.70194WO00 13324217.2PE7

[0173] 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

[0174] 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: MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLG NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMA KVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDST DKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI NASGVDAKAILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYID GGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLRKQRTFDNGSIPHQIHLGE LHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKV KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEIS GVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA NRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKV VDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKD DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVY GDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIET NGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKL IARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYT STKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKR KVSGGSSGGSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQA PLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGT NDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPT SQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQ YVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEG QRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTL FNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWR 58 / 189 B1195.70194WO00 13324217.2RPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALV KQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILA EAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGT SAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKN KDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL IENSSPSGGSKRTADGSEFESPKKKRKVGSGPAAKRVKLD (SEQ ID NO: 350) 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 BIPARTITE SV40NLS Other linker sequence c-Myc NLS

[0175] In some embodiments, a PEmax protein is a PE4max protein. In some embodiments, a PE4max protein comprises a Cas9 protein that recognizes a non-canonical PAM sequence (e.g., Cas9-NRCH). In some embodiments, PE4max-NRCH comprises the amino acid sequence:MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFK VLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMA KVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKA DLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD AKAILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVK RYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILE KMDGTEELLVKLKREDLLRKQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREK IEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTN FDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGI RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL AGSPAIKKGILQTVKVVDELVKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKR IEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDA IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREV 59 / 189 B1195.70194WO00 13324217.2KVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVY GDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGE TGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDKLIARKKD WDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL EAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNELALPSKYVNFLYLA SHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTINRKQYNTTKEVLDATLIRQSITGLY ETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSTLNIEDEYRLHE TSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQE ARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHP TVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLT WTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTR ALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT PRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLT APALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPC LRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALL LDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTW YTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKK LNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCP GHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPKRTADGSEFESPKKKR KVPAAKRVKLD (SEQ ID NO: 359); or the amino acid sequence (which comprises a C-terminally truncated reverse transcriptase domain):

[0176] PEmax∆RNaseH-NRCH:

[0177] MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKV LGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAK VDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA KAILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKR YDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK MDGTEELLVKLKREDLLRKQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKI EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNF 60 / 189 B1195.70194WO00 13324217.2DKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGI RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL AGSPAIKKGILQTVKVVDELVKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKR IEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDA IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREV KVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVY GDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGE TGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDKLIARKKD WDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL EAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNELALPSKYVNFLYLA SHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTINRKQYNTTKEVLDATLIRQSITGLY ETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSTLNIEDEYRLHE TSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQE ARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHP TVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLT WTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTR ALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT PRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLT APALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPC LRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALL LDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDKRTADGSEFESPKKKRKVPAAKR VKLD (SEQ ID NO: 360)

[0178] In some embodiments, the prime editor is PE2, PE3, PE4, PE5, or a variant thereof (including PE2max, PE3max, PE4max, and PE5max). In certain embodiments, the prime editor is PE4max-NRCH, or a variant thereof. In some embodiments, the prime editor is a PE6 prime editor, or a variant thereof. In certain embodiments, the prime editor is PE6b- NRCH, PE6c-NRCH, PE6d-NRCH, or a variant thereof.

[0179] Additional prime editors known in the art may also be used in the present disclosure. Such prime editors include, but are not limited to, the following: 61 / 189 B1195.70194WO00 13324217.2Other Prime Editors

[0180] A prime editor also includes any modified prime editor that is available to the public, e.g., as published in scientific literature or in patent literature.

[0181] Prime editors and / or pegRNAs are described in the following literature and may be implemented in the disclosed methods and compositions. The content of each reference is incorporated herein by reference.62 / 189 B1195.70194WO00 13324217.263 / 189 B1195.70194WO00 13324217.264 / 189 B1195.70194WO00 13324217.265 / 189 B1195.70194WO00 13324217.266 / 189 B1195.70194WO00 13324217.267 / 189 B1195.70194WO00 13324217.268 / 189 B1195.70194WO00 13324217.269 / 189 B1195.70194WO00 13324217.270 / 189 B1195.70194WO00 13324217.2

[0182] In addition, prime editors contemplated herein may include any improved version of a prime editor and / or pegRNA, for example, those described in the following references (each of which is incorporated by reference): (1) Truong et al., “Exonuclease-enhanced prime editors,” Nature Methods, February 1, 2024, 21: 455-464 (which describes an exonuclease- recruiting prime editor called Exo-PE which facilitates removal of the 5′ end endogenous flap); (2) Pengpeng Liu et al., “Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice,” Nature Communications, April 9, 2021, 12, 2121 (2021) (which describes a NLS-optimized SpCas9-based prime editor named PE2* that improves genome editing efficiency in both fluorescent reporter cells and at endogenous loci in cultured cell lines); (3) Ferreira da Silva, J., Oliveira, G.P., Arasa-Verge, E.A. et al., Prime 71 / 189 B1195.70194WO00 13324217.2editing efficiency and fidelity are enhanced in the absence of mismatch repair. Nat Commun 13, 760 (2022) (which describes cell-based work showing that the ablation of mismatch repair (MMR) affords a 2–17 fold increase in PE efficiency in modified cells); (4) Chen PJ, Hussmann JA, Yan J, Knipping F, Ravisankar P, Chen PF, Chen C, Nelson JW, Newby GA, Sahin M, Osborn MJ, Weissman JS, Adamson B, Liu DR. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell.2021 Oct 28;184(22):5635- 5652 (which developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit / indel ratios by 3.4-fold in MMR- proficient cell types); (5) Yan, J., Oyler-Castrillo, P., Ravisankar, P. et al. Improving prime editing with an endogenous small RNA-binding protein. Nature 628, 639–647 (2024) (which reports the use of the small RNA-binding exonuclease protection factor La to enhance prime editing); (6) Liu, Y., Yang, G., Huang, S. et al. Enhancing prime editing by Csy4-mediated processing of pegRNA. Cell Res 31, 1134–1136 (2021) (which reports on several modifications to pegRNAs and PE to modify editing efficiency); (7) Qian, Y., Wang, D., Niu, W. et al. Development of a highly efficient prime editor system in mice and rabbits. Cell. Mol. Life Sci.80, 346 (2023) (which reports on the development of a prime editor variant referred to as ePE3max which contains various modifications to the pegRNA and PE); (8) Zhang, G., Liu, Y., Huang, S. et al. Enhancement of prime editing via xrRNA motif-joined pegRNA. Nat Commun 13, 1856 (2022) (which describes an engineered PE called “xrPE” that is formed by appending a viral exoribonuclease-resistant RNA motif (xrRNA) to the 3′- extended portion of pegRNAs for their increased resistance against degradation); (9) Chauhan, Vikash P. et al. “Engineered prime editors with minimal genomic errors.” bioRxiv (2024): n. pag. (which describes an engineered prime editor called “vPE” which engineers the Cas9 nickase to reposition prime editor nicks to favor the installation of the 3’ new strands); and (10) Liu, Bin et al. “A split prime editor with untethered reverse transcriptase and circular RNA template.” Nature Biotechnology 40 (2022): 1388 – 1393 (which describes a split PE (sPE) in which the Cas9 nickase (nCas9) remains untethered from the reverse transcriptase (RT) showed similar efficiencies in installing precise edits as the parental unsplit PE3 and no increase in insertion–deletion (indel) byproducts). 72 / 189 B1195.70194WO00 13324217.2

[0183] Prime editors and / or pegRNA are also described in the following patent literature, each of which is incorporated herein by reference and which may be utilized in accordance with the present disclosure.73 / 189 B1195.70194WO00 13324217.274 / 189 B1195.70194WO00 13324217.275 / 189 B1195.70194WO00 13324217.276 / 189 B1195.70194WO00 13324217.277 / 189 B1195.70194WO00 13324217.278 / 189 B1195.70194WO00 13324217.279 / 189 B1195.70194WO00 13324217.280 / 189 B1195.70194WO00 13324217.281 / 189 B1195.70194WO00 13324217.282 / 189 B1195.70194WO00 13324217.2Methods of Treatment and Uses

[0184] In another aspect, the present disclosure provides methods for correcting a pathogenic mutation in the CDKL5 gene by prime editing, including, but not limited to, the correction of a c.1412delA mutation in the CDKL5 gene by prime editing, and therefore treating CDKL5 deficiency disorder. 83 / 189 B1195.70194WO00 13324217.2

[0185] The methods of the present disclosure use a prime editor in combination with a pegRNA (and optionally an ngRNA) to carry out prime editing to directly correct mutations in the CDKL5 gene that cause CDKL5 deficiency disorder. In one aspect, the present disclosure provides methods of correcting a 1412delA mutation in a CDKL5 gene by prime editing comprising contacting a target DNA sequence comprising the CDKL5 gene with a prime editor and a prime editing guide RNA (pegRNA), wherein the pegRNA comprises a spacer sequence of GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5), or TAGCTATGCCGACT (SEQ ID NO: 6), or a spacer 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 GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5), or TAGCTATGCCGACT (SEQ ID NO: 6), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5), or TAGCTATGCCGACT (SEQ ID NO: 6). In certain embodiments, the pegRNA comprises the sequence of any of the pegRNAs provided in Tables 1, 2, and 4 herein, 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 any of the pegRNAs provided in Tables 1, 2, and 4 herein.

[0186] In some embodiments, the contacting further comprises using a nicking guide RNA (ngRNA). In some embodiments, the ngRNA comprises a spacer sequence of GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13), or a spacer 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 GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ 84 / 189 B1195.70194WO00 13324217.2ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13). In certain embodiments, the ngRNA comprises the sequence of any one of any one of the ngRNAs provided in Table 3 herein, 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 any one of the ngRNAs provided in Table 3 herein.

[0187] In some embodiments, the prime editor comprises (i) a napDNAbp domain, and (ii) a polymerase domain. In certain embodiments, the napDNAbp domain has a nickase activity. In some embodiments, the napDNAbp domain is a Cas9 protein or a variant thereof (e.g., Cas9-NRCH). In certain embodiments, the napDNAbp domain is a Cas9 nickase (nCas9). In certain embodiments, the napDNAbp domain comprises a sequence of any one of the napDNAbps provided herein, 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 any one of the napDNAbps provided herein. In some embodiments, the polymerase domain is a reverse transcriptase domain. In certain embodiments, the polymerase domain comprises a sequence of any one of the polymerases provided herein, 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 any one of the polymerases provided herein.

[0188] In some embodiments, the prime editor is PE2, PE3, PE4, PE5, or a variant thereof (including PE2max, PE3max, PE4max, and PE5max). In certain embodiments, the prime editor is PE4max-NRCH, or a variant thereof. In some embodiments, the prime editor is a PE6 prime editor, or a variant thereof. In certain embodiments, the prime editor is PE6b- NRCH, PE6c-NRCH, PE6d-NRCH, or a variant thereof.

[0189] In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in a subject. In certain embodiments, the subject is a human. In some embodiments, the method is a method of treating CDKL5 deficiency disorder in the subject. In certain embodiments, the method achieves greater than 20%, greater than 25%, greater than 30%, greater than 35%, or greater than 40% correction of the 1412delA mutation. In certain embodiments, the method results in introduction of less than 2%, less than 3%, less than 4%, or less than 5% indel byproducts in the target DNA sequence. 85 / 189 B1195.70194WO00 13324217.2

[0190] In another aspect, the present disclosure provides methods for correcting a c.826-1 G- to-A point mutation in a CDKL5 gene by prime editing. In some embodiments, the method comprises contacting a target DNA sequence comprising the CDKL5 gene with a prime editor and a prime editing guide RNA (pegRNA), wherein the pegRNA comprises a spacer sequence of GCTGTCAAGTATCTGTCAGCT (SEQ ID NO: 7), or a spacer 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 GCTGTCAAGTATCTGTCAGCT (SEQ ID NO: 7), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCTGTCAAGTATCTGTCAGCT (SEQ ID NO: 7). In certain embodiments, the pegRNA comprises the sequence GCTGTCAAGTATCTGTCAGCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATTTCCTAAGAAC CTGTTGAAGTTGGACCCAGCTGACAGATACTCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 39), 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 GCTGTCAAGTATCTGTCAGCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATTTCCTAAGAAC CTGTTGAAGTTGGACCCAGCTGACAGATACTCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 39), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCTGTCAAGTATCTGTCAGCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATTTCCTAAGAAC CTGTTGAAGTTGGACCCAGCTGACAGATACTCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 39). In some embodiments, the method further comprises contacting the target DNA sequence with a nicking guide RNA. In certain embodiments, the nicking guide RNA comprises the sequence GATTTCAAACCCAGAGACTTC (SEQ ID NO: 15), GTTCTGGATCGTTCTCCTTCA (SEQ ID NO: 16), or GTACTGAAGTTGGACCCAGCT (SEQ ID NO: 17), 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 GATTTCAAACCCAGAGACTTC (SEQ ID NO: 15), GTTCTGGATCGTTCTCCTTCA (SEQ ID NO: 16), or GTACTGAAGTTGGACCCAGCT (SEQ ID NO: 17), or a sequence comprising one, two, three, four, or five mutations, 86 / 189 B1195.70194WO00 13324217.2insertions, or deletions relative to GATTTCAAACCCAGAGACTTC (SEQ ID NO: 15), GTTCTGGATCGTTCTCCTTCA (SEQ ID NO: 16), or GTACTGAAGTTGGACCCAGCT (SEQ ID NO: 17).

[0191] In another aspect, the present disclosure provides methods for correcting a cluster of mutations in exon 8 of the CDKL5 gene by prime editing. In some embodiments, the method comprises contacting a target DNA sequence comprising the CDKL5 gene with a prime editor and a prime editing guide RNA (pegRNA), wherein the pegRNA comprises a spacer sequence of CAGAGTACGTTGCCACCAGA (SEQ ID NO: 8), or a spacer 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 CAGAGTACGTTGCCACCAGA (SEQ ID NO: 8), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to CAGAGTACGTTGCCACCAGA (SEQ ID NO: 8). In certain embodiments, the pegRNA comprises the sequence CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGTAAGGGCTCT AGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTTAAAC CAACTAGAATTTTTTT (SEQ ID NO: 40) or CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCACCCAAGTAAGG GCTCTAGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 41), 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 CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGTAAGGGCTCT AGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTTAAAC CAACTAGAATTTTTTT (SEQ ID NO: 40) or CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCACCCAAGTAAGG GCTCTAGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 41), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG 87 / 189 B1195.70194WO00 13324217.2CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGTAAGGGCTCT AGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTTAAAC CAACTAGAATTTTTTT (SEQ ID NO: 40) or CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCACCCAAGTAAGG GCTCTAGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 41). In some embodiments, the method further comprises contacting the target DNA sequence with a nicking guide RNA. In certain embodiments, the nicking guide RNA comprises the sequence GTGGAAATGTCATTCTATTTT (SEQ ID NO: 18), 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 GTGGAAATGTCATTCTATTTT (SEQ ID NO: 18), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GTGGAAATGTCATTCTATTTT (SEQ ID NO: 18).

[0192] In another aspect, the present disclosure provides methods for treating CDKL5 deficiency disorder (CDD) by prime editing using the prime editor PE6b-NRCH, PE6c- NRCH, or PE6d-NRCH. In some embodiments, the method comprises contacting a target DNA sequence comprising a CDKL5 gene comprising a pathogenic mutation with a prime editor, or a nucleotide sequence encoding a prime editor, and a pegRNA, or a nucleotide sequence encoding a prime editor, wherein the prime editor comprises an amino acid sequence of any one of SEQ ID NOs: 355-357, 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 an amino acid sequence of any one of SEQ ID NOs: 355-357. In some embodiments, the pegRNA is capable of correcting a c.1412delA mutation in the CDKL5 gene. In some embodiments, the pegRNA is any of the pegRNAs described herein.

[0193] In another aspect, the present disclosure provides for the use of any of the pegRNAs, compositions, polynucleotides, vectors, AAV particles, VLPs, or pharmaceutical compositions provided herein in the treatment of CDKL5 deficiency disorder.

[0194] In another aspect, the present disclosure provides for the use of any of the pegRNAs, compositions, polynucleotides, vectors, AAV particles, VLPs, or pharmaceutical compositions provided herein in in the manufacture of a medicament for the treatment of CDKL5 deficiency disorder. 88 / 189 B1195.70194WO00 13324217.2Complexes, Polynucleotides, Vectors, Cells, and Kits

[0195] The present disclosure provides, in some aspects, complexes comprising any of the pegRNAs or ngRNAs disclosed herein bound to any of the prime editors disclosed herein (e.g., PE6b-NRCH, PE6c-NRCH, and PE6d-NRCH). The present disclosure also provides, in some aspects, polynucleotides and vectors encoding any of the pegRNAs, ngRNAs, prime editors, complexes, systems, and / or compositions described herein. In some aspects, the present disclosure provides polynucleotides and vectors encoding a pegRNA and a prime editor as disclosed herein. In some embodiments, the polynucleotides and vectors provided herein comprise DNA (e.g., plasmid DNA or viral DNA). In some embodiments, the polynucleotides and vectors provided herein comprise RNA (e.g., mRNA or viral RNA).

[0196] Cells that may contain any of the pegRNAs, ngRNAs, prime editors, complexes, systems, and / or compositions described herein are also provided by the present disclosure. The methods described herein may be used to deliver a pegRNA and prime editor (and optionally an ngRNA) into a eukaryotic cell (e.g., a mammalian cell, such as a human cell, for example a human nerve cell). In some embodiments, the cell is in vitro (e.g., a cultured cell). In some embodiments, the cell is in vivo (e.g., in a subject, such as a human subject). In some embodiments, the cell is ex vivo (e.g., isolated from a subject and may be administered back to the same or a different subject).

[0197] In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. In some embodiments, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with the components of a prime editing system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a prime editing complex, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence.

[0198] The pegRNAs, ngRNAs, prime editors, systems, complexes, compositions, polynucleotides, and / or vectors described herein may also be assembled into kits. In some embodiments, the kit comprises polynucleotides for expression of the pegRNAs, ngRNAs, prime editors, complexes, systems, and / or compositions described herein. In some 89 / 189 B1195.70194WO00 13324217.2embodiments, the kit comprises appropriate pegRNAs or nucleic acid vectors for the expression of such pegRNAs to target the Cas9 protein of a prime editor to a desired target sequence, e.g., in CDKL5. In some embodiments, the pegRNAs in the kit are useful for correcting a c.1412delA mutation in a CDKL5 gene.

[0199] The kits described herein may include one or more containers housing components for performing the methods described herein, and optionally instructions for use. Any of the kits described herein may further comprise components needed for performing the prime editing methods described herein. Each component of the kits, where applicable, may be provided in liquid form (e.g., in solution) or in solid form, (e.g., a dry powder). In certain cases, some of the components may be reconstitutable or otherwise processible (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water), which may or may not be provided with the kit.

[0200] In some embodiments, the kits may optionally include instructions and / or promotion for use of the components provided. As used herein, “instructions” can define a component of instruction and / or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and / or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which can also reflect approval by the agency of manufacture, use, or sale for animal administration. As used herein, “promoted” includes all methods of doing business including methods of education, hospital and other clinical instruction, scientific inquiry, drug discovery or development, academic research, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral, and electronic communication of any form, associated with the disclosure. Additionally, the kits may include other components depending on the specific application, as described herein.

[0201] The kits may contain any one or more of the components described herein in one or more containers. The components may be prepared sterilely, packaged in a syringe, and shipped refrigerated. Alternatively, they may be housed in a vial or other container for storage. A second container may have other components prepared sterilely. Alternatively, the kits may include the active agents premixed and shipped in a vial, tube, or other container. 90 / 189 B1195.70194WO00 13324217.2

[0202] The kits may have a variety of forms, such as a blister pouch, a shrink-wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box, or a bag. The kits may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kits may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration, etc. EXAMPLES Example 1. Prime Editing for CDD Creation of a mouse cell line:

[0203] An epegRNA and nicking guide capable of installing the c.1412delA mutation in an N2a cell line were designed. Synthetic guide sequences were ordered from an oligonucleotide synthesis company, and 90 pmol of synthetic epegRNA and 60 pmol of synthetic nicking sgRNA were electroporated into 2 x 105wildtype N2a cells with 1000 ng each of in vitro transcribed PEmax mRNA and MLH1dn mRNA. The cells were allowed to grow for three days before being diluted to 1 cell per well of a 96 well plate. After growing up single cell colonies for two weeks, colonies were sequenced, and growth of heterozygous cell lines with the c.1412delA mutation and without indels was continued for use in further experiments. Creation of a human cell line:

[0204] 16 epegRNAs and 4 nicking guides capable of installing the c.1412delA mutation in a HEK293T cell line were designed and screened. After the most efficient PE5max editing strategy was determined, approximately 3 x 104cells were transfected and allowed to grow for three days before being diluted into a single cell per well of a 96 well plate. After growing single cell colonies for two weeks, colonies were sequenced, and growth of heterozygous and homozygous cell lines with the c.1412delA mutation and without indels was continued for use in further experiments. Cloning of epegRNAs:

[0205] An isothermal assembly method was used to clone epegRNAs. Cloning of PEmax, PEmax∆RNaseH and PE6b-d with SpCas9-NRCH: 91 / 189 B1195.70194WO00 13324217.2

[0206] PCR primers were designed to amplify SpCas9-NRCH and to amplify the backbone, or the reverse transcriptase, antibiotic resistance cassette, and other non-Cas features of the editing plasmids. Gibson assembly protocol was then used to clone plasmids with SpCas9- NRCH and the desired reverse transcriptase from each prime editor. Transfections to correct c.1412delA in the N2a and HEK293T disease cell lines:

[0207] All transfections were completed with Lipofectamine 2000 and included 60 ng of epegRNA, 200 ng of prime editor, 22 ng of nicking guide, and 100 ng of MLH1dn.

[0208] All pegRNAs were encoded using the promoter sequence TACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAA AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATC TTGTGGAAAGGACGAAACACC (SEQ ID NO: 403). Tested pegRNAs and gRNAs

[0209] pegRNAs and nicking gRNAs of the following sequences were tested: Table 1. epegRNAs for SpCas9-NRCH in N2a mouse model cell line (All sequences are shown 5′ to 3′) (All pegRNAs encode a +2 G>T seed edit and the +6T correction (corresponding to the A that needs inserting to correct the A deletion on the opposite strand))92 / 189 B1195.70194WO00 13324217.293 / 189 B1195.70194WO00 13324217.294 / 189 B1195.70194WO00 13324217.295 / 189 B1195.70194WO00 13324217.296 / 189 B1195.70194WO00 13324217.297 / 189 B1195.70194WO00 13324217.298 / 189 B1195.70194WO00 13324217.299 / 189 B1195.70194WO00 13324217.2100 / 189 B1195.70194WO00 13324217.2101 / 189 B1195.70194WO00 13324217.2102 / 189 B1195.70194WO00 13324217.2103 / 189 B1195.70194WO00 13324217.2Table 2. epegRNAs for SpCas9-NRCH in HEK293T model cell line (All sequences are shown 5′ to 3′) 104 / 189 B1195.70194WO00 13324217.2105 / 189 B1195.70194WO00 13324217.2106 / 189 B1195.70194WO00 13324217.2107 / 189 B1195.70194WO00 13324217.2108 / 189 B1195.70194WO00 13324217.2109 / 189 B1195.70194WO00 13324217.2110 / 189 B1195.70194WO00 13324217.2111 / 189 B1195.70194WO00 13324217.2112 / 189 B1195.70194WO00 13324217.2113 / 189 B1195.70194WO00 13324217.2114 / 189 B1195.70194WO00 13324217.2115 / 189 B1195.70194WO00 13324217.2116 / 189 B1195.70194WO00 13324217.2117 / 189 B1195.70194WO00 13324217.2118 / 189 B1195.70194WO00 13324217.2119 / 189 B1195.70194WO00 13324217.2120 / 189 B1195.70194WO00 13324217.2121 / 189 B1195.70194WO00 13324217.2122 / 189 B1195.70194WO00 13324217.2Table 3. Nicking guide RNAs (All sequences are shown 5’ to 3')123 / 189 B1195.70194WO00 13324217.2Example 2. Prime editing strategies for the correction of CDKL5 deficiency disorder mutations

[0210] CDKL5 deficiency disorder (CDD) is an X-linked, neurological disorder caused by pathogenic mutations in the cyclin-dependent kinase-like 5 (CDKL5) gene173. CDKL5 is a serine / threonine kinase with key roles in brain development and function174including dendritic microtubule regulation175and maintenance of dendritic spine morphology176,177. Though originally thought of as an early-onset form of Rett syndrome178, CDD is now considered a distinct disorder179,180with hundreds of causative mutations in the CDKL5 gene identified in patients178,181–191. While most mutations that cause the disorder are spontaneous de novo mutations, at least one case of a maternally-inherited pathogenic variant of CDKL5 has been described192. Pathogenic mutations in CDD typically result in haploinsufficiency either through full or partial loss of CDKL5 catalytic activity193,194, though at least one example of a hypermorphic mutation has been described195, and some lesser-studied CDD mutations may behave similarly to dominant negative mutations found via functional and biochemical assays176,196,197.

[0211] Symptoms of CDD begin in early postnatal life, correlating with peak CDKL5 expression173,176,196. A major clinical manifestation of CDD is infantile-onset seizures which are treatment-resistant, however patients also experience developmental delay, intellectual disability, extreme difficulty sleeping, visual impairment, gastrointestinal issues, and respiratory difficulties, among other symptoms173,194. Treatment for CDD is limited to symptom management. Symptom management options include broad-range anti-seizure medications (ASMs) like levetiracetam, the only FDA-approved ASM specifically for CDD called ganaxolone, ketogenic diet, and non-FDA approved cannabidiol derivatives198,199. Currently, there are no curative therapies available for CDD, though investigational disease- modifying therapeutics have been pursued with varying effectiveness198,199. Current investigational therapeutic strategies include epigenetic reactivation of CDKL5 expression200and a gene therapy to deliver a functional copy of the CDKL5 protein to the brain201. Though each of these strategies have unique advantages, none of them directly address the underlying genetic mutations that cause CDD. In contrast, development of a genome editing therapeutic has the potential to be curative by directly correcting pathogenic CDKL5 mutations. 124 / 189 B1195.70194WO00 13324217.2

[0212] Because CDD mutations can be caused by a suite of genetic changes such as deletions, duplications, and transition or transversion mutations178,181–191,195, development of a genome editing strategy for CDD must utilize a genome editing tool capable of performing precise changes of any variation. Traditional CRISPR nuclease-mediated genome editing results in uncontrolled mixtures of insertion and deletion (indel) alleles5,10, a strategy best suited for disruption of a locus, but not the correction of specific insertions, deletions, or other small mutations that cause CDD. Additionally, nuclease-based strategies require double-strand DNA breaks (DSBs), which can result in p53 activation41,43and undesirable chromosomal abnormalities like large deletions or chromothripsis37,42,202.

[0213] In contrast, prime editing can encode deletions, insertions, point mutations, and any combination thereof across hundreds of base pairs at a targeted locus in living cells without requiring a DSB intermediate3,86,89,157–160, making it an optimal choice for the development of corrective genome editing strategies for CDD. As presented herein, three uniquely prime editable (i.e., difficult or impossible to correct precisely with base editing53,54or Cas9 nuclease editing) pathogenic mutations in CDKL5 were focused on, namely a single nucleotide deletion (c.1412delA), a splice acceptor variant surrounded by additional adenines (c.826-1 G > A), and the installation of clustering pathogenic mutations in the eighth exon of CDKL5181. The studies presented herein demonstrate that prime editing can efficiently correct multiple classes of pathogenic CDKL5 variants.

[0214] Reported herein is the development of prime editing strategies for all three regions of interest with high on-target efficiency and with minimal indel by-products. The pathogenic allele harboring the equivalent c.1412delA mutation was specifically targeted in murine CDKL5 with correction efficiencies up to 40% by utilizing a bespoke prime editor comprised of a SpCas9-NRCH DNA targeting domain60and PE6 variants (PE6b-d) with evolved and / or engineered reverse transcriptase (RT) domains88.

[0215] A previously reported c.1412delA correction strategy86was optimized, and 42.3% and 36.2% desired correction was achieved with high specificity in patient primary fibroblasts using PE3max and PE6c88respectively. Finally, the applicability of prime editing to various other pathogenic mutations in CDKL5, including correction of c.826-1 G > A at an average of 8.8% and installing a series of seven pathogenic mutations or seed edits for pathogenic mutations in exon 8, a known hotspot for CDKL5 mutations181, is demonstrated at nearly 30% efficiency. Taken together, these results show that prime editing systems enable the efficient and precise correction of multiple types of pathogenic CDKL5 mutations, even 125 / 189 B1195.70194WO00 13324217.2simultaneously with a single prime editing guide RNA (pegRNA), and without requiring DSBs. Results Optimizing prime editing to correct c.1412delA in murine Cdkl5

[0216] Nuclease-based editing strategies for the correction of specific nucleotides could result in more indel alleles than alleles with the desired correction26,30–32. This editing strategy is of limited therapeutic benefit for c.1412delA as it is located in the catalytic domain where both in-frame and out-of-frame mutations can manifest in disease199. Indel alleles created by nuclease editing are predictable but uncontrollable18, making it hard to specify a particular indel allele as the desired outcome. Though base editors are more precise and cause fewer indel alleles than nuclease-mediated editing strategies, deletions like c.1412delA are not addressable with base editing technology. Base editors enable efficient A•T-to-G•C editing54or C•G-to-T•A53editing and vice versa, but base editors cannot revert a deletion mutation.

[0217] c.1412delA represents a uniquely prime-editable pathogenic variant of CDKL5. A pegRNA was previously optimized to correct c.1412delA via electroporation of in vitro transcribed PE mRNA and synthetic pegRNA into patient-derived induced pluripotent stem cells (iPSCs)86. It was sought to further improve this editing strategy with the use of engineered pegRNAs89and a suite of newly evolved and engineered PE6s88at both mouse and human CDKL5.

[0218] Because the mouse model for the c.1412delA mutation is not humanized, a prime editing strategy to correct the equivalent c.1412delA mutation in the murine Cdkl5 gene was first developed to enable future in vivo studies. The c.1412delA mutation occurred in a female patient, so the patient still has one healthy CDKL5 gene intact. Consequently, it was reasoned that an optimal genome editing strategy would preferentially target the disease allele while bypassing the healthy allele. To this end, an allele-specific prime editing strategy that incorporates recent prime editing advances was developed. Engineered pegRNAs (epegRNAs) that included a structured 3' motif that protects the reverse transcriptase template (RTT) from endogenous exonuclease degradation89were designed and screened. A dominant negative version of MLH1 (MLH1dn) was included to downregulate mismatch repair (MMR) as inhibition of MMR leads to improved prime editing outcomes86,87. PE6 variants88, or next- generation prime editors with evolved and / or rationally designed mutations in the Cas9 or reverse transcriptase domains, were also utilized. In order to specifically target the disease allele, the SpCas9- NRCH60DNA-targeting domain was used to create bespoke PEmax- 126 / 189 B1195.70194WO00 13324217.2NRCH and PE6-NRCH variants that recognize NRCH protospacer adjacent motifs (PAMs), where N is any nucleotide, R is adenine or guanine, and H is any nucleotide except guanine. The pathogenic c.1412delA mutation allows the creation of an NRCH PAM (TGCA), and correction of the mutation disrupts the NRCH PAM (TGTCA) (FIG.11A).

[0219] First, a monoclonal heterozygous murine Neuro-2a (N2a) cell line was created with 50% of alleles harboring the murine equivalent of c.1412delA. All combinations of epegRNAs with primer binding site (PBS) lengths of 11-15 nt and RTT lengths of 14-21 nt were then designed and screened for efficient correction of the equivalent c.1412delA mutation in Cdkl5 (FIG.11B). In addition to correcting the pathogenic mutation (+6 T insertion), the epegRNAs were also designed to incorporate a silent mutation in the spacer region (seed edit, +2 G > T) as this kind of edit has been shown to improve editing outcomes86. The cells were then transfected with a PE4max strategy constituting plasmid DNA for PEmax-NRCH, MLH1dn, and each epegRNA.

[0220] Genomic DNA was collected from transfected cells without enriching for edited cells 3 days post-transfection, and on-target editing was quantified with high-throughput sequencing (HTS). The most efficient desired editing was observed in the 50% of targetable alleles containing the pathogenic mutation was 18.6 ± 1.5% desired editing without indels using an epegRNA with a PBS of 11 nt and an RTT of 15 nt (FIG.11B).

[0221] To further optimize allele-specific correction, epegRNAs were designed with shorter PBS lengths of 9 nt and 10 nt while keeping the RTT template the same length (15 nt). EpegRNAs were also designed with the same PBS and RTT parameters, but with a “flip and extension” (F+E) scaffold203to determine whether the F+E scaffold could further improve prime editing as previously demonstrated when used with epegRNAs at other loci89. The F+E guide RNA scaffold mutates the fourth base pair of the direct repeat in the standard scaffold from U•A to A•U and extends the direct repeat by five base pairs, removing a potential pol III terminator and improving Cas9 binding respectively203. Using a PE4max strategy, plasmid DNA was transfected for PEmax-NRCH, MLH1dn, and each of the newly designed epegRNAs into heterozygous N2a cells (FIG.11C). Amongst the epegRNAs designed with the standard guide RNA scaffold, an increase in desired editing was observed when using a PBS length of 9 nt or 10nt (23.7 ± 2.4% and 25 ± 1.3% respectively, FIG.11C). Furthermore, no increase in desired editing outcomes was observed when using the F+E scaffold for the shortened PBS epegRNAs. 127 / 189 B1195.70194WO00 13324217.2

[0222] Because it achieved the highest level of on-target editing, the epegRNA with a standard scaffold and a PBS length of 10 nt was used for all further studies. Taken together, the epegRNA optimization efforts reveal that development of an allele-specific correction strategy for the murine equivalent of c.1412delA in CDKL5 is achievable with PEmax- NRCH and does not require an engineered guide RNA scaffold for efficient editing. Next, it was sought to optimize the prime editing system used in the correction strategy presented herein.

[0223] Next-generation prime editor systems, the PE6 variants, have shown equal or improved editing efficiency compared to PEmax while taking advantage of size-minimizing evolution and engineering efforts88. In particular, editing strategies were developed that included PE6b, whose reverse transcriptase (RT) was evolved from a yeast RT, PE6c, which was derived from PE6b with the addition of rationally engineered RT mutations, and PE6d, an RNaseH-truncated version of the M-MLV RT utilized in PEmax with the addition of evolved and rationally engineered mutations88.

[0224] To determine if the PE6 variants were applicable to the allele-specific editing strategy for the murine equivalent of c.1412delA in CDKL5, NRCH variants PE6b-NRCH, PE6c- NRCH, and PE6d-NRCH were created. PE6d(1904)-NRCH, a variant of PE6d-NRCH only with mutations T128F and V223M, was also created, and PEmax-∆RNaseH-NRCH, an RNaseH-truncated version of PEmax-NRCH with no other evolved or engineered mutations, was additionally created. The optimized epegRNA, MLH1dn, and each prime editor was transfected via plasmid DNA transfection, and allele-specific correction was quantified with HTS (FIG.11D). It was found that PEmax-NRCH (17 ± 3.4%), PE6b-NRCH (18 ± 2%), and PE6c-NRCH (19 ± 2%) had comparable levels of desired editing, all of which were higher levels of editing than that achieved by PE6d-NRCH (11.6 ± 2.2%), PE6d(1904)-NRCH (13.8 ± 1.5%), and PEmax- ∆RNaseH-NRCH (11.4 ± 1%) (FIG.11D). These results suggest that PE6 variants, particularly PE6b and PE6c, are amenable to the NRCH-based allele-specific prime editing strategy.

[0225] To further optimize the allele-specific prime editing strategy, three different nicking single guide RNAs (sgRNAs) were screened with opposite strand nick-to-nick distances of 87 nt (NG1), 63 nt (NG2), or 47 nt (NG3). Consistent with the previous prime editor screen, it was observed that PE5max-NRCH, PE6b-NRCH, and PE6c-NRCH out-performed PE6d- NRCH, PE6d(1901)-NRCH, and PE5max-∆RNaseH-NRCH across all nicking sgRNAs (FIG.11D). 128 / 189 B1195.70194WO00 13324217.2

[0226] It was found that for each prime editing system, the desired editing efficiencies were lowest when using NG3 and highest with NG1 and NG2 (FIG.11D). For all screened prime editors, indel formation was minimally increased when paired with NG2, so it was determined that NG1 was the most therapeutically-relevant nicking sgRNA. While the desired editing efficiency for PE5max-NRCH (38.3 ± 8.3%), PE6b-NRCH (38.7 ± 4.9%), and PE6c-NRCH (38.3 ± 6.7%) were similar on average when using NG1, average indel rates of 3.7 ± 0.7% with PE6c-NRCH were 2× and 1.5× higher than average indel rates with PE5max-NRCH (1.8 ± 0.2%) and PE6b-NRCH (2.4 ± 0.1%), respectively (FIG.11D). As such, it was determined that both PE5max-NRCH and PE6b-NRCH with NG1 were optimal systems to use in future studies for in vivo correction of c.1412delA in Cdkl5. Overall, these results establish an allele-specific correction strategy for c.1412delA in Cdkl5 with PEmax or PE6 variants, an optimized epegRNA, and optimized nicking sgRNA placement. Optimizing prime editing to correct c.1412delA in human CDKL5

[0227] Next, it was sought to optimize a prime editing strategy to correct the pathogenic c.1412delA mutation in human CDKL5. Monoclonal homozygous HEK293T cell lines harboring the mutation of interest were created. A corrective strategy similar to the allele- specific editing strategy was then developed by designing epegRNAs to be used with NRCH variants of the prime editors (FIG.12A). Of all the 80+ epegRNAs screened, the epegRNAs that exhibited detectable editing with NRCH editors had a non-silent seed edit encoding I470L (FIGs.12A-12B). Furthermore, additional PE and nicking sgRNA screening with the optimal I470L-encoding epegRNA achieved 4.9% editing with the most optimal conditions utilizing PE6b-NRCH and a nicking sgRNA with a nick-to-nick distance of 76 nt (NG6) (FIG.12C). This data suggests that development of a prime editing strategy with an SpCas9- NRCH DNA targeting domain for human CDKL5 requires a unique set of optimizations that cannot be directly inferred from development of an analogous strategy in murine Cdkl5.

[0228] Correction of c.1412delA in CDKL5 has been previously optimized for PEmax (which utilizes an SpCas9 binding domain that recognizes the canonical NGG PAM) and a pegRNA encoding a silent seed edit (+1 C > T) in addition to the corrective edit (+14 T insertion, FIG.12A)86. Given that epegRNAs are the state-of-the-art guide RNA for prime editors85,89, the pegRNA was converted to an epegRNA by adding a structured 3' trimmed evopreQ1 motif89. In order to determine if an optional 8 nt linker designed to minimize interactions between the protective 3' motif and the RTT89would improve editing efficiencies over not including the linker, epegRNAs were designed both with and without the linker to 129 / 189 B1195.70194WO00 13324217.2test in a head-to-head comparison (FIG.12D). Plasmid DNA was transfected for either the linker-containing or non-linker epegRNA along with the previously optimized nicking sgRNA86, MLH1dn, and PEmax constituting a PE5max editing strategy. PE5max desired editing was observed with the non-linker epegRNA at 3.3 ± 0.5%, which minimally outperformed the desired editing achieved with the linker-containing epegRNA at 2.6 ± 1.2% (FIG.12D). These results suggested that the presence of the optional 8 nt linker does not improve prime editing outcomes at the desired locus when using PEmax.

[0229] To fully optimize the prime editing strategy and determine whether the linker- containing epegRNA might improve editing with other PE systems, desired editing was compared across both epegRNAs when used with the previously optimized nicking sgRNA86, MLH1dn, and one of six prime editor variants—PEmax, PEmax-∆RNaseH, PE6b, PE6c, PE6d, and PE6e, a PE6 variant with a Cas9 domain harboring evolved and engineered

[0230] Across all PE systems, it was found that the non-linker epegRNA achieved higher desired editing than the linker-containing epegRNA (FIG.12D). In particular, it was observed that using the non-linker epegRNA with PE6c resulted in the highest level of correction at 32.9 ± 2.7% desired editing with 12 ± 2.5% indels while PE6c with the linker- containing epegRNA resulted in 22.8 ± 9.2% desired editing with 6.3 ± 2.5% indels. PE6d editing outcomes were modestly decreased with 29.6 ± 3% desired correction with the non- linker epegRNA and 24.7 ± 4.7% desired correction with the linker-containing epegRNA (FIG.12D). In contrast to the highly efficient PE6b-NRCH editing optimized for murine Cdkl5 (FIG.11D), PE6b desired editing was among the lowest for human CDKL5 at just 6 ± 2.1% for the non-linker epegRNA (FIG.12D). Because it achieved the highest on-target editing, PE6c is the optimal candidate for future studies in therapeutically relevant models such as neurons or cortical organoids differentiated from patient-derived iPSCs. c.1412delA correction in patient primary fibroblasts

[0231] Next, it was determined whether the PE6c strategy could correct the c.1412delA mutation in primary patient cells. Efficient editing via electroporation of in vitro transcribed PE mRNA and synthetic pegRNA has been previously demonstrated in patient-derived iPSCs up to 17% with PE3 or 34% with PE586. To determine if c.1412delA correction with PE6c or PEmax was efficient in primary patient cells, PE6c or PEmax mRNA was electroporated with 130 / 189 B1195.70194WO00 13324217.2synthetic non-linker-containing epegRNA and nicking sgRNA into patient fibroblasts isolated from a female patient heterozygous for the pathogenic mutation.

[0232] These electroporations yielded 36.2% correction of editable alleles and 1.9% indel products with PE6c, and 42.8% correction of editable alleles with 1.6% indel products with PE3max (FIG.13). Notably, this level of correction did not require the addition of MLH1dn.

[0233] Given PE6c’s outsized c.1412delA correction compared to PE5max in HEK293T cells homozygous for c.1412delA (FIG.12D), it was anticipated that PE6c would outperform PE3max in primary patient cells. Additionally, the HEK293T experiments utilized MLH1dn for a PE6c and PE5max comparison, thus PE6c editing outcomes may benefit more from the presence of MLH1dn than PEmax. Genome-wide off-target editing analysis in patient primary fibroblasts

[0234] After optimizing the prime editing strategy for corrective editing of c.1412delA in CDKL5 and quantifying its efficacy in patient primary fibroblasts, it was next sought to quantify any off-target editing. Prime editing has widely been reported to have substantially lower levels of off-target editing compared to other CRISPR-based gene editing methods79,97–105, reflecting the multiple steps at which an off-target sequence can be rejected without modification3. To assess off-target prime editing outcomes from the PEmax and PE6c correction strategies in patient primary fibroblasts, Cas-OFFinder, a computational method for nominating potential off-target loci128, was used. The Cas-OFFinder algorithm was used to nominate off-target loci with up to three mismatches to the target protospacer sequences of the epegRNA and the nicking sgRNA. For the epegRNA, 15 off-target loci were nominated (Peg OT1-15). Just three off-target loci were nominated for the nicking sgRNA (Nick OT1- 3). In both cases, all nominated off-target loci had exactly three mismatches.

[0235] HTS was used to quantify off-target editing at each of the nominated loci. For the epegRNA off-targets, the mutation frequency was quantified at the position of the first substitution that would be encoded by the epegRNA RTT as this position is the most likely nucleotide to be changed during prime editing3,79. Since the probability of an incorrect base call for the HTS method is 1 in 1000 bases204, a threshold of 0.1% was established in the analysis for bona fide epegRNA-dependent off-target substitution. It was observed that epegRNA-dependent off-target substitutions in PE3max-treated or PE6c-treated fibroblasts were largely similar to that of untreated fibroblasts (FIG.14A). Only at Peg OT9 was any editing above the 0.1% threshold observed. PE6c-treated fibroblasts accumulated an average of 0.13 ± 0.06% substitutions at Peg OT9, a range that overlaps with the observed 131 / 189 B1195.70194WO00 13324217.2substitution rate for the untreated samples at the same locus (0.09 ± 0.01%). The initial analysis in primary patient fibroblasts is consistent with previous reports that prime editing leads to minimal pegRNA-dependent off-target editing79,97–105.

[0236] The indel frequency at each of the epegRNA nominated sites was also quantified. It was found that off-target indel formation in PE3max-treated or PE6c-treated fibroblasts was similar to that of untreated fibroblasts at nearly every site (FIG.14B). Though off-target indel formation at Peg OT5 for PE6c-treated samples was minimally increased compared to untreated samples, the rate of indel formation was quite low (0.08%). Overall, these results are consistent with previous reports of low PE-mediated off-target editing79,97–105.

[0237] To quantify off-target editing at Cas-OFFinder-nominated sites for the nicking sgRNA, the indel frequency was quantified at two of the three nominated sites via HTS (FIG. 14B). Editing at Nick OT3 could not be quantified as it is located on the Y chromosome and the fibroblasts were derived from a female patient. Compared to untreated control fibroblasts, there was no nicking sgRNA-dependent indel formation at either of the remaining 2 nominated loci in treated fibroblasts (FIG.14B). Collectively, these results suggest that PE3max-edited and PE6c-edited c.1412delA fibroblasts experience minimal PE-mediated off-target editing, and the prime editing strategy could be used to correct c.1412delA with minimal detectable off-target editing in the human genome. Corrective prime editing of alternative pathogenic CDKL5 mutations

[0238] There are hundreds of pathogenic mutations known to cause CDD178,181–192. Because of this, it was sought to apply prime editing strategies to other loci of interest in CDKL5. One locus of interest is a pathogenic mutation at a splice site acceptor, c.826-1 G > A. This CDKL5 variant is uniquely prime-editable given the short-comings of nuclease-based editing and the presence of multiple other adenines in the editing window that could result in bystander A > G editing if the locus were targeted by a base editor.

[0239] In order to begin initial optimizations for a corrective c.826-1 G > A prime editing strategy, two heterozygous HEK293T cell lines were first created harboring the pathogenic mutation about 30% of alleles. After establishing the cell lines, epegRNAs were then designed and screened that encode the desired corrective edit (+24 A > G) and multiple silent mutations near the desired corrective edit (+17-21 TTTAC > CCTGT) (FIG.15A). These benign mutations help the prime editing intermediate to naturally evade MMR, a DNA repair process known to decrease desired prime editing outcomes86,87. An epegRNA screen was conducted by transfecting PEmax-∆RNaseH, MHL1dn, and various epegRNAs into the 132 / 189 B1195.70194WO00 13324217.2model cell lines (FIG.15A). While editing efficiencies were low, several epegRNAs were observed with desired editing percentages above 0.7% across all alleles. The epegRNA with PBS and RTT lengths of 11 and 33 nt respectively were elected to be used, reaching an average of 0.75 ± 0.32% editing for additional optimization.

[0240] Next, to determine the most optimal prime editor for the corrective strategy (FIG. 15C), the most optimal nicking sgRNA position with 3 nicking sgRNAs in total (NG1, NG2, NG3, FIG.15C) was also screened. NG3 was designed for edit-specific complementary nicking in which nicking does not occur until after the desired edit has been installed. This edit-specific nicking sgRNA design leads to minimal indel formation3. Unlike NG1 and NG2, NG3 utilizes an NGA PAM instead of the canonical NGG PAM because SpCas9 can have residual catalytic activity on NGA PAMs44,205. It was observed that PE6c resulted in the highest correction efficiency with up to 9.8% desired editing when paired with NG2 (FIG. 15C). Average indel formation was minimal for all samples, with indel formation for all nicking guides occurring at less than 0.11% in all samples. Therefore, corrective editing with PE6c and NG2 currently represents the strongest prime editing strategy for further studies. Overall, these data suggest that the c.826-1 G > A, like c.1412delA, is amenable to prime editing correction, most notably with PE6c.

[0241] Most patients with CDD have unique pathogenic mutations, and these mutations occur throughout the CDKL5 gene, but pathogenic missense occur almost exclusively the catalytic domain of CDKL5181,187,199. The ability to use a single epegRNA to correct pathogenic mutations for multiple patients is ideal. This strategy would allow for the development of a single composition of matter and a single clinical trial for a group of patients as opposed to the development of multiple compositions of matter for each individual patient, and consequently multiple clinical trials. Thus, exon 8 was chosen as a model site for demonstrating the ability of a single epegRNA to modify multiple bases in a region like the catalytic domain where multiple pathogenic mutations cluster. As a proof-of- principle, two epegRNAs were designed to install five known pathogenic mutations (p.R175S (+3 A > C), p.W176G (+4 T > C), p.Y177C (+8 A > G), p.P180L (+17 C > T), and p.L182P (+23 T > C))181,191and two additional MMR evading mutations (+12 G > C and +21 A > G) in exon 8 (FIG.16A). These epegRNAs were not fully optimized for PBS and RTT lengths. The epegRNAs were transfected into wildtype HEK293T cells with PEmax and a +50 nicking sgRNA, and editing efficiency of all seven edits being incorporated into the target locus at once was quantified. It was found that on average, the first four desired modifications 133 / 189 B1195.70194WO00 13324217.2were installed at an editing efficiency of 29.4%, and all seven modifications were installed at 25.4% editing efficiency (FIG.16B). The decreased editing efficiency for the final three modifications suggests potential degradation of the newly synthesized DNA strand by endogenous exonucleases before resolution of editing via DNA repair. Having a longer RTT that extends more bases past the last desired edit could remediate this issue. These findings suggest the potential for a therapeutic strategy to correct multiple pathogenic CDD mutations across several amino acids with a single epegRNA. Discussion

[0242] The lack of approved disease-modifying therapeutics for CDD highlights the urgent unmet medical need of CDD patients and their caregivers. While a gene therapy that would deliver a functional copy of CDKL5 is being developed, this therapeutic strategy may be intractable for patients with dominant negative mutations206. Additionally, the dosage sensitivity of many genetic epilepsy disorders, including CDD where overexpression of CDKL5 has been reported to manifest in disease207, limits the applicability of a gene replacement therapy since endogenous transcriptional control of the target gene would be lost206,208. The most physiological approach to treating CDD would be to revert the disease alleles back to wildtype.

[0243] PEmax-NRCH and PE6-NRCH strategies capable of enabling 38% allele-specific correction of c.1412delA in heterozygous murine N2a cells were developed. Though development of a PE-NRCH correction strategy in human CDKL5 was not initially successful without the addition of a non-silent seed edit, nearly 40% correction of the c.1412delA allele in patient primary fibroblasts was demonstrated with PE6c and a previously reported pegRNA converted to an epegRNA. Initial off-target editing analyses with Cas-OFFinder- nominated loci revealed that the prime editing strategy was highly-specific. Initial experiments for the correction of c.826-1 G > A resulted in up to 9.8% prime editing with PE6c, and prime editing at a mutational hotspot in exon 8 resulted in 25.4% installation of seven additional CDKL5 mutations or relevant seed edits. These results demonstrate that the development of prime editing strategies, including those with the recently described PE6 variants, lead to robust levels of editing across the CDKL5 gene.

[0244] Another important question in the development of CDD therapeutics is timing of drug administration. Since CDD is early-onset, and clinical symptoms occur within the first few months of life, there have historically been concerns about the therapeutic window at which a patient must be treated208. A recent study has shown that restoration of CDKL5 expression in 134 / 189 B1195.70194WO00 13324217.2adult mice rescued CDD-associated phenotypes177. These findings suggest that the ability to treat CDD beyond early infancy may be possible. Methods Culture of cell lines and primary patient fibroblasts

[0245] HEK293T cells and Neuro-2a (N2a) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus GlutaMAX (Thermo Fisher Scientific) supplemented with 10% (v / v) fetal bovine serum (FBS) (Thermo Fisher Scientific). Primary fibroblasts were obtained from a patient sample215and cultured in DMEM supplemented with 20% fetal bovine serum. Generation of cell lines with CDKL5 mutations

[0246] For c.1412delA installation, 1.2-1.5 × 104N2a or HEK293T cells were transfected with 200 ng of PEmax, 60 ng of an appropriate epegRNA installing the desired mutation (c.1412delA in murine or human CDKL5), 22 ng of an appropriate nicking sgRNA, and 100 ng of MHL1dn. For c.826-1 G > A installation, 1.5 × 104HEK293T cells were transfected with 200ng of CBE6b67and 50 ng of sgRNA. All DNA transfections were completed with 0.5μl Lipofectamine 2000 (Thermo Fisher Scientific) and 5.75 μl OptiMEM combined with an appropriate DNA plasmid mixture.

[0247] Transfected cells were then diluted to approximately 1 cell / well and subsequently allowed to grow into single cell colonies for 7-14 days. Colonies were split evenly for HTS verification, and cell culture was continued. Sequence verified cells were expanded until a confluent T-75 flask could be established. From confluent flasks, several vials of 1 × 106cells were cryopreserved for future experiments, while 2 × 105cells were seeded for continued culture and corrective editing experiments. Transfections for epegRNA, nicking sgRNA, and prime editor screens

[0248] 1.5 × 104N2a or HEK293T cells per well in a 96-well tissue culture plate were seeded 1 day before transfection. All DNA transfections were completed with 0.5μl Lipofectamine 2000 (Thermo Fisher Scientific) and 5.75 μl OptiMEM combined with an appropriate DNA plasmid mixture. The DNA plasmid mixture included 200 ng of prime editor plasmid, 60 ng of epegRNA plasmid, and in experiments where nicking guides or MLH1dn are also included, 22 ng of nicking sgRNA plasmid and / or 100 ng of MLH1dn plasmid were also transfected. After 3 days, genomic DNA was extracted from the cells and desired editing was measured via HTS. Sequences of relevant epegRNAs and nicking sgRNAs:

[0249] Newly described guide RNA sequences are in Tables 4 and 5: 135 / 189 B1195.70194WO00 13324217.2Table 4. Additional pegRNAs targeting CDKL5 c.1412delA used in Example 2:136 / 189 B1195.70194WO00 13324217.2137 / 189 B1195.70194WO00 13324217.2138 / 189 B1195.70194WO00 13324217.2139 / 189 B1195.70194WO00 13324217.2140 / 189 B1195.70194WO00 13324217.2141 / 189 B1195.70194WO00 13324217.2142 / 189 B1195.70194WO00 13324217.2143 / 189 B1195.70194WO00 13324217.2144 / 189 B1195.70194WO00 13324217.2145 / 189 B1195.70194WO00 13324217.2146 / 189 B1195.70194WO00 13324217.2147 / 189 B1195.70194WO00 13324217.2148 / 189 B1195.70194WO00 13324217.2Table 5: EpegRNA and nicking sgRNA sequences for described mutations

[0250] Plasmids were cloned via current best practices85. Corrective pegRNA (GAGGGACTCCTAGAGGACTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG 149 / 189 B1195.70194WO00 13324217.2GCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATATTGACACAA TTCCCCAATCCTCTAGGAGTCTTT SEQ ID NO: 346) and nicking sgRNA (GCAGAACCGCCACTCATTCA SEQ ID NO: 20) for CDKL5 were previously described86. The epegRNA, described in Table 5, includes the 3’ tevo-preQ1 motif89. In vitro transcription of PEmax and PE6c

[0251] In vitro transcribed PEmax and PE6c mRNA were ordered from GenScript with full substitution of uridine for N1-methylpseudouridine. The final mRNA products were diluted in 1mM Sodium Citrate (pH 6.5) at a concentration of 0.5 mg / ml. Synthetic guide RNAs

[0252] Synthetic pegRNA was ordered from Integrated DNA Technologies. Each epeg contained 2′-O-methyl modifications at the first and last three nucleotides and phosphorothioate linkages between the three first and last nucleotides. Synthetic nicking sgRNAs were ordered from Synthego and included 2′-O-methyl modifications at the first and last three nucleotides and phosphorothioate bonds between the first three and last two bases. Electroporation of patient fibroblasts

[0253] Electroporations were performed with the Lonza 4D Nucleofector.2.5 × 105fibroblasts per condition were electroporated with the SE Cell Line X Kit S (Lonza, V4XC) according to manufacturer instructions using pulse code CM-130. Electroporations included 2000 ng of either PEmax or PE6c mRNA, 180 pmol of synthetic pegRNA, and 120 pmol of synthetic nicking sgRNA. Cells underwent media exchange on day 3 post-transfection and genomic DNA was collected 6 days post-transfection. HTS

[0254] Genomic DNA (gDNA) was extracted from N2a cells, HEK293T cells, and primary fibroblasts using a lysis buffer (10 mM Tris-HCl, pH 8.0, 0.05% SDS, 25 μg ml−1proteinase K (Qaigen)) followed by incubation at 37°C for an hour and heat inactivation at 80°C for 30 minutes. Primers for the amplification of c.1412delA in Cdkl5 were forward: ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGCTGGTGGGAGAGTTCA AG (SEQ ID NO: 361) and reverse: TGGAGTTCAGACGTGTGCTCTTCCGATCTGGTCCAGGCACAAAGTACCTC (SEQ ID NO: 362).

[0255] Primers for the amplification of c.1412delA in CDKL5 were forward: ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCTTCAGAAGGCCCAGGGA CAAAG (SEQ ID NO: 363) and reverse: 150 / 189 B1195.70194WO00 13324217.2TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGGTACTGGGCTCCGCAATTT (SEQ ID NO: 364).

[0256] Primers for the amplification of c.826-1 G > A in CDKL5 were forward: ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTGTTTACTTGATATTCTG CAATGACTG (SEQ ID NO: 365) and reverse: TGGAGTTCAGACGTGTGCTCTTCCGATCTCCAACACTGCATCTGTATTTCCTTAAA AG (SEQ ID NO: 366).

[0257] Primers for the amplification of mutations in exon 8 were forward: ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAGCCCATGCGAGAACAGT CA (SEQ ID NO: 367) and reverse: TGGAGTTCAGACGTGTGCTCTTCCGATCTTGACAATAGAATCAGCAGATGTGGA (SEQ ID NO: 368).

[0258] Subsequent HTS was performed via Crispresso2. Briefly, fastq files were aligned and editing frequency was quantified in Crispresso2 in batch mode with a window with spanning at least 10nt past each nick site as previously described79. Cas-OFFinder off-target nomination, amplicon sequencing, and analysis

[0259] Computational nomination of potential off-target sites with three or fewer mismatches to the epegRNA or nicking sgRNA protospacers of the c.1412delA prime editing strategy were nominated using Cas-OFFinder128. HTS sequencing primers for each site were designed as follows: Table 6: HTS sequences for Cas-OFFinder-nominated loci151 / 189 B1195.70194WO00 13324217.2

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[0261] In the articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Embodiments or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0262] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claims that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements and / or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub–range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0263] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a 168 / 189 B1195.70194WO00 13324217.2conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the embodiments. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any embodiment, for any reason, whether or not related to the existence of prior art.

[0264] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended embodiments. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following embodiments. 169 / 189 B1195.70194WO00 13324217.2

Claims

CLAIMS What is claimed is:

1. A method of correcting a 1412delA mutation in a CDKL5 gene by prime editing comprising contacting a target DNA sequence comprising the CDKL5 gene with a prime editor and a prime editing guide RNA (pegRNA), wherein the pegRNA comprises a spacer sequence of GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5), or TAGCTATGCCGACT (SEQ ID NO: 6), or a spacer 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 GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5), or TAGCTATGCCGACT (SEQ ID NO: 6), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5) or TAGCTATGCCGACT (SEQ ID NO: 6).

2. The method of claim 1, wherein the pegRNA comprises the sequence of any of the pegRNAs provided in Tables 1, 2, and 4, 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 any of the pegRNAs provided in Tables 1, 2, and 4.

3. The method of claim 1 or 2, wherein the pegRNA comprises a gRNA scaffold sequence of GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 576) or GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTT 170 / 189 B1195.70194WO00 13324217.2ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 577), 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 GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 576) or GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 577), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 576) or GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 577).

4. The method of any one of claims 1-3, wherein the pegRNA comprises a reverse transcription template (RTT) comprising the sequence of any one of SEQ ID NOs: 404-563, 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 of any one of SEQ ID NOs: 404-563, or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to of any one of SEQ ID NOs: 404-563.

5. The method of any one of claims 1-4, wherein the pegRNA comprises a primer binding site comprising the sequence TTCCCCAGT, TTCCCCAGTC (SEQ ID NO: 29), TTCCCCAGTCC (SEQ ID NO: 30), TTCCCCAGTCCT (SEQ ID NO: 31), TTCCCCAGTCCTC (SEQ ID NO: 32), TTCCCCAGTCCTCC (SEQ ID NO: 33), TTCCCCAGTCCTCCC (SEQ ID NO: 34), TAGCTATGC, TAGCTATGCC (SEQ ID NO: 35), TAGCTATGCCG (SEQ ID NO: 36), TAGCTATGCCGA (SEQ ID NO: 37), TAGCTATGCCGAC (SEQ ID NO: 38), or TAGCTATGCCGACT (SEQ ID NO: 6), 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 TTCCCCAGT, TTCCCCAGTC (SEQ ID NO: 29), TTCCCCAGTCC (SEQ ID NO: 30), TTCCCCAGTCCT (SEQ ID NO: 31), TTCCCCAGTCCTC (SEQ ID NO: 32), TTCCCCAGTCCTCC (SEQ ID NO: 33), TTCCCCAGTCCTCCC (SEQ ID NO: 34), TAGCTATGC, TAGCTATGCC (SEQ ID NO: 35), TAGCTATGCCG (SEQ ID NO: 36), TAGCTATGCCGA (SEQ ID NO: 37), 171 / 189 B1195.70194WO00 13324217.2TAGCTATGCCGAC (SEQ ID NO: 38), or TAGCTATGCCGACT (SEQ ID NO: 6), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to TTCCCCAGT, TTCCCCAGTC (SEQ ID NO: 29), TTCCCCAGTCC (SEQ ID NO: 30), TTCCCCAGTCCT (SEQ ID NO: 31), TTCCCCAGTCCTC (SEQ ID NO: 32), TTCCCCAGTCCTCC (SEQ ID NO: 33), TTCCCCAGTCCTCCC (SEQ ID NO: 34), TAGCTATGC, TAGCTATGCC (SEQ ID NO: 35), TAGCTATGCCG (SEQ ID NO: 36), TAGCTATGCCGA (SEQ ID NO: 37), TAGCTATGCCGAC (SEQ ID NO: 38), or TAGCTATGCCGACT (SEQ ID NO: 6).

6. The method of any one of claims 1-5, wherein the pegRNA comprises a 3' structured motif of the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 14), 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 CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 14), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 14).

7. The method of any one of claims 1-6, wherein the pegRNA comprises a terminator sequence of TTTTTT, 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 TTTTTT, or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to 8. The method of any one of claims 1-7, wherein the pegRNA comprises the structure 5'-[spacer]-[gRNA scaffold]-[RTT]-[PBS]-[3' motif]-[terminator]-3' or 5'-[spacer]-[gRNA scaffold]-[RTT]-[PBS]-[3' motif]- 3'.

9. The method of any one of claims 1-8, wherein the contacting further comprises using a nicking guide RNA (ngRNA).

10. The method of claim 9, wherein the ngRNA comprises a spacer sequence of GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or 172 / 189 B1195.70194WO00 13324217.2GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13), or a spacer 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 GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13).

11. The method of claim 9 or 10, wherein the ngRNA comprises the sequence of any one of the ngRNAs provided in Table 3, 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 any one of the ngRNAs provided in Table 3.

12. The method of any one of claims 9-11, wherein the ngRNA comprises a gRNA scaffold sequence of GTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 578), 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 GTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 578), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 578).

13. The method of any one of claims 9-12, wherein the ngRNA comprises a terminator sequence of TTTTTT, 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 TTTTTT, or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to 173 / 189 B1195.70194WO00 13324217.

214. The method of any one of claims 9-13, wherein the ngRNA comprises the structure 5'-[spacer]-[gRNA scaffold]-[terminator]- 3' or 5'-[spacer]-[gRNA scaffold]- 3'.

15. The method of any one of claims 1-14, wherein the prime editor comprises (i) a napDNAbp domain, and (ii) a polymerase domain.

16. The method of claim 15, wherein the napDNAbp domain has a nickase activity.

17. The method of claim 15 or 16, wherein the napDNAbp domain is a Cas9 protein or a variant thereof.

18. The method of any one of claims 15-17, wherein the napDNAbp domain is a Cas9 nickase (nCas9).

19. The method of any one of claims 15-18, wherein the napDNAbp domain comprises a sequence of any one of the napDNAbps provided herein, 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 any one of the napDNAbps provided herein.

20. The method of any one of claims 15-19, wherein the polymerase domain is a reverse transcriptase domain.

21. The method of any one of claims 15-20, wherein the polymerase domain comprises a sequence of any one of the polymerase domains provided herein, 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 any one of the polymerase domains provided herein.

22. The method of any one of claims 1-21, wherein the prime editor is PE2, PE3, PE4, PE5, or a variant thereof.

23. The method of any one of claims 1-22, wherein the prime editor is PE4max-NRCH, or a variant thereof. 174 / 189 B1195.70194WO00 13324217.

224. The method of any one of claims 1-21, wherein the prime editor is a PE6 prime editor.

25. The method of any one of claims 1-21 or 24, wherein the prime editor is PE6b- NRCH, PE6c-NRCH, or PE6d-NRCH.

26. The method of any one of claims 1-25, wherein the method is performed in vitro.

27. The method of any one of claims 1-25, wherein the method is performed in vivo.

28. The method of any one of claims 1-25 or 27, wherein the method is performed in a subject.

29. The method of claim 28, wherein the subject is a human.

30. The method of claim 28 or 29, wherein the method is a method of treating CDKL5 deficiency disorder in the subject.

31. The method of any one of claims 1-30, wherein the method achieves greater than 20%, greater than 25%, greater than 30%, greater than 35%, or greater than 40% correction of the 1412delA mutation.

32. The method of any one of claims 1-31, wherein the method results in introduction of less than 2%, less than 3%, less than 4%, or less than 5% indel byproducts in the target DNA sequence.

33. A prime editing guide RNA (pegRNA) comprising a spacer sequence of GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5), or TAGCTATGCCGACT (SEQ ID NO: 6), or a spacer 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 GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG 175 / 189 B1195.70194WO00 13324217.2(SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5), or TAGCTATGCCGACT (SEQ ID NO: 6), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCCGGGAGGACTGGGGAATGG (SEQ ID NO: 1), GCCAGATCTCAGCAGAACAGG (SEQ ID NO: 2), GCATTCATGGAAAGCTCCCAG (SEQ ID NO: 3), GCCAGAGCAAAGCTGGGACCC (SEQ ID NO: 4), GCAGAGTCGGCATAGCTATAT (SEQ ID NO: 5), TAGCTATGCCGACT (SEQ ID NO: 6).

34. The pegRNA of claim 33, wherein the pegRNA comprises the sequence of any of the pegRNAs provided in Tables 1, 2, and 4 herein, 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 any of the pegRNAs provided in Tables 1, 2, and 4 herein.

35. The pegRNA of claim 33 or 34, wherein the pegRNA comprises a gRNA scaffold sequence of GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 576) or GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 577), 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 GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 576) or GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 577), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 576) or GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 577). 176 / 189 B1195.70194WO00 13324217.

236. The pegRNA of any one of claims 33-35, wherein the pegRNA comprises a reverse transcription template (RTT) comprising the sequence of any one of SEQ ID NOs: 404-563, 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 of any one of SEQ ID NOs: 404-563, or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to of any one of SEQ ID NOs: 404-563.

37. The pegRNA of any one of claims 33-36, wherein the pegRNA comprises a primer binding site comprising the sequence TTCCCCAGT, TTCCCCAGTC (SEQ ID NO: 29), TTCCCCAGTCC (SEQ ID NO: 30), TTCCCCAGTCCT (SEQ ID NO: 31), TTCCCCAGTCCTC (SEQ ID NO: 32), TTCCCCAGTCCTCC (SEQ ID NO: 33), TTCCCCAGTCCTCCC (SEQ ID NO: 34), TAGCTATGC, TAGCTATGCC (SEQ ID NO: 35), TAGCTATGCCG (SEQ ID NO: 36), TAGCTATGCCGA (SEQ ID NO: 37), TAGCTATGCCGAC (SEQ ID NO: 38), or TAGCTATGCCGACT (SEQ ID NO: 6), 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 TTCCCCAGT, TTCCCCAGTC (SEQ ID NO: 29), TTCCCCAGTCC (SEQ ID NO: 30), TTCCCCAGTCCT (SEQ ID NO: 31), TTCCCCAGTCCTC (SEQ ID NO: 32), TTCCCCAGTCCTCC (SEQ ID NO: 33), TTCCCCAGTCCTCCC (SEQ ID NO: 34), TAGCTATGC, TAGCTATGCC (SEQ ID NO: 35), TAGCTATGCCG (SEQ ID NO: 36), TAGCTATGCCGA (SEQ ID NO: 37), TAGCTATGCCGAC (SEQ ID NO: 38), or TAGCTATGCCGACT (SEQ ID NO: 6), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to TTCCCCAGT, TTCCCCAGTC (SEQ ID NO: 29), TTCCCCAGTCC (SEQ ID NO: 30), TTCCCCAGTCCT (SEQ ID NO: 31), TTCCCCAGTCCTC (SEQ ID NO: 32), TTCCCCAGTCCTCC (SEQ ID NO: 33), TTCCCCAGTCCTCCC (SEQ ID NO: 34), TAGCTATGC, TAGCTATGCC (SEQ ID NO: 35), TAGCTATGCCG (SEQ ID NO: 36), TAGCTATGCCGA (SEQ ID NO: 37), TAGCTATGCCGAC (SEQ ID NO: 38), or TAGCTATGCCGACT (SEQ ID NO: 6).

38. The pegRNA of any one of claims 33-37, wherein the pegRNA comprises a 3' structured motif of the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 14), or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at 177 / 189 B1195.70194WO00 13324217.2least 98%, or at least 99% identical to CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 14), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 14).

39. The pegRNA of any one of claims 33-38, wherein the pegRNA comprises a terminator sequence of TTTTTT, 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 TTTTTT, or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to TTTTTT.

40. The pegRNA of any one of claims 33-39, wherein the pegRNA comprises comprise the structure 5'-[spacer]-[gRNA scaffold]-[RTT]-[PBS]-[3' motif]-[terminator]-3' or 5'- [spacer]-[gRNA scaffold]-[RTT]-[PBS]-[3' motif]-3'.

41. A composition comprising the pegRNA of any one of claims 33-40.

42. The composition of claim 41 further comprising a nicking guide RNA (ngRNA).

43. The composition of claim 42, wherein the ngRNA comprises a spacer sequence of GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13), or a spacer 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 GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCTGGGGAATTGTGTCAAGAT (SEQ ID NO: 10), GGCCTTGGTCCTGTAGGAGG (SEQ ID NO: 11), GTTGCTCACAGACTTGGAGT (SEQ ID NO: 12), or GCTGGGGAATTGTGTGAGGAT (SEQ ID NO: 13). 178 / 189 B1195.70194WO00 13324217.

244. The composition of claim 42 or 43, wherein the ngRNA comprises the sequence of any one of the ngRNAs provided in Table 3, 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 any one of the ngRNAs provided in Table 3.

45. The composition of any one of claims 42-44, wherein the ngRNA comprises a gRNA scaffold sequence of GTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 578), 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 GTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 578), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 578).

46. The composition of any one of claims 42-45, wherein the ngRNA comprises a terminator sequence of TTTTTT, 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 TTTTTT, or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to TTTTTT.

47. The composition of any one of claims 42-46, wherein the ngRNA comprises the structure 5'-[spacer]-[gRNA scaffold]-[terminator]- 3'.

48. The composition of any one of claims 41-47 further comprising a prime editor.

49. The composition of claim 48, wherein the prime editor comprises (i) a napDNAbp domain, and (ii) a polymerase domain.

50. The composition of claim 49, wherein the napDNAbp domain has a nickase activity. 179 / 189 B1195.70194WO00 13324217.

251. The composition of claim 49 or 50, wherein the napDNAbp domain is a Cas9 protein or a variant thereof.

52. The composition of any one of claims 49-51, wherein the napDNAbp domain is a Cas9 nickase (nCas9).

53. The composition of any one of claims 49-52, wherein the napDNAbp domain comprises a sequence of any one of the napDNAbps provided herein, 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 any one of the napDNAbps provided herein.

54. The composition of any one of claims 49-53, wherein the polymerase domain is a reverse transcriptase domain.

55. The composition of any one of claims 49-54, wherein the polymerase domain comprises a sequence of any one of the polymerases provided herein, 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 any one of the polymerases provided herein.

56. The composition of any one of claims 48-55, wherein the prime editor is PE2, PE3, PE4, PE5, or a variant thereof.

57. The composition of any one of claims 48-56, wherein the prime editor is PE4max- NRCH, or a variant thereof.

58. The composition of any one of claims 48-55, wherein the prime editor is a PE6 prime editor, or a variant thereof.

59. The composition of any one of claims 48-55 or 58, wherein the prime editor is PE6b- NRCH, PE6c-NRCH, PE6d-NRCH, or a variant thereof.

60. One or more polynucleotides encoding the pegRNA of any one of claims 33-40 and / or the ngRNA of the composition of any one of claims 41-59. 180 / 189 B1195.70194WO00 13324217.

261. The one or more polynucleotides of claim 60, wherein the one or more polynucleotides encode the prime editor of the composition of any one of claims 48-59.

62. One or more vectors comprising the one or more polynucleotides of claim 60 or 61.

63. The one or more vectors of claim 62, wherein the vectors are AAV vectors.

64. One or more AAV particles comprising the pegRNA of any one of claims 33-40, the composition of any one of claims 41-59, the one or more polynucleotides of claim 60 or 61, or the one or more vectors of claim 62 or 63.

65. A virus-like particle (VLP) comprising the pegRNA of any one of claims 33-40, the composition of any one of claims 41-59, the one or more polynucleotides of claim 60 or 61, or the one or more vectors of claim 62 or 63.

66. A pharmaceutical composition comprising the pegRNA of any one of claims 33-40, the composition of any one of claims 41-59, the one or more polynucleotides of claim 60 or 61, the one or more vectors of claim 62 or 63, the one or more AAV particles of claim 64, or the VLP of claim 65, and a pharmaceutically acceptable excipient.

67. A cell comprising the pegRNA of any one of claims 33-40, the composition of any one of claims 41-59, the one or more polynucleotides of claim 60 or 61, the one or more vectors of claim 62 or 63, the one or more AAV particles of claim 64, or the VLP of claim 65.

68. A kit comprising the pegRNA of any one of claims 33-40, the composition of any one of claims 41-59, the one or more polynucleotides of claim 60 or 61, the one or more vectors of claim 62 or 63, the one or more AAV particles of claim 64, or the VLP of claim 65.

69. Use of the pegRNA of any one of claims 33-40, the composition of any one of claims 41-59, the one or more polynucleotides of claim 60 or 61, the one or more vectors of claim 62 181 / 189 B1195.70194WO00 13324217.2or 63, the one or more AAV particles of claim 64, the VLP of claim 65, or the pharmaceutical composition of claim 66 in the treatment of CDKL5 deficiency disorder.

70. Use of the pegRNA of any one of claims 33-40, the composition of any one of claims 41-59, the one or more polynucleotides of claim 60 or 61, the one or more vectors of claim 62 or 63, the one or more AAV particles of claim 64, the VLP of claim 65, or the pharmaceutical composition of claim 66 in the manufacture of a medicament for the treatment of CDKL5 deficiency disorder.

71. A system comprising a pegRNA of Table 1 or Table 2, an ngRNA of Table 3, and a prime editor.

72. The system of claim 71, wherein the prime editor is PE6b-NRCH, PE6c-NRCH, or PE6d-NRCH.

73. A method of correcting a 1412delA mutation in a CDKL5 gene by prime editing comprising contacting a target DNA sequence comprising the CDKL5 gene with a prime editor and a prime editing guide RNA (pegRNA), wherein the pegRNA comprises a reverse transcription template (RTT) of the sequence of any one of SEQ ID NOs: 404-563, or a RTT 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 of any one of SEQ ID NOs: 404-563, or a RTT comprising one, two, three, four, or five mutations, insertions, or deletions relative to the sequence of any one of SEQ ID NOs: 404-563.

74. A prime editing guide RNA (pegRNA) comprising a reverse transcription template (RTT) of the sequence of any one of SEQ ID NOs: 404-563, or a RTT 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 of any one of SEQ ID NOs: 404-563, or a RTT comprising one, two, three, four, or five mutations, insertions, or deletions relative to the sequence of any one of SEQ ID NOs: 404-563.

75. A method of correcting a c.826-1 G-to-A point mutation in a CDKL5 gene by prime editing comprising contacting a target DNA sequence comprising the CDKL5 gene with a 182 / 189 B1195.70194WO00 13324217.2prime editor and a prime editing guide RNA (pegRNA), wherein the pegRNA comprises a spacer sequence of GCTGTCAAGTATCTGTCAGCT (SEQ ID NO: 7), or a spacer 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 GCTGTCAAGTATCTGTCAGCT (SEQ ID NO: 7), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCTGTCAAGTATCTGTCAGCT (SEQ ID NO: 7).

76. The method of claim 75, wherein the pegRNA comprises the sequence GCTGTCAAGTATCTGTCAGCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATTTCCTAAGAAC CTGTTGAAGTTGGACCCAGCTGACAGATACTCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 39), 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 GCTGTCAAGTATCTGTCAGCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATTTCCTAAGAAC CTGTTGAAGTTGGACCCAGCTGACAGATACTCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 39), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCTGTCAAGTATCTGTCAGCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATTTCCTAAGAAC CTGTTGAAGTTGGACCCAGCTGACAGATACTCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 39).

77. The method of claim 75 or 76 further comprising contacting the target DNA sequence with a nicking guide RNA.

78. The method of claim 77, wherein the nicking guide RNA comprises the sequence GATTTCAAACCCAGAGACTTC (SEQ ID NO: 15), GTTCTGGATCGTTCTCCTTCA (SEQ ID NO: 16), or GTACTGAAGTTGGACCCAGCT (SEQ ID NO: 17), 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 GATTTCAAACCCAGAGACTTC (SEQ ID NO: 15), GTTCTGGATCGTTCTCCTTCA (SEQ ID NO: 16), or GTACTGAAGTTGGACCCAGCT 183 / 189 B1195.70194WO00 13324217.2(SEQ ID NO: 17), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GATTTCAAACCCAGAGACTTC (SEQ ID NO: 15), GTTCTGGATCGTTCTCCTTCA (SEQ ID NO: 16), or GTACTGAAGTTGGACCCAGCT (SEQ ID NO: 17).

79. A pegRNA comprising a spacer sequence of GCTGTCAAGTATCTGTCAGCT (SEQ ID NO: 7), or a spacer 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 GCTGTCAAGTATCTGTCAGCT (SEQ ID NO: 7), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCTGTCAAGTATCTGTCAGCT (SEQ ID NO: 7).

80. The pegRNA of claim 79, wherein the pegRNA comprises the sequence GCTGTCAAGTATCTGTCAGCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATTTCCTAAGAAC CTGTTGAAGTTGGACCCAGCTGACAGATACTCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 39), 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 GCTGTCAAGTATCTGTCAGCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATTTCCTAAGAAC CTGTTGAAGTTGGACCCAGCTGACAGATACTCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 39), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GCTGTCAAGTATCTGTCAGCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCATTTCCTAAGAAC CTGTTGAAGTTGGACCCAGCTGACAGATACTCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 39).

81. A nicking guide RNA comprising the sequence GATTTCAAACCCAGAGACTTC (SEQ ID NO: 15), GTTCTGGATCGTTCTCCTTCA (SEQ ID NO: 16), or GTACTGAAGTTGGACCCAGCT (SEQ ID NO: 17), 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% 184 / 189 B1195.70194WO00 13324217.2identical to GATTTCAAACCCAGAGACTTC (SEQ ID NO: 15), GTTCTGGATCGTTCTCCTTCA (SEQ ID NO: 16), or GTACTGAAGTTGGACCCAGCT (SEQ ID NO: 17), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GATTTCAAACCCAGAGACTTC (SEQ ID NO: 15), GTTCTGGATCGTTCTCCTTCA (SEQ ID NO: 16), or GTACTGAAGTTGGACCCAGCT (SEQ ID NO: 17).

82. A method of correcting a cluster of mutations in exon 8 of CDKL5 by prime editing comprising contacting a target DNA sequence comprising the CDKL5 gene with a prime editor and a prime editing guide RNA (pegRNA), wherein the pegRNA comprises a spacer sequence of CAGAGTACGTTGCCACCAGA, or a spacer 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 CAGAGTACGTTGCCACCAGA, or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to CAGAGTACGTTGCCACCAGA.

83. The method of claim 82, wherein the pegRNA comprises the sequence CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGTAAGGGCTCT AGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTTAAAC CAACTAGAATTTTTTT (SEQ ID NO: 40) or CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCACCCAAGTAAGG GCTCTAGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 41), 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 CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGTAAGGGCTCT AGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTTAAAC CAACTAGAATTTTTTT (SEQ ID NO: 40) or CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCACCCAAGTAAGG 185 / 189 B1195.70194WO00 13324217.2GCTCTAGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 41), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGTAAGGGCTCT AGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTTAAAC CAACTAGAATTTTTTT (SEQ ID NO: 40) or CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCACCCAAGTAAGG GCTCTAGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 41).

84. The method of claim 82 or 83 further comprising contacting the target DNA sequence with a nicking guide RNA.

85. The method of claim 84, wherein the nicking guide RNA comprises the sequence GTGGAAATGTCATTCTATTTT (SEQ ID NO: 18), 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 GTGGAAATGTCATTCTATTTT (SEQ ID NO: 18), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GTGGAAATGTCATTCTATTTT (SEQ ID NO: 18).

86. A pegRNA comprising a spacer sequence of CAGAGTACGTTGCCACCAGA (SEQ ID NO: 8), or a spacer 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 CAGAGTACGTTGCCACCAGA (SEQ ID NO: 8), or a spacer sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to CAGAGTACGTTGCCACCAGA (SEQ ID NO: 8).

87. The pegRNA of claim 86, wherein the pegRNA comprises the sequence CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGTAAGGGCTCT AGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTTAAAC 186 / 189 B1195.70194WO00 13324217.2CAACTAGAATTTTTTT (SEQ ID NO: 40) or CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCACCCAAGTAAGG GCTCTAGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 41), 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 CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGTAAGGGCTCT AGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTTAAAC CAACTAGAATTTTTTT (SEQ ID NO: 40) or CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCACCCAAGTAAGG GCTCTAGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 41), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGTAAGGGCTCT AGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTTAAAC CAACTAGAATTTTTTT (SEQ ID NO: 40) or CAGAGTACGTTGCCACCAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCACCCAAGTAAGG GCTCTAGGGAGCGACACCGGCTGGTGGCAACCGCGGTTCTATCTAGTTACGCGTT AAACCAACTAGAATTTTTTT (SEQ ID NO: 41).

88. A nicking guide RNA comprising the sequence GTGGAAATGTCATTCTATTTT (SEQ ID NO: 18), 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 GTGGAAATGTCATTCTATTTT (SEQ ID NO: 18), or a sequence comprising one, two, three, four, or five mutations, insertions, or deletions relative to GTGGAAATGTCATTCTATTTT (SEQ ID NO: 18). 187 / 189 B1195.70194WO00 13324217.

289. A method for treating CDKL5 deficiency disorder (CDD) by prime editing comprising contacting a target DNA sequence comprising a CDKL5 gene comprising a pathogenic mutation with a prime editor, or a nucleotide sequence encoding a prime editor, and a pegRNA, or a nucleotide sequence encoding a prime editor, wherein the prime editor comprises an amino acid sequence of any one of SEQ ID NOs: 355-357, 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 an amino acid sequence of any one of SEQ ID NOs: 355-357.

90. The method of claim 89, wherein the pegRNA is capable of correcting a c.1412delA mutation in the CDKL5 gene.

91. The method of claim 89 or 90, wherein the pegRNA is a pegRNA of any one of claims 33-40.

92. A complex comprising a pegRNA of any one of claims 1-40 and a prime editor. 188 / 189 B1195.70194WO00 13324217.2

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