Base editing methods and compositions for treating dravet syndrome

Abstract

The present disclosure provides a method for correcting SCN1A mutations linked to Dravet syndrome that uses adenine base editors. The disclosure provides compositions and methods, including adenine base editors (ABEs), guide RNAs, complexes, nucleic acid molecules, vectors, delivery systems, and pharmaceutical compositions aimed at targeting SCN1A mutations.

Description

BASE EDITING METHODS AND COMPOSITIONS FOR TREATING DRAVET SYNDROMERELATED APPLICATIONS

[0001] This application claims priority under 35 U. S. C. § 119(e) to U. S. Provisional Application, U. S. S. N. 63 / 749,384, filed January 24, 2025, and U. S. Provisional Application, U. S. S. N. 63 / 807,070, filed May 16, 2025, each of which is incorporated herein by reference.GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant Nos. AI142756, HG009490, and GM 118062 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (B1195.70210WO00-SEQ-TNG.xml; Size: 716,677 bytes; and Date of Creation: January 23, 2026) are herein incorporated by reference in their entirety.BACKGROUND

[0004] Dravet syndrome (DS) is a lifelong severe neurodevelopmental disorder and epileptic encephalopathy defined by treatment-resistant epilepsy, temperature-sensitive seizures, developmental delay / intellectual disability, features of autism spectrum disorder, and high rate of sudden unexpected death in epilepsy (SUDEP). DS generally begins in infancy and proceeds with accumulating morbidity that significantly impacts individuals throughout their lifetime. DS is caused by heterozygous loss-of-function variants in SCN1A, which encodes the voltage-gated sodium channel a subunitNavl.l. This gene is preferentially expressed in GABAergic inhibitory neurons (INs). Loss of one functional copy of Nav1.1 impairs the generation and propagation of action potentials in these cells, especially in fast-spiking parvalbumin-expressing inhibitory neurons (PV-INs).

[0005] Current treatment options for DS are limited and there are no cures. Also, some medications have significant side effects, thereby limiting their medical use. Standard of care treatment currently involves regimens of medications for inhibiting and / or regulating seizure activity. Further, DS is a spectrum disorder, meaning patients present with a wide range of severity and seizure types, and no two patients respond to treatment the same way. Common 1 / 224Bl 195.70210WO00#14840465vlmedications include administration of anti-seizure agents valproate, fenfluramine, stiripentol, clobazam, cannabidiol, topiramate. Medications which exacerbate seizures due to their effects on sodium ion channels include carbamazepine, oxcarbazepine, lamotrigine, and phenytoin. Even with standard of care medications, most patients will not achieve complete seizure freedom.

[0006] Thus, there is an unmet medical need to develop new treatment options for DS.Treatment options that provide a more durable treatment with limited side effects, such as by gene editing, for DS would significantly advance the art.SUMMARY

[0007] The present disclosure relates, in part, to the discovery that adenine base editing is an effective approach for correcting DS-causing mutations in SCN1A. Thus, as described herein, the present disclosure provides adenine base editing compositions — including adenine base editors (ABEs), guide RNAs effective for targeting and correcting SCN1A mutations, complexes comprising ABEs and guide RNAs effective for targeting and correcting SCN1A mutations, nucleic acid molecules encoding ABEs and guide RNAs effective for targeting and correcting SCN1A mutations, vectors and delivery systems comprising nucleic acid molecules encoding ABEs and guide RNAs effective for targeting and correcting SCN1A mutations, cells and tissues comprising ABEs and guide RNAs targeting SCN1A mutations and / or comprising nucleic acid molecules encoding such ABEs and guide RNAs effective for targeting and correcting SCN1A mutations, and pharmaceutical compositions and kits comprising ABEs and guide RNAs effective for targeting and correcting SCN1A mutations or nucleic acid molecules encoding such ABEs and guide RNAs effective for targeting and correcting SCN1A mutations or delivery systems / vectors comprising nucleic acid molecules encoding such ABEs and guide RNAs effective for targeting SCN1A mutations — and methods of correcting one or more SCN1A mutations in an SCN1A mutant allele (such as those listed in Table 1, including the SCN1A (R613X) mutant allele) under in vivo, ex vivo, or in vivo conditions using the herein disclosed ABEs, guide RNAs, and compositions and / or vectors comprising or encoding same.

[0008] Further, as described in the Examples, the present inventors have tested adenine base editing in a mouse model comprising an exemplary SCN1A mutant allele, namely the SCN1A-p. R613X mutant allele, which is a recurrent variant identified in several DS patients. The 2 / 224Bl 195.70210WO00#14840465vlmouse model is an SCN1AR613X / +mouse model which closely replicates the key molecular and phenotypic features of the disease in humans. The inventors have shown effective editing and / or restoration of the SCN1A-p.R613X mutant allele to wildtype in the mouse model and that as a result adenine base editing can be an effective approach for correcting and / or restoring any ABE-targetable SCN1A mutation in humans, such as any of those human mutations provided in Table 1. The SCN1A-p.R613X mutant allele is an example of a nonsense SCN1A mutation. Such mutations cause premature truncations (nonsense, splicesite, and frameshifting) and exhibit the highest penetrance6,7. These variants cause truncated or misfolded proteins, and their pathogenicity is compounded by nonsense mediated decay of the dysfunctional transcript8. In the ClinVar database, about 10% of listed DS-causing nonsense variants are Arg> Ter (i.e., Arg to stop codons) codons, which are caused by CGA> TGA transition mutations.

[0009] Adenine base editing (ABE) is a system capable of converting A T base pairs to G C base pairs (or T A base pairs to C G base pairs) at specific locations in the genome without the formation of double-stranded DNA breaks. ABEs comprise a nucleic acid-programmable DNA-binding domain (such as a Cas9 nickase) fused to an adenosine deaminase, and this fusion enables modularity to expand the editor’s scope. ABEs have been used with a variety of Cas domains to change both the editing window and PAM preference9–12. These modifications more precisely position the target base for deamination and prevent editing of unwanted bystander bases. The deaminase domain can be similarly interchanged to narrow or broaden the editing window, increase enzyme efficiency, or reduce Cas domain-independent deamination events13. Since their invention, ABEs have been used in ex vivo and in vivo animal applications such as the treatment of sickle cell disease14, Hutchinson-Gilford progeria syndrome15, acute lymphoblastic leukemia16, spinal muscular atrophy17, and other genetic diseases. Presented herein is the ABE-mediated correction of SCN1AR613X / +rescuing mice from DS pathology, thereby demonstrating that any ABE-targetable SCN1A mutant allele, such as any of those listed in Table 1, can be corrected. Thus, ABE editing may be used as a treatment for DS or symptoms thereof.

[0010] Mouse models of SCN1A haploinsufficiency recapitulate many aspects of the human phenotype, including febrile seizures, SUDEP, and cognitive deficits1–5. Symptom onset in DS occurs between 4-8 months of age, coinciding with expression of SCN1A. This is suggestive of a window for disease prevention which is also recapitulated in mouse models 3 / 224Bl 195.70210WO00#14840465vl(-P18-21 in mice). Correcting DS-causing mutations prior to the onset of SCN1A expression could offer complete disease rescue. The human and mouse SCN1A genes have 98.2% amino acid identity and 90.5% coding sequence identity, potentially allowing direct translation of preclinical compounds.

[0011] Thus, in one aspect, the present disclosure provides methods of correcting a SCN1A mutant allele comprising at G> A or C> T mutation relative to a wildtype SCN1A nucleotide sequence, said method comprising contacting a nucleic acid sequence comprising the SCN1A mutant allele with an adenine base editor (ABE) and a gRNA targeting the ABE to the SCN1A mutant allele, thereby restoring the G> A or C> T mutation to wildtype to produce a corrected SCN1A mutant allele. In some embodiments, the wildtype SCN1A nucleotide sequence comprises SEQ ID NO: 114 [human] or SEQ ID NO: 116 [mouse]. In some embodiments, the wildtype SCN1A nucleotide sequence encodes wildtype voltage-gated sodium channel a subunit (NAV1.1) comprising the amino acid sequence of SEQ ID NO: 106 [human] or SEQ ID NO: 108 [mouse]. In certain embodiments, the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a G>A or C>T mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114 [human]. In certain embodiments, the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a G>A mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114 [human]. In certain embodiments, the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a C>T mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114 [human]. In certain embodiments, the SCN1A mutant allele comprises a T mutation at position 1837 (1837C>T) relative to wildtype SCN1A nucleotide sequence of SEQ ID NO: 114, thereby introducing a stop codon. In some embodiments, the SCN1A mutant allele encodes a variant of voltage-gated sodium channel a subunit (NAV1.1) having the amino acid change specified in Table 1 relative to wildtype NAV1.1 of SEQ ID NO: 106. In some embodiments, the SCN1A mutant allele encodes an NAV 1.1 variant comprising an R613X substitution relative to NAV 1.1 wildtype amino acid sequence of SEQ ID NO: 106, wherein X is a termination codon.

[0012] In some embodiments, the gRNA targets a strand at the SCN1A mutant allele. In some embodiments, the gRNA comprises a spacer having a nucleotide sequence that is the inverse complement of a target strand at the SCN1A mutant allele, wherein the SCN1A mutant allele 4 / 224Bl 195.70210WO00#14840465vlis any one of the SCN1A mutant alleles identified in Table 1. In some embodiments, the gRNA comprises a nucleotide sequence of any one of the gRNA sequences of Tables 4A or 4B, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the nucleotide sequences of Tables 4A or 4B. In some embodiments, the gRNA comprises a spacer comprising a nucleotide sequence of any one of the gRNA spacers of Tables 4A or 4B, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the nucleotide sequences of Tables 4A or 4B.

[0013] In some embodiments, the adenine base editor (ABE) comprises a nucleic acid-programmable DNA-binding protein (napDNAbp) and an adenosine deaminase. In some embodiments, the nucleic acid-programmable DNA-binding protein (napDNAbp) comprises a Cas9 protein. In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease-inactive Cas9 (dCas9). In some embodiments, the Cas9 protein is a Streptococcus pyogenes Cas9 protein or a variant thereof. In some embodiments, the napDNAbp comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1-23, 139-140. In some embodiments, the napDNAbp comprises the amino acid sequence of any one of SEQ ID NOs: 1-23, 139-140. In some embodiments, the napDNAbp is the napDNAbp of ABE7.10, ABE8e, ABE8e-(V106W)-SaCas9, ABE8e-(V106W)-VRQR / NG, or ABE8e-(V106W)-SpCas9. In some embodiments, the adenosine deaminase comprises the amino acid sequence of any one of SEQ ID NOs: 24-70. In some embodiments, the adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 24-70. In some embodiments, the adenosine deaminase is the adenosine deaminase of ABE7.10, ABE8e, ABE8e-(V106W)-SaCas9, ABE8e-(V106W)-VRQR / NG, or ABE8e-(V106W)-SpCas9. In certain embodiments, the adenine base editor (ABE) is selected from the group consisting of ABE7.10, ABE8e, ABE8e-(V106W)-SaCas9, ABE8e-(V106W)-VRQR / NG, or ABE8e-(V106W)-SpCas9. In certain embodiments, the adenine base editor (ABE) comprises the amino acid sequence of any one of SEQ ID NOs: 99-105, or a sequence having at least 80%, at least 85%, at least 5 / 224Bl 195.70210WO00#14840465vl90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 99-105.

[0014] In some embodiments, the adenine base editor (ABE) further comprises one or more nuclear localization sequences (NLS). In certain embodiments, the one or more NLS comprises the amino acid sequence of any one of SEQ ID NOs: 71-82, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 71-82. In some embodiments, the adenine base editor (ABE) is a fusion protein further comprising a linker between the napDNAbp and the adenosine deaminase. In certain embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 83-98, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 83-98. In some embodiments, the fusion protein comprises in the N-to-C terminal direction the [napDNAbp]-[linker]-[adenosine deaminase]. In some embodiments, the fusion protein comprises in the N-to-C terminal direction the [adenosine deaminase]-[linker]-[napDNAbp]. In some embodiments, the adenine base editor (ABE) is split fusion protein comprising an N-terminal portion and a C-terminal protein. In certain embodiments, the N-terminal portion comprises an N-intein and the C-terminal portion comprises a C-intein.

[0015] In some embodiments, the method results in correction of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the SCN1A mutant allele in a population of cells. In some embodiments, the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a G> A or C> T mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114. In some embodiments, the SCN1A mutant allele comprises a C> T mutation at position 1837 (1837C> T) relative to wildtype SCN1A nucleotide sequence of SEQ ID NO: 114, thereby introducing a stop codon. In some embodiments, the SCN1A mutant allele encodes a variant of voltage-gated sodium channel a subunit (NAV1.1) having the amino acid change specified in Table 1 relative to wildtype NAV1.1 of SEQ ID NO: 106. In some embodiments, the SCN1A mutant allele encodes an NAVI.1 variant comprising an R613X substitution relative to NAVI.1 wildtype amino acid sequence of SEQ ID NO: 106, wherein X is a termination codon.6 / 224Bl 195.70210WO00#14840465vl

[0016] In some embodiments, one or more polynucleotides encoding the adenine base editor (ABE) and the gRNA are delivered to the SCN1A mutant allele in one or more delivery vehicles. In some embodiments, the one or more delivery vehicles comprise a viral vector or a non-viral vector, or a combination thereof. In certain embodiments, the viral vector is an AAV vector or lentivirus vector. In certain embodiments, the non-viral vector is a virus-like particle (VLP). In certain embodiments, the non-viral vector is a nanoparticle. In certain embodiments, the nanoparticle is a lipid nanoparticle (LNP), polymeric nanoparticle, inorganic nanoparticle, liposome, or a nanostructured lipid carrier. In some embodiments, the one or more delivery vehicles is a targeted delivery vehicle. In some embodiments, the targeted delivery vehicle comprises at least one targeting agent capable of targeting the delivery vehicle to a desired cell. In certain embodiments, the desired cell is a GABA interneuron. In some embodiments, the targeting agent is an antibody or antigen binding domain which is capable of binding to an antigen of the desired cell.

[0017] In some embodiments, the adenine base editor (ABE) and the gRNA are encoded on the same polynucleotide. In some embodiments, the adenine base editor (ABE) and the gRNA are encoded on different polynucleotides. In some embodiments, the N-terminal portion and C-terminal portion of the split adenine base editor (ABE) are encoded by different polynucleotides. In some embodiments, the different polynucleotides are encapsulated by the same delivery vehicle. In certain embodiments, the delivery vehicle is an AAV or lentivirus vector. In some embodiments, the different polynucleotides are encapsulated by different delivery vehicles. In certain embodiments, each of the different delivery vehicles comprise an AAV vector or lentivirus vector.

[0018] In some embodiments, the method results in the treatment, reduction, or prevention of Dravet Syndrome or at least one symptom or condition associated with a SCN1A mutant allele in a subject. In some embodiments, the at least one symptom or condition associated with the SCN1A mutant allele comprises epilepsy, seizures, developmental delay / intellectual disability, autism spectrum disorder, SUDEP (high rate of sudden unexpected death in epilepsy), and / or severe encephalopathy. In some embodiments, the step of contacting corrects a G> A transition mutation in a SCN1A mutant allele. In certain embodiments, the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a G> A mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114. In certain embodiments, the step of contacting corrects a C>T transition 7 / 224Bl 195.70210WO00#14840465vlmutation in a SCN1A mutant allele. In certain embodiments, the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a C>T mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114. In some embodiments, correction of the C>T transition mutation in the SCN1A gene results in correction of an R613X mutation.

[0019] In some embodiments, the step of contacting a nucleic acid sequence comprising the SCN1A mutant allele is performed in a cell. In some embodiments, the cell is a neuronal cell. In certain embodiments, the neuronal cell is a GABA (gamma-aminobutyric acid) interneuron. In some embodiments, the step of contacting a nucleic acid sequence comprising the SCN1A mutant allele is performed in vivo. In some embodiments, the step of contacting a nucleic acid sequence comprising the SCN1A mutant allele is performed in vitro. In some embodiments, the step of contacting a nucleic acid sequence comprising the SCN1A mutant allele is performed ex vivo. In some embodiments, the step of contacting a nucleic acid sequence comprising the SCN1A mutant allele is performed in a subject. In certain embodiments, the subject is a human. In certain embodiments, the subject is a mouse.

[0020] In another aspect, the present disclosure provides methods of increasing expression of SCN1A, said method comprising contacting a nucleic acid sequence comprising SCN1A with an adenine base editor (ABE) and a gRNA targeting the ABE to a splice acceptor sequence upstream of exon 20N in SCN1A. In some embodiments, introduction of an A-to-G mutation in the splice acceptor sequence by the ABE prevents nonproductive splicing of SCN1A. In some embodiments, the method is a method for treating Dravet syndrome (e.g., in a subject, such as a human) comprising contacting a nucleic acid sequence comprising SCN1A with an adenine base editor (ABE) and a gRNA targeting the ABE to a splice acceptor sequence upstream of exon 20N in SCN1A and introducing an A-to-G mutation in the splice acceptor sequence, thereby preventing nonproductive splicing of SCN1A.

[0021] In another aspect, the present disclosure provides complexes for correcting a SCN1A mutant allele having a G>A or C>T mutation in a SCN1A gene, said complex comprising an adenine base editor (ABE) and a gRNA targeting the ABE to the SCN1A mutant allele, thereby restoring the G>A or C>T mutation to wildtype to produce a corrected SCN1A mutant allele. In some embodiments, the wildtype SCN1A nucleotide sequence comprises SEQ ID NO: 114 [human] or SEQ ID NO: 116 [mouse]. In some embodiments, the wildtype SCN1A nucleotide sequence encodes wildtype voltage-gated sodium channel a subunit 8 / 224Bl 195.70210WO00#14840465vl(NAV1.1) comprising the amino acid sequence of SEQ ID NO: 106 [human] or SEQ ID NO: 108 [mouse]. In certain embodiments, the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a G>A or C>T mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114. In certain embodiments, the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a G>A mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114. In certain embodiments, the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a C>T mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114. In some embodiments, the SCN1A mutant allele comprises a T mutation at position 1837 (1837C>T) relative to wildtype SCN1A nucleotide sequence of SEQ ID NO: 114, thereby introducing a stop codon. In some embodiments, the SCN1A mutant allele encodes a variant of voltage-gated sodium channel a subunit (NAV1.1) having the amino acid change specified in Table 1 relative to wildtype NAV1.1 of SEQ ID NO: 106. In some embodiments, the SCN1A mutant allele encodes an NAV1.1 variant comprising an R613X substitution relative to NAV1.1 wildtype amino acid sequence of SEQ ID NO: 106, wherein X is a termination codon.

[0022] In some embodiments, the gRNA targets a strand at the SCN1A mutant allele. In some embodiments, the SCN1A mutant allele encodes an NAV 1.1 variant comprising an R613X substitution relative to NAV1.1 wildtype amino acid sequence of SEQ ID NO: 106, wherein X is a termination codon. In some embodiments, the gRNA comprises a spacer having a nucleotide sequence that is the inverse complement of a target strand at the SCN1A mutant allele, wherein the SCN1A mutant allele is any one of the SCN1A mutant alleles identified in Table 1. In certain embodiments, the gRNA comprises a nucleotide sequence of any one of the gRNA sequences of Tables 4A or 4B, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the nucleotide sequences of Tables 4A or 4B. In certain embodiments, the gRNA comprises a spacer comprising a nucleotide sequence of any one of the gRNA spacers of Tables 4A or 4B, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the nucleotide sequences of Tables 4A or 4B.9 / 224Bl 195.70210WO00#14840465vl

[0023] In some embodiments, the adenine base editor (ABE) comprises a nucleic acid-programmable DNA-binding protein (napDNAbp) and an adenosine deaminase. In some embodiments, the nucleic acid-programmable DNA-binding protein (napDNAbp) comprises a Cas9 protein. In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9) or a nuclease-inactive Cas9 (dCas). In certain embodiments, the napDNAbp comprises 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: 1-23, 139-140. In certain embodiments, the napDNAbp is the napDNAbp of ABE7.10, ABE8e, ABE8e-(V106W)-SaCas9, ABE8e-(V106W)-VRQR / NG, or ABE8e-(V106W)-SpCas9. In certain embodiments, the adenosine deaminase comprises 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: 24-70. In certain embodiments, the adenosine deaminase is the adenosine deaminase of ABE7.10, ABE8e, ABE8e-(V106W)-SaCas9, ABE8e-(V106W)-VRQR / NG, or ABE8e-(V106W)-SpCas9. In certain embodiments, the adenine base editor (ABE) is selected from the group consisting of ABE7.10, ABE8e, ABE8e-(V106W)-SaCas9, ABE8e-(V106W)-VRQR / NG, and ABE8e-(V106W)-SpCas9. In some embodiments, the adenine base editor (ABE) comprises the sequence of any one of SEQ ID NOs: 99-105, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of any one of SEQ ID NOs: 99-105. In certain embodiments, the adenine base editor (ABE) comprises the sequence of SEQ ID NO: 99-105. In some embodiments, the adenine base editor is ABE8e-NRRH, ABE8e-V106W-NRRH, ABE8e-SpyMAC, or ABE8e-iSpyMAC.

[0024] In some embodiments, the adenine base editor (ABE) further comprises one or more nuclear localization sequences (NLS). In certain embodiments, the one or more NLS comprise the sequence of any one of SEQ ID NOs: 71-82, 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 of any one of SEQ ID NOs: 71-82. In some embodiments, the adenine base editor (ABE) is a fusion protein further comprising a linker between the napDNAbp and the adenosine deaminase. In certain embodiments, the linker comprises the sequence of any one of SEQ ID NOs: 83-98, 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 of any one of SEQ ID NOs: 83-98. In some embodiments, the fusion protein 10 / 224Bl 195.70210WO00#14840465vlcomprises in the N-to-C terminal direction the [napDNAbp]-[linker]-[adenosine deaminase]. In some embodiments, the fusion protein comprises in the N-to-C terminal direction the [adenosine deaminase]-[linker]-[napDNAbp], In some embodiments, the adenine base editor (ABE) is split fusion protein comprising an N-terminal portion and a C-terminal protein. In certain embodiments, the N-terminal portion comprises an N-intein and the C-terminal portion comprises a C-intein.

[0025] In another aspect, the present disclosure provides guide RNAs (gRNAs) comprising (a) a spacer sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence GGUCAUCGGGGCACAAACAA (SEQ ID NO: 124) [human], or a spacer sequence comprising one, two, three, four, or five nucleotide substitutions relative to the sequence GGUCAUCGGGGCACAAACAA [human] (SEQ ID NO: 124) or (b) a spacer sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence GCGUGUCAUCGAGGAACGAACA (SEQ ID NO: 122) [mouse] or a spacer sequence comprising one, two, three, four, or five nucleotide substitutions relative to the sequence GCGUGUCAUCGAGGAACGAACA (SEQ ID NO: 122) [mouse],

[0026] In another aspect, the present disclosure provides gRNAs comprising a spacer sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence GUAUAGGAUAAUCUUGCUCC (SEQ ID NO: 240), GGUAUAGGAUAAUCUUGCUC (SEQ ID NO: 241), GAUAUAGGAUAAUCUUGCUC (SEQ ID NO: 242), GUAUAGGAUAAUCUUGCUC (SEQ ID NO: 243), or GUAUAGGAUAAUCUUGCUC (SEQ ID NO: 243).

[0027] In another aspect, the present disclosure provides polynucleotide molecules comprising a nucleotide sequence encoding a gRNA as provided herein. In some embodiments, the polynucleotide molecule further comprises a nucleotide sequence encoding an adenine base editor (ABE). In some embodiments, the adenine base editor is a split adenine base editor.

[0028] In another aspect, the present disclosure provides compositions comprising a first polynucleotide molecule encoding any of the gRNAs provided herein and one or more11 / 224Bl 195.70210WO00#14840465vladditional polynucleotide molecules encoding an adenine base editor (ABE). In some embodiments, the adenine base editor (ABE) is a split adenine base editor (ABE).

[0029] In another aspect, the present disclosure provides vectors comprising a polynucleotide molecule as disclosed herein or a composition as disclosed herein. In some embodiments, each of the vectors is a viral or non-viral vector. In certain embodiments, the viral vector is an AAV or a lentivirus vector. In certain embodiments, the non-viral vector is a nanoparticle. In certain embodiments, the nanoparticle is a lipid nanoparticle (LNP), polymeric nanoparticle, inorganic nanoparticle, liposome, or a nanostructured lipid carrier. In certain embodiments, the non-viral particle is a virus-like particle (VLP).

[0030] In another aspect, the present disclosure provides pharmaceutical compositions comprising any of the complexes, gRNAs, polynucleotide molecules, compositions, or vectors provided herein, or any combinations thereof, and at least one pharmaceutically acceptable excipient.

[0031] In another aspect, the present disclosure provides cells comprising any of the complexes, gRNAs, polynucleotide molecules, compositions, or vectors provided herein, or any combinations thereof, and at least one pharmaceutically acceptable excipient. In some embodiments, the cell is a GABA interneuron.

[0032] In another aspect, the present disclosure provides tissues comprising any of the complexes, gRNAs, polynucleotide molecules, compositions, or vectors provided herein, or any combinations thereof, and at least one pharmaceutically acceptable excipient. In some embodiments, the tissue is a neuronal or CNS tissue.

[0033] In another aspect, the present disclosure provides kits comprising any of the complexes, gRNAs, polynucleotide molecules, compositions, or vectors provided herein, or any combinations thereof, and at least one pharmaceutically acceptable excipient, optionally an administration device, and optionally a set of instructions for use.

[0034] In another aspect, the present disclosure provides for the use of any of the complexes, gRNAs, polynucleotide molecules, compositions, or vectors provided herein, or any combinations thereof, in the manufacture of a medicament for the treatment of Dravet Syndrome or at least one symptom or condition associated with a SCN1A mutant allele in a subject. In some embodiments, the at least one symptom or condition associated with the SCN1A mutant allele comprises epilepsy, seizure, developmental delay / intellectual disability,12 / 224Bl 195.70210WO00#14840465vlautism spectrum disorder, SUDEP (high rate of sudden unexpected death in epilepsy), and / or severe encephalopathy.

[0035] In another aspect, any of the complexes, gRNAs, polynucleotide molecules, compositions, or vectors provided herein, or any combinations thereof for use in the treatment of Dravet Syndrome or at least one symptom or condition associated with a SCN1A mutant allele in a subject. In some embodiments, the at least one symptom or condition associated with the SCN1A mutant allele comprises epilepsy, seizure, developmental delay / intellectual disability, autism spectrum disorder, SUDEP (high rate of sudden unexpected death in epilepsy), and / or severe encephalopathy.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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.

[0037] FIG. 1 shows background on Dravet Syndrome (DS).

[0038] FIG. 2 shows a DS molecular mechanism.

[0039] FIGs. 3A-3B show current DS treatment modalities.

[0040] FIGs. 4A-4C show a description of a DS mouse model of interest. Figure discloses “HGER” as SEQ ID NO: 289.

[0041] FIGs. 5A-5B provide sequence context for the R613X mouse model.

[0042] FIG. 6 shows initial in vitro correction of SCN1AR613Xin Neuro-2A (N2A) cells. N2A cells engineered to harbor a homozygous SCN1AR6I3Xmutation were transfected with ABE plasmid and sgRNA plasmid. After 72 hours, editing efficiency was assessed by HTS. All three assayed editing strategies demonstrated efficient on-target editing efficiency and detectable, nonsilent bystander editing at V610 and R612. Because R612Q is reported as a benign mutation in the gnomAD database, ABE8e-VRQR and ABE8e-NG were prioritized for further studies.

[0043] FIGs. 7A-7B show optimization of editing strategy with ABE8e-V106W. The mutation of V106W in the TadA* deaminase domain has been shown to decrease off-target transcriptome deamination and constrict the ABE editing window. This ABE8e variant substantially decreased indel formation and bystander editing at C7 while maintaining 13 / 224Bl 195.70210WO00#14840465vlefficient stop codon correction. This ABE8e-V106W editing strategy performs with similar efficiency and purity when targeting the concomitant SCN1AR6I3Xmutation in HEK293T cells engineered to harbor the homozygous mutation.

[0044] FIG. 8 shows Cas9-dependent off-target nomination with CIRCLE-seq. CIRCLE-seq is an in vitro method for nominating genomic loci which may be altered by Cas9-dependent off-target editing. In all, 89 off-target sites were nominated by CIRCLE-seq.

[0045] FIGs. 9A-9C show in vivo editing efficiency analysis. P0 and Pl SCN1AR613X / +mice were treated with a three-virus ABE: GFP (~10: 1 viral genomes) AAV9 system by intracerebroventricular (ICV) injection. From resected brain tissues, nuclei were isolated and sorted by GFP+ signal. From both bulk and GFP+ nuclei, both gDNA and RNA were isolated in linked samples, and editing efficiency was measured by HTS.

[0046] FIGs. 10A-10B show molecular rescue of SCN1A transcripts and Navl.l protein. Transcripts containing premature stop codons (PTCs) are known to undergo nonsense mediated decay (NMD). P0 ABE8e-V106W-VRQR ICV treatment rescues SCN1A transcripts from NMD in SCN1AR613X / +mice at P21 in whole cell lysates from brain hemispheres. SCN1AR613X / +mice are also known to have decreased Nav1.1 expression in isolated hippocampi.

[0047] FIG. 11 shows physiological modulation of base edited inhibitory neurons (INs). Loss of functional Navl.l protein leads to hypoexcitability and lowered action potential (AP) frequency in inhibitory neurons. In INs from ABE-treated SCN1AR613X / +mice, increased AP frequency in GFP+ nuclei compared to GFP- nuclei was observed with increasing current.

[0048] FIGs. 12A-12B show gross phenotypic rescue of key DS symptoms. DS mice experience febrile seizures, similar to DS patients. Following ABE-treatment, 10 of \2SCN1AR613X / +mice remained seizure free, and two experienced seizures at temperatures >42.5°C (-108 °F), whereas untreated SCN1AR613X / +mice experienced seizures at an average core temperature of 40.3 °C. Additionally, ABE treatment saved SCN1AR613X / +mice from sudden unexpected death in epilepsy.

[0049] FIGs. 13A-13E show in vitro optimization of the ABE for the correction of ScnlctR6i3X. FIG. 13A shows sequence alignment of SCN1A in the region containing R613X. Sequence differences between the mouse (Jackson laboratories strain #034129) and human (GRCh37) disease allele nucleotide sequences are bolded. Figure discloses SEQ ID NOS 152-156, 152, 157-158, 255, and 156, respectively, in order of appearance. FIG. 13B shows 14 / 224Bl 195.70210WO00#14840465vlediting efficiencies of ABE8e correction strategies in N2A 5cw7a-p. R613X cells. Samples were sequenced 3 days post-plasmid transfection. FIG. 13C shows a comparison of ABE8e editing outcomes with and without the addition of V106W to the deaminase domain. Samples were sequenced 3 days post-plasmid transfection. FIG. 13D shows ABE8e-V106W editing outcomes in HEK293T £GVL4-p. R613X cells, sequenced 3 days after plasmid transfection. FIG. 13E shows the percentage of sequencing reads with A»T-to-G»C substitutions in the editing window of CIRCLE-seq nominated genomic loci (GROG 7) above background (untreated samples) in HEK293T 5 VA4-p. R613X cells. Genomic DNA was extracted from cells untreated (N=3) or 3 days post transfection with ABE8e-V106W-VRQR and SCN1A R613X sgRNA (N=3). Off-target editing with P > 0.01 compared to untreated control are labeled with hollow circles, and those with P < 0.01 are labeled with solid circles. Each dot represents the mean values of three biological replicates. Bar values and error bars in FIGs.13B-13D show mean±SD, respectively, ofN>3 independent biological samples (shown as black dots).

[0050] FIGs. 14A-14F show base editing in Scn1aR613X / +mice. FIG. 14A provides a schematic of dual-AAV9 ABE editing strategy. Mice were injected at P0 by ICV injection of lei 1 total vg of ABE8e-V106W-AAV or 7.2el 1 total vg of ABE8e-AAV, each of which contained equal amounts of N- and C-terminal halves ABE AAVs, and lelO vg GFP-KASH. FIG. 14B shows sequencing analysis of Senia in genomic DNA and in nuclear RNA from bulk nuclei and GFP+nuclei. N=8 mice in ABE-treated groups; N=3 mice in the uninjected group. All samples were isolated from bulk cortices. Genomic DNA and cDNA samples were isolated from identical nuclei populations in parallel. FIG. 14C shows genomic DNA sequencing data from bulk and GFP+nuclei from four brain regions in P45 Sen laR6iR:':mice treated with either ABE8e-V106W-VRQR or PBS vehicle (N>5 / group). FIG. 14D shows cDNA sequencing data from identical nuclei samples as in FIG. 14C. FIG. 14E shows Senia expression quantification by qPCR from whole cell lysates of brain hemispheres. Relative expression of Senia is equivalent to (2'AACt) normalized to wild-type ABE8e-V106W-VRQR-treated mice. Statistical analyses were performed by unpaired students T-test between groups (*P<0.05; **P<0.01). Bar values and error bars in FIGs. 14B-14E show mean±SD, respectively, of N>3 independent biological samples (shown as black dots). FIG. 14F shows a western blot analysis performed for Nav1.1 protein expression in isolated P21 mouse neocortices.15 / 224Bl 195.70210WO00#14840465vl

[0051] FIGs. 15A-15I show rescue of DS neocortical parvalbumin-positive GABAergic inhibitory interneuron (PVIN) excitability by ABE-V106W-VRQR. FIG. 15A shows experimental design. Sen lei'6'333, PV-tdTomato mice were randomly assigned to the GFP-KASH only or ABE + GFP-KASH treatments. At Pl 8-21, mice were sacrificed, and coronal brain slices were prepared from dissected brains. Whole-cell recordings of ascending-amplitude square-wave current injections were taken from PVINs in the primary somatosensory cortex in neocortical layer 2 / 3. From mice treated with GFP-KASH only, GFP+ / tdT+(unedited) PVINs were recorded. From mice treated with ABE + GFP-KASH, GFP+ / tdT+(edited) PVINs were recorded. FIG. 15B shows differential interference contrast (DIC), GFP-KASH, and tdTomato images of an example recorded cell. Scale bar, 20 pm. FIG. 15C shows example traces from unedited and edited PVINs in DS mice treated with ABE. For each cell, voltage responses to -100, +500, and +1000 pA are shown. Inset traces correspond to the first 10 ms of the same +500 pA voltage responses. FIG. 15D shows inputoutput curves for edited and unedited PVINs as a function of current injection divided by rheobase (fold rheobase). A mixed effects model was fitted to the data (Treatment F = 555.9, P < 0.0001; Fold rheobase F = 33.16, P < 0.0001; interaction F = 12.62, P < 0.0001). The asterisk indicates significance of post-hoc Sidak’s multiple comparisons tests (0.0001 < P < 0.05). Shading represents mean ± SEM. FIGs. 15E-15I show maximum steady-state firing frequency (FIG. 15E), maximum instantaneous firing frequency (FIG. 15F), input resistance (FIG. 15G), maximum upstroke velocity of the first action potential (AP) at rheobase (FIG.15H), and AP half-width of the first AP at rheobase (FIG. 151). -values indicate the significance of a linear mixed effects model that accounts for the random effects of recording multiple cells from the same animal. Bars and error bars show mean±SD of individual values (shown as dots of different shades of gray).

[0052] FIGs. 16A-16C show phenotypic rescue of DS by in vivo base editing. FIG. 16A shows overall study design, with mice assayed for either 45-day survival, febrile seizures, or acute brain slice electrophysiology (shown in FIGs. 15A-15I). Mice evaluated in febrile seizure assays were excluded from survival studies. FIG. 16B shows that heated cage assays increase the core body temperature of Sen Id'61333mice. After reaching a core body temperature of 42.5 °C, mice are removed from the cage and placed on ice for recovery. WT mice did not experience febrile seizures in this temperature range. FIG. 16C shows P45 survival for Sen Id'6133mice in male and female mice (N=15 / group / sex). In FIGs. 16B-16C,16 / 224Bl 195.70210WO00#14840465vlsignificance was determined by Mantel-Cox test of survival curves **P < 0.01; ****p < 0.0001.

[0053] FIGs. 17A-17B show HEK293T cell editing using a human genome-specific ABE8e-V106W-SpCas9 PAM strategy. FIG. 17A shows sequence alignment of SCN1A in the region containing R613 X. Figure discloses SEQ ID NOS 152, 157-158, 255, and 156, respectively, in order of appearance. FIG. 17B shows editing efficiencies of ABE8e and ABE8e-V106W correction strategies in HEK293T 5'C7V7H-p. R613X cells. Samples were sequenced 3 days after plasmid transfection. Bar values and error bars represent the mean±SD of three independent biological samples (shown as black dots).

[0054] FIG. 18 shows off-target editing in HEK293T £GV 4-p. R613X cells after plasmid transfection of ABE8e-V106W-VRQR and SCN1A R613X sgRNA. The percentage of A»T-to-G»C substitution at each of the 89 CIRCLE-seq-nominated off-target sites in the human genome (GRCh37) is shown; see FIG. 13E. Genomic DNA was extracted from HEK293T 5 fM-p. R613X cells untreated (n=3) or after 3 days following transfection of plasmids encoding ABE8e-V106W-VRQR and the SCN1A R613X sgRNA (n=3). In both untreated and ABE8e-V106W-VRQR treated samples, OT54 contains one A-to-G mutation relative to GROG 7 at position 9 of the putative protospacer sequence nominated by CIRCLE-seq. No additional A»T-to-G»C substitutions in this off-target site were observed. Dots represent individual biological replicates, and error bars represent standard deviations.

[0055] FIGs. 19A-19B show representative flow cytometry gating for brain nuclei sorting of GFP-KASH AAV treated samples. Plots show 100,000 events. Nuclei were gated sequentially on the basis of BSC-A and FSC-A, DyeCycle Ruby signal, and GFP / DyeCycle Ruby signal, as shown above. FIG. 19A shows representative untreated cortex isolated at P21. FIG. 19B shows representative GFP-KASH AAV-treated cortex isolated at P21.

[0056] FIGs. 20A-20L show functional rescue of DS PVINs by ABE-V106W-VRQR. FIG.20A shows example traces from WT, unedited, and edited PVINs. For each cell, voltage responses to -100, rheobase, and 3x rheobase stimulation are shown. FIG. 20B shows magnification of the first action potential (AP) at rheobase from the cells in FIG. 20A. FIG.20C shows input-output curves for WT, unedited, and edited PVINs as a function of current injection. Unedited cells were significantly less excitable than WT or edited cells (WT vs unedited: p = 0.0017, WT vs edited: p = 0.0045, unedited vs edited: p < 0.0001). Asterisks indicate the significance of post-hoc multiple comparison tests between WT and unedited 17 / 224Bl 195.70210WO00#14840465vlcells. FIGs. 20D-20H show intrinsic properties of PVINs. FIG. 201 shows example sodium current recordings from WT, unedited, and edited nucleated macropatches pulled from GFP+ / tdT+PVINs. FIG. 20J shows current-voltage relationship of sodium currents from PVIN nucleated macropatches. Unedited cells had significantly less sodium current than WT or edited cells (WT vs unedited: p < 0.0001, WT vs edited: p= 0.0665, unedited vs edited: p < 0.0001). FIG. 20K shows maximum sodium current from PVIN nucleated macropatches. FIG. 20L shows voltage dependences of activation and inactivation of sodium currents from PVIN nucleated macropatches. Unedited and edited voltage dependences were not significantly different from WT (activation, / ? = 0.0833 unedited vs WT,? = 0.2445 edited vs WT; inactivation, / ? = 0.1790 unedited vs WT, / ? = 0.1559 edited vs WT). All / ?-values indicate the significance of generalized linear mixed-effects models that account for the random effects of age, sex, and recording multiple cells from the same animal. Error bars show mean ± SD, and each dot represents one cell.

[0057] FIGs. 21A-21C show phenotypic rescue of DS by in vivo base editing. FIG. 21 A (Left) shows proportion analysis of spontaneous seizures observed in a 24-hour period by treatment group. Significance was determined by Fisher’s exact test. (Right) The number of seizures observed for each mouse over the 24-hour period. Significance was determined by unpaired T-test. (* / ? < 0.05) FIG. 21B shows P45 survival for ScnlaR613X / +male and female mice (N=15 / group / sex). FIG. 21C shows P60 survival for ScnlaR613X / +mice. The black, vertical dotted line at day 12 denotes the time of ICV injection for both ABE- and vehicle-treated mice. (FIG. 16B, FIG. 2 IB, and FIG. 21C) Significance was determined by Mantel-Cox test of survival curves **p < 0.01; *** / ? < 0.001; ****p < 0.0001.

[0058] FIGs. 22A-22C show electrophysiological characterization of 5cw7a-p. R612Q.HEK293T cells were transfected with WT or p. R612Q Senia plasmids. FIG. 22A shows current-voltage relationship (WT vs R612Q / ?= 0.0900). FIG. 22B shows voltage dependence of activation (WT vs R612Q / ? = 0.5243). FIG. 22C shows voltage dependence of inactivation (WT vs R612Q / ? = 0.2007). All / ?-values indicate the significance of generalized linear mixed-effects models that account for the random effects of age, sex, and recording multiple cells from the same animal. Error bars show mean ± SD. WT n = 11 cells; R612Q n = 12 cells.

[0059] FIG. 23 shows HEK293T 5 VA4-p. R613X cell off-target time course. Genomic DNA was collected from HEK293T 5 VA4-p. R613X cells either three, six, or nine days 18 / 224Bl 195.70210WO00#14840465vlfollowing treatment with ABE8e-V106W-VRQR targeting V A74-p. R6l3X, and the specified locus was amplified from each genomic DNA sample. Each dot represents the mean values of three biological replicates. Bar values and error bars show mean±SD, respectively, ofN>3 independent biological samples (shown as black dots).

[0060] FIGs. 24A-24B shows representative flow cytometry gating for brain nuclei sorting of GFP-KASH AAV treated samples (FIG. 24A and FIG. 24B). Plots show 100,000 events. Nuclei were gated sequentially on the basis of BSC-A and FSC-A, DyeCycle Ruby signal, and GFP / DyeCycle Ruby signal, as shown above. FIG. 24A shows representative untreated cortex isolated at P21. FIG. 24B shows representative GFP-KASH AAV-treated cortex isolated at P21.

[0061] FIG. 25 shows Pl ICV injection causes widespread expression of AAV9-GFP reporter. WT mice treated with AAV9-GFP and AAV9-ABE were dissected on P20. GFP was expressed in a high proportion of cells in cortex and hippocampus, and in a lower proportion of cells in cerebellum, brainstem, thalamus, and striatum.

[0062] FIGs. 26A-26B shows base editing by ABE8e-V106W-VRQR in ScrilaR6i~::mice at P21. FIG. 26 A shows genomic DNA sequencing data from bulk and GFP+nuclei from five brain regions in P21 ScnlaR6nxmice treated at Pl with either ABE8e-V106W-VRQR or PBS vehicle (N=3 / sex / group). FIG. 26B shows cDNA sequencing data from identical nuclei samples as in (FIG. 26A). Bar values and error bars in FIGs. 26A-26B show mean±SD, respectively, of independent biological samples (shown as black dots).

[0063] FIGs. 27A-27B show cDNA allele analysis of Pl ABE8e-V106W-VRQR treated P21 and P45 mouse brain tissues. Analysis for R613 codon reads was performed on the same sequencing data as that collected in FIG. 26A (FIG. 27A) and FIG. 14D (FIG. 27B). Stacked bars represent a parts-of-whole analysis summing to the total measured 5c«7a-p. R613 reads from cDNA. Bar values and error bars show mean±SD, respectively, of N>5 biological samples. (P = PBS-injected mice; bulk nuclei / B = ABE-treated mice; bulk nuclei / G = ABE-treated mice; GFP+ nuclei).

[0064] FIGs. 28A-28J show RT-qPCR sodium channel mRNA analysis of mouse neocortices. FIGs. 28A-28J show whole cell mRNA expression of each given sodium channel gene (specified by the graph title) isolated from the neocortex of Pl 8-21 male mice. All samples are normalized to their Ct value observed for Gapdh. Due to low expression of some sodium channel genes (namely, Scn4a, Sen 5a, Scn9a, Sen 10a, and Scnl la), all 19 / 224Bl 195.70210WO00#14840465vlnormalization is expressed as ACt normalized to Gapdh rather than fold-expression change. All relationships are not significant unless otherwise noted. Significance was determined by one-way ANOVA with follow-up unpaired students T-tests between groups, adjusting for multiple comparisons. Bar values and error bars show mean±SD, respectively, of N=3 independent biological samples (shown as black dots).

[0065] FIG. 29 shows uncropped western blot for Navl.l protein expression analysis.Dashed boxes indicate the blot regions used for densitometry analysis.

[0066] FIGs. 30A-30J show normal electrophysiology in WT, unedited, and edited excitatory pyramidal cells. FIG. 30A shows experimental design. Litters of Sen la1'613'' mice were randomly assigned to the GFP-KASH only or ABE + GFP-KASH treatments. At Pl 8-21, GFP+pyramidal cells were recorded. FIG. 30B shows differential interference contrast (DIC) and GFP-KASH images of an example recorded cell. Scale bar, 20 um. FIG. 30C shows example traces from WT, unedited, and edited pyramidal cells. For each cell, voltage responses to -100, rheobase, and 2x rheobase stimulation are shown. FIG. 30D shows magnification of the first action potential (AP) at rheobase from the cells in FIG. 30C. FIG.30E shows input-output curves for WT, unedited, and edited pyramidal cells as a function of current injection. Unedited cells were significantly less excitable than WT or edited cells (WT vs unedited: p = 0.0946, WT vs edited: p = 0.1006, unedited vs edited: p= 0.2025). FIGs. 30F-30J show intrinsic properties of pyramidal cells. All / i-values indicate the significance of generalized linear mixed-effects models that account for the random effects of age, sex, and recording multiple cells from the same animal. Error bars show mean ± SD, and each dot represents one cell.

[0067] FIGs. 31A-31H show normal spontaneous synaptic transmission in WT, unedited, and edited excitatory pyramidal cells. FIG. 31A shows experimental design. Litters of Scn1aR613X / +:mice were randomly assigned to the GFP-KASH only or ABE + GFP-KASH treatments. At P18-21, GFP+pyramidal cells were recorded. FIG. 3 IB shows that pyramidal cells were recorded using pipettes filled with CsMeSCh-based internal solution containing 2 mM QX-314 to block spikes. Spontaneous excitatory post-synaptic currents (sEPSCs) were recorded in voltage-clamp (VC) gap-free mode while holding the cell at -70 mV.Spontaneous inhibitory post-synaptic currents (sIPSCs) were recorded in voltage-clamp gap-free mode while holding the cell at 0 mV. FIG. 31C shows example sEPSC traces. FIGs.20 / 224Bl 195.70210WO00#14840465vl31D-31E show properties of sEPSCs. FIG. 3 IF shows example sIPSC traces. FIGs. 31G-31H shows properties of sIPSCs.

[0068] FIGs. 32A-32C show base editing by ABE8e-V106W-VRQR in P12 treatedR613X / +mice at P60. FIG. 32A shows genomic DNA sequencing data from bulk and GFP+nuclei from four brain regions in P60R613X / +mice treated with either ABE8e-V106W-VRQR or PBS vehicle (N>3 / group). Only male PBS-treated mice survived to P60 for tissue collection. FIG. 32B shows cDNA sequencing data from identical nuclei samples as in (FIG. 32A). Bar values and error bars in FIGs. 32A-32B show mean±SD, respectively, of independent biological samples (shown as black dots). FIG. 32C shows analysis for R613 codon reads was performed on the same sequencing data as that collected in (FIG. 32B). Stacked bars represent a parts-of-whole analysis summing to the total measured Scn1a-p.R613 reads from cDNA. Bar values and error bars show mean±SD, respectively, of N>3 biological samples. (P = PBS-injected mice; bulk nuclei / B = ABE-treated mice; bulk nuclei / G = ABE-treated mice; GFP+ nuclei).

[0069] FIG. 33 shows spontaneous seizure monitoring of P12 treatedR613X / +mice at P22-26. (Left) Proportion analysis of spontaneous seizures observed in a 24-hour period by treatment group. Significance was determined by Fisher’s exact test. (Right) The number of seizures observed for each mouse over the 24-hour period.

[0070] FIGs. 34A-34C show that Stoke ASO targets a naturally-occurring, nonproductive splicing variant.

[0071] FIGs. 35A-35B show SCN1A intronic variants in epileptic patients, highlighting “poison exon” 20N. FIG. 35A shows the 20N “poison exon” with its cryptic splice acceptor and splice donor sequences. Figure discloses SEQ ID NO: 256. FIG. 35B shows the aberrant splicing event, which occurs in “nonproductive” splicing. Figure discloses SEQ ID NOS 257-258, 257, and 259-260, respectively, in order of appearance. This occurs sporadically in both healthy and Dravet syndrome-affected humans.

[0072] FIGs. 36A-36C show ASO screens specifically for productive splicing.

[0073] FIGs. 37A-37B show that A> G base editing is predicted to ablate exon 20N inclusion by in silico methods.

[0074] FIGs. 38A-38D show that ABE8e-NRRH efficiently ablates the exon 20N splice acceptor in N2A cells. Figure 38A discloses SEQ ID NO: 261 and Figure 38C discloses SEQ ID NO: 262.21 / 224Bl 195.70210WO00#14840465vl

[0075] FIG. 39 shows that the “A12” bystander edit is co-occurring with the bystander edit designated “Al 1”. Figure discloses SEQ ID NOS 263, and 263-288, respectively, in order of appearance.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.Adenosine deaminase

[0077] As used herein, the term “adenosine deaminase” or “adenosine deaminase domain” refers to a protein or enzyme that catalyzes a deamination reaction of an adenosine (or adenine). The terms are used interchangeably. In certain embodiments, the disclosure provides nucleobase editor fusion proteins comprising one or more adenosine deaminase domains (e.g., fused to a napDNAbp such as a Cas9 protein). For instance, an adenosine deaminase domain may comprise a heterodimer of a first adenosine deaminase and a second deaminase domain, connected by a linker. Adenosine deaminases (e.g., engineered adenosine deaminases or evolved adenosine deaminases) provided herein may be enzymes that convert adenine (A) to inosine (I) in DNA or RNA. Such adenosine deaminases can lead to an A: T to G: C base pair conversion. In some embodiments, the deaminase is a variant of a naturally-occurring deaminase from an organism (e.g., bacteria, such as A. coli). In some embodiments, the deaminase does not occur in nature. For example, in some embodiments, the deaminase is 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 a naturally-occurring deaminase.

[0078] In some embodiments, the adenosine deaminase is derived from a bacterium, such as, E. coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, C. jejuni, or C. crescentus. In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is an E. coli TadA deaminase (ecTadA). In some embodiments, the TadA 22 / 224Bl 195.70210WO00#14840465vldeaminase is a truncated E. coli TadA deaminase. For example, the truncated ecTadA may be missing one or more N-terminal amino acids relative to a full-length ecTadA. In some embodiments, the truncated ecTadA may be missing 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 N-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the truncated ecTadA may be missing 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 C-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the ecTadA deaminase does not comprise an N-terminal methionine. In some embodiments, the adenosine deaminase comprises ecTadA(8e) ( / .<?., as used in the base editor ABE8e) as described further herein. Adenosine deaminases are further described, for example, in International Patent Application Publication No. WO 2018 / 027078, which is incorporated herein by reference.ABE-targetable mutation

[0079] As used herein, the term “ABE-targetable” (or “ABE-correctable”) mutation (e.g., SCN1A mutation or SCN1A mutant allele) is a mutation which is capable of being targeted by an adenine base editor complexed with an appropriately designed guide RNA to result in a desired edit to correct a OT or G> A mutation at a specified nucleotide residue position relative to a wildtype nucleotide sequence. Thus, an ABE-targetable mutation is either T: A base pair installed in place of a wildtype C: G base pair, or an A: T base pair installed in place of a wildtype G: C base pair. As a result of the edit by the ABE, the T: A pair is converted back to a C: G base pair, and the A: T pair is converted back to the G: C pair. The person having ordinary skill in the art will appreciate that an ABE-targetable mutation further requires that the target sequence comprising the mutation to be corrected comprises a suitable PAM (protospacer adjacent motif) sequence that is recognized and bound by the particular napDNAbp domain utilized as part of the ABE editor complex, and an editing window comprising the mutation to be corrected. Exemplary ABE-targetable SCN1A mutations are provided in Table 1.Base editing

[0080] “Base editing” refers to a genome editing technology that involves the conversion of a specific nucleic acid base into another at a targeted genomic locus. In certain embodiments, this can be achieved without requiring double-stranded DNA breaks (DSB), or single stranded breaks ( / .<?., nicking). Many other genome editing techniques, including CRISPR-based systems, begin with the introduction of a DSB at a locus of interest. Subsequently,23 / 224Bl 195.70210WO00#14840465vlcellular DNA repair enzymes mend the break, commonly resulting in random insertions or deletions (indels) of bases at the site of the DSB. However, when the introduction or correction of a point mutation at a target locus is desired rather than stochastic disruption of the entire gene, these genome editing techniques are unsuitable, as correction rates are low (e.g., typically 0.1% to 5%), with the major genome editing products being indels. In order to increase the efficiency of gene correction without simultaneously introducing random indels, the CRISPR system is modified to directly convert one DNA base into another without DSB formation. See, Komor, A. C., et al., Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424 (2016), the entire contents of which is incorporated by reference herein. In some embodiments, base editing is accomplished using a fusion protein comprising a deaminase and napDNAbp (e.g., a Cas9 protein).

[0081] In principle, there are 12 possible base-to-base changes that may occur via individual or sequential use of transition (z.e., a purine-to-purine change or pyrimidine-to-pyrimidine change) or transversion (z.e., a purine-to-pyrimidine or pyrimidine-to-purine) editors. These include transition base editors such as the cytosine base editor (“CBE”), also known as a C-to-T base editor (or “CTBE”). This type of editor converts a C: G Watson-Crick nucleobase pair to a T: A Watson-Crick nucleobase pair. Because the corresponding Watson-Crick paired bases are also interchanged as a result of the conversion, this category of base editor may also be referred to as a guanine base editor (“GBE”) or G-to-A base editor (or “GABE”). Other transition base editors include the adenine base editor (or “ABE”), also known as an A-to-G base editor (“AGBE”). This type of editor converts an A: T Watson-Crick nucleobase pair to a G: C Watson-Crick nucleobase pair. Because the corresponding Watson-Crick paired bases are also interchanged as a result of the conversion, this category of base editor may also be referred to as a thymine base editor (or “TBE”) or T-to-G base editor (“TGBE”).Base editors

[0082] The terms “base editor (BE)” and “nucleobase editor,” which are used interchangeably herein, refer to an agent comprising a polypeptide that is capable of making a modification to a base (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA) that converts one base to another (e.g., A to G, A to C, A to T, C to T, C to G, C to A, G to A, G to C, G to T, T to A, T to C, or T to G). In some embodiments, the base editor is capable of deaminating a base within a nucleic acid, such as a base within a DNA molecule.24 / 224Bl 195.70210WO00#14840465vlIn some embodiments, a base editor is an adenine base editor. In the case of an adenine base editor, the base editor is capable of deaminating an adenine (A) in DNA. Such base editors may include a nucleic acid programmable DNA binding protein (napDNAbp) fused to an adenosine deaminase. Some base editors include CRISPR-mediated fusion proteins that are utilized in the base editing methods described herein. In some embodiments, the base editor comprises a Cas9 protein fused to a deaminase that binds a nucleic acid in a guide RNA-programmed manner via the formation of an R-loop, but does not cleave the nucleic acid.

[0083] In some embodiments, a base editor is a macromolecule or macromolecular complex that results primarily (e.g., more than 80%, more than 85%, more than 90%, more than 95%, more than 99%, more than 99.9%, or 100%) in the conversion of a nucleobase in a polynucleotide sequence into another nucleobase ( / .<?., a transition or transversion) using a combination of 1) a nucleotide-, nucleoside-, or nucleobase-modifying enzyme, and 2) a nucleic acid binding protein that can be programmed to bind to a specific nucleic acid sequence.

[0084] In some embodiments, the base editor comprises a DNA binding domain (e.g., a programmable DNA binding domain, such as a Cas9 protein) that directs it to a target sequence. In some embodiments, the base editor comprises a nucleobase modification domain fused to a programmable DNA binding domain (e.g., a Cas9 protein). The terms “nucleobase modifying enzyme” and “nucleobase modification domain,” which are used interchangeably herein, refer to an enzyme that can modify a nucleobase and convert one nucleobase to another (e.g., a deaminase, such as a cytidine deaminase or an adenosine deaminase). In some embodiments, A to G editing is carried out by a deaminase, e.g., an adenosine deaminase.

[0085] In some embodiments, a base editor converts an A to a G. In some embodiments, the base editor comprises an adenine deaminase. An “adenine deaminase” is an enzyme involved in purine metabolism. It is needed for the breakdown of adenosine from food and for the turnover of nucleic acids in tissues. Its primary function in humans is the development and maintenance of the immune system. An adenine deaminase catalyzes hydrolytic deamination of adenosine (forming inosine, which base pairs as G) in the context of DNA. There are no known natural adenine deaminases that act on DNA. Instead, known adenine deaminase enzymes only act on RNA (tRNA or mRNA). Evolved deoxy adenosine deaminase enzymes that accept DNA substrates and deaminate dA to deoxyinosine have been described, e.g., in 25 / 224Bl 195.70210WO00#14840465vlInternational Patent Application No. PCT / US2017 / 045381, filed August 3, 2017, which published as WO 2018 / 027078, International Patent Application No. PCT / US2019 / 033848, which published as WO 2019 / 226953, International Patent Application No PCT / US2019 / 033848, filed May 23, 2019, which published as WO 2019226953, and International Patent Application No. PCT / US2020 / 028568, filed April 17, 2020, which published as WO 2020214842; each of which is incorporated herein by reference.

[0086] Exemplary adenine base editors are also described in Rees & Liu, “Base editing: precision chemistry on the genome and transcriptome of living cells,” Nat. Rev. Genet.2018;19 (12):770-788; as well as U. S. Patent Application Publication No. 2018 / 0073012, published March 15, 2018, which issued as U. S. Patent No. 10,113,163 on October 30, 2018; U. S. Patent Application Publication No. 2017 / 0121693, published May 4, 2017, which issued as U. S. Patent No. 10,167,457 on January 1, 2019; PCT Application Publication No. WO 2017 / 070633, published April 27, 2017; U. S. Patent Application Publication No.2015 / 0166980, published June 18, 2015; U. S. Patent No. 9,840,699, issued December 12, 2017; and U. S. Patent No. 10,077,453, issued September 18, 2018, each of which is incorporated herein by reference.Cas9

[0087] The term “Cas9” or “Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 domain, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and / or the gRNA binding domain of Cas9). A “Cas9 domain,” as used herein, is a protein fragment comprising an active or fully or partly inactive cleavage domain of Cas9 and / or the gRNA binding domain of Cas9. A “Cas9 protein” is a full length Cas9 protein. A Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (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 26 / 224Bl 195.70210WO00#14840465vlcomplementary to the spacer. The strand in the target DNA not complementary to crRNA is first cut endonucleolytically, then trimmed 3 '-5' exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the contents of which are incorporated herein by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an Ml strain of Streptococcus pyogenes"' Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U. S. A.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.

[0088] 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 etal., Science.337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell. 28; 152(5): 1173-83, the entire 27 / 224Bl 195.70210WO00#14840465vlcontents 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 RuvCl subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvCl subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek etal., Science. 337:816-821(2012); Qi etal., Cell. 28; 152(5): 1173-83 (2013)). In some embodiments, a Cas9 protein comprises one or more mutations to inactivate the nuclease activity of only one of the HNH subdomain or the RuvCl subdomain.

[0089] 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 (e.g., SpCas9 of SEQ ID NO: 1). In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes compared to wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 1). In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 1). In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 1).28 / 224Bl 195.70210WO00#14840465vlDeaminase

[0090] The term “deaminase” or “deaminase domain” refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA) to inosine.

[0091] The deaminases provided herein may be from any organism, such as a bacterium. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is 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 a naturally occurring deaminase.Dravet Syndrome

[0092] Dravet syndrome (DS) is a lifelong severe neurodevelopmental disorder and epileptic encephalopathy defined by treatment-resistant epilepsy, temperature-sensitive seizures, developmental delay / intellectual disability, features of autism spectrum disorder, and high rate of sudden unexpected death in epilepsy (SUDEP). DS generally begins in infancy and proceeds with accumulating morbidity that significantly impacts individuals throughout their lifetime. DS is caused by heterozygous loss-of-function variants in SCN1A, which encodes the voltage-gated sodium channel a subunitNavl.l. This gene is preferentially expressed in GABAergic inhibitory neurons (INs). Loss of one functional copy of Nav1.1 impairs the generation and propagation of action potentials in these cells, especially in fast-spiking parvalbumin-expressing inhibitory neurons (PV-INs). Nonsense mutations in SCN1A (such as SCN1A (R613X)) are thought to result in decreases in sodium currents and impairment of GABAergic interneurons of the hippocampus. Dravet syndrome is further described, for example, in Anwar et al. Cureus 2019, 11(6): e5006 and Gao et al., J. Clin. Med. 2023, 12(7): 2532, each of which is incorporated herein by reference. The amino acid and nucleotide sequences of the human and mouse SCN1A gene and Navl.l product are provided herein in the Sequences section. In addition, the amino acid and nucleotide sequences of the human and mouse SCN1A R613X mutant allele and mutant Nav1.1R613Xproduct are provided herein in the Sequences section. Lastly, additional ABE-targetable SCN1A mutant alleles are29 / 224Bl 195.70210WO00#14840465vlprovided in Table 1, wherein each of the listed mutant alleles is relative to the wildtype human SCN1A gene.

[0093] As used herein, “a symptom or condition associated with a SCN1A mutant allele” refers to any DS-associated symptom or condition arising due to the underlying disease, and may include but is not limited to the following symptoms or conditions: epilepsy, seizures, developmental delay / intellectual disability, autism spectrum disorder, SUDEP (high rate of sudden unexpected death in epilepsy), and / or severe encephalopathy.Fusion protein

[0094] The term “fusion protein” as used herein refers to a hybrid polypeptide that comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy -terminal (C-terminal) protein, thus forming an “amino-terminal fusion protein” or a “carboxy -terminal fusion protein,” respectively. A protein may comprise different domains, for example, a Cas9 protein fused to a deaminase ( / .<?., a base editor). Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2012)), the entire contents of which is incorporated herein by reference.Guide RNA (“gRNA”)

[0095] As used herein, the term “guide RNA” is a particular type of guide nucleic acid which is commonly associated with a Cas protein (e.g., a Cas9 protein), directing the Cas protein to a specific sequence in a DNA molecule that includes complementarity to the protospacer sequence of the guide RNA. For example, a gRNA may direct a Cas protein (e.g., as part of a base editor) to a target site in the SCN1A gene. However, this term also embraces the equivalent guide nucleic acid molecules that associate with Cas protein equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and which otherwise program the Cas protein equivalent to localize to a specific target nucleotide sequence. The Cas protein equivalents may include other napDNAbps from any type of CRISPR system (e.g., type II, V, VI), including Cpfl (a type-V CRISPR-Cas system), C2cl (a type V CRISPR-Cas system), C2c2 (a type VI 30 / 224Bl 195.70210WO00#14840465vlCRISPR-Cas system), and C2c3 (a type V CRISPR-Cas system). Further Cas-equivalents are described in Makarova etal., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299), which is incorporated herein by reference. Exemplary sequences and structures of guide RNAs are provided herein.

[0096] Functionally, guide RNAs associate with a Cas protein, directing (or programming) the Cas protein to a specific sequence in a DNA molecule that includes a sequence complementary to the protospacer sequence for the guide RNA. A gRNA is a component of the CRISPR / Cas system. The sequence specificity of a Cas DNA-binding protein is determined by gRNAs, which have nucleotide base-pairing complementarity to target DNA sequences. The native gRNA comprises a 20 nucleotide (nt) spacer, which specifies the DNA sequence to be targeted, and is immediately followed by an 80 nt scaffold sequence, which associates the gRNA with the Cas protein. In some embodiments, an SDS of the present disclosure has a length of 15 to 100 nucleotides, or more. For example, an SDS may have a length of 15 to 90, 15 to 85, 15 to 80, 15 to 75, 15 to 70, 15 to 65, 15 to 60, 15 to 55, 15 to 50, 15 to 45, 15 to 40, 15 to 35, 15 to 30, or 15 to 20 nucleotides. In some embodiments, the SDS is 20 nucleotides long. For example, the SDS may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. At least a portion of the target DNA sequence is complementary to the SDS of the gRNA. For a Cas protein to successfully bind to the DNA target sequence, a region of the target sequence is complementary to the SDS of the gRNA sequence and is immediately followed by the correct protospacer adjacent motif (PAM) sequence. In some embodiments, an SDS is 100% complementary to its target sequence. In some embodiments, the SDS sequence is less than 100% complementary to its target sequence and is, thus, considered to be partially complementary to its target sequence. For example, a targeting sequence may be 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% complementary to its target sequence. In some embodiments, the SDS of template DNA or target DNA may differ from a complementary region of a gRNA by 1, 2, 3, 4, or 5 nucleotides.

[0097] In some embodiments, the guide RNA is about 15-120 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence (e.g., a target sequence in SCN1A). In some embodiments, the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,31 / 224Bl 195.70210WO00#14840465vl92, 93, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 nucleotides long. In some embodiments, the guide RNA comprises a sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides that is complementary to a target sequence. Sequence complementarity refers to distinct interactions between adenine and thymine (DNA) or uracil (RNA), and between guanine and cytosine. In any of the guide RNA sequences provided herein, thymines and uracils may be used interchangeably, and a person of ordinary skill in the art will appreciate that the sequences comprise uracils because they are RNA.Linker

[0098] The term “linker,” as used herein, refers to a molecule linking two other molecules or moieties. The linker can be an amino acid sequence in the case of a linker joining two components of a fusion protein. For example, a napDNAbp (e.g., a Cas9 protein) can be fused to a deaminase (e.g., an adenosine deaminase) by an amino acid linker sequence. The linker can also be a nucleotide sequence in the case of joining two nucleotide sequences together (e.g., in a gRNA). In other embodiments, the linker is a non-peptide linker. In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-200 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.napDNAbp

[0099] As used herein, the term “nucleic acid programmable DNA binding protein” or “napDNAbp,” of which Cas proteins such as Cas9 and variants thereof are examples, refers to a protein that uses RNA: DNA hybridization to target and bind to specific sequences in a DNA molecule. Each napDNAbp is associated with at least one guide nucleic acid (e.g., guide RNA), which localizes the napDNAbp to a DNA sequence that comprises a DNA strand ( / .<?., a target strand) that is complementary to the guide nucleic acid, or a portion thereof (e.g., the protospacer of a guide RNA). In other words, the guide nucleic-acid “programs” the napDNAbp (e.g., Cas9, or a variant thereof) to localize and bind to a complementary sequence.

[0100] 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 32 / 224Bl 195.70210WO00#14840465vlthe 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, / .<?., the DNA is “nicked” on one strand.Nickase

[0101] As used herein, a “nickase” refers to a napDNAbp (e.g., a Cas9 protein) that is capable of cleaving only one of the two complementary strands of a double-stranded target DNA sequence, thereby generating a nick in that strand. In some embodiments, the nickase cleaves a non-target strand of a double stranded target DNA sequence. In some embodiments, the nickase comprises an amino acid sequence with one or more mutations in a catalytic domain of a canonical napDNAbp (e.g., a Cas9 protein), wherein the one or more mutations reduces or abolishes nuclease activity of the catalytic domain. In some embodiments, the nickase is a Cas9 that comprises one or more mutations in a RuvC-like domain relative to a wild type Cas9 sequence or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 that comprises one or more mutations in an HNH-like domain relative to a wild type Cas9 sequence or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 that comprises an aspartate-to-alanine substitution (D10A) in the RuvCl 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 N863 A 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.33 / 224Bl 195.70210WO00#14840465vl

[0102] In some embodiments, the napDNAbp of a base editor is a Cas9 nickase (nCas9) that nicks only a single strand. In other embodiments, the napDNAbp can be selected from the group consisting of: Cas9, Casl2e, Casl2d, Casl2a, Casl2bl, Casl2b2, Casl3a, Casl2c, Cast 2d, Casl2e, Casl2h, Casl2i, Cast 2g, Casl2f (Cast 4), Casl2fl, Casl2j (Cas ), and Argonaute and optionally has a nickase activity such that only one strand is cut. In some embodiments, the napDNAbp is selected from Cas9, Casl2e, Casl2d, Casl2a, Casl2bl, Casl2b2, Cast 3 a, Cast 2c, Cast 2d, Casl2e, Casl2h, Casl2i, Cast 2g, Casl2f (Cast 4), Casl2fl, Casl2j (Cas ), and Argonaute and optionally has a nickase activity such that one DNA strand is cut preferentially to the other DNA strand.Nuclear localization sequence (NLS)

[0103] The term “nuclear localization sequence” or “NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., international PCT application, PCT / EP2000 / 011690, filed November 23, 2000, published as WO / 2001 / 038547 on May 31, 2001, the contents of which are incorporated herein by reference for its disclosure of exemplary nuclear localization sequences. In some embodiments, a base editor comprises one or more NLS as described herein.Nucleic acid molecule

[0104] The term “nucleic acid,” as used herein, (also referred to as a “polynucleotide”) refers to a polymer of nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxy cytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyl uridine, C5 propynyl cytidine, C5 methylcytidine, 7 deazaadenosine, 7 deazaguanosine, 8 oxoadenosine, 8 oxoguanosine, 0(6) methylguanine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1-methyl adenosine, 1 -methyl guanosine, N6-methyl adenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, 2'-O-methyl cytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5' N phosphoramidite linkages).34 / 224Bl 195.70210WO00#14840465vlProtein, peptide, and polypeptide

[0105] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein, or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2012)), the contents of which are incorporated herein by reference.Protospacer

[0106] As used herein, the term “protospacer” refers to the sequence (~20 bp) in DNA adjacent to the PAM (protospacer adjacent motif) sequence. The protospacer shares the same sequence as the spacer sequence of the guide RNA. The guide RNA anneals to the complement of the protospacer sequence on the target DNA (specifically, one strand thereof, / .<?., the “target strand” versus the “non-target strand” of the target DNA sequence). The skilled person will appreciate that the literature in the state of the art sometimes refers to the “protospacer” as the ~20-nt target-specific guide sequence on the guide RNA itself, rather than referring to it as a “spacer.” Thus, in some cases, the term “protospacer” as used herein may be used interchangeably with the term “spacer.” The context of the description surrounding the appearance of either “protospacer” or “spacer” will help inform the reader as to whether the term is in reference to the gRNA or the DNA target.35 / 224Bl 195.70210WO00#14840465vlSpacer sequence

[0107] As used herein, the term “spacer sequence” in connection with a guide RNA refers to the portion of the guide RNA of about 20 nucleotides that contains a nucleotide sequence that shares the same sequence as the protospacer sequence in the target DNA sequence. The spacer sequence anneals to the complement of the protospacer sequence to form a ssRNA / ssDNA hybrid structure at the target site and a corresponding R loop ssDNA structure of the endogenous DNA strand.Subject

[0108] 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

[0109] The term “target site” refers to a sequence within a nucleic acid molecule that is modified (e.g., edited) by a fusion protein disclosed herein (e.g., a base editor). The target site further refers to the sequence within a nucleic acid molecule (e.g., a nucleic acid molecule comprising SCN1A) to which a complex of, for example, a base editor and a gRNA binds. Treatment

[0110] The terms “treatment,” “treat,” and “treating,” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder (e.g., Dravet syndrome), or one or more symptoms thereof, as described herein. As used herein, the terms “treatment,” “treat,” and “treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder (e.g., Dravet syndrome), 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 36 / 224Bl 195.70210WO00#14840465vlabsence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease (e.g., Dravet syndrome). 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[OHl] As used herein, the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant Cas9 is a Cas9 comprising one or more changes in amino acid residues (i.e., “substitutions”) as compared to a wild type Cas9 amino acid sequence. The term “variant” encompasses homologous proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence that display the same or substantially the same functional activity or activities as the reference sequence. Vector

[0112] The term “vector,” as used herein, refers to a nucleic acid that can be modified to encode a gene of interest and that is able to enter a host cell, mutate, and replicate within the host cell, and then transfer a replicated form of the vector into another host cell. Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages and filamentous phage, and conjugative plasmids. Additional suitable vectors will be apparent to those of skill in the art based on the instant disclosure.DETAILED DESCRIPTION

[0113] The present disclosure describes the discovery that adenine base editing (ABE) is an effective method for correcting mutations in the SCN1A gene that cause Dravet Syndrome (DS). This disclosure includes various compositions such as adenine base editors (ABEs), guide RNAs designed to target and correct SCN1A mutations, and complexes that consist of ABEs and guide RNAs. It also encompasses nucleic acid molecules encoding these ABEs and guide RNAs, as well as vectors and delivery systems that facilitate their use.Additionally, the disclosure covers cells and tissues containing ABEs and guide RNAs aimed 37 / 224Bl 195.70210WO00#14840465vlat SCN1A mutations, along with pharmaceutical compositions and kits containing these components for targeting SCN1 A mutations. Methods for correcting one or more SCN1 A mutations in mutant alleles, including the SCN1A (R613X) mutant allele, are disclosed herein under in vivo, ex vivo, or in vitro conditions using the disclosed ABEs, guide RNAs, and associated compositions or vectors.

[0114] Furthermore, the inventors have tested adenine base editing in a mouse model featuring the SCNlA-p. R613X mutant allele (“the SCN1AR613X / +mouse model”), a common variant found in DS patients. This SCN1AR613X / +mouse model closely mirrors the molecular and phenotypic characteristics of the disease in humans. The inventors demonstrated effective editing and restoration of the SCNlA-p. R613X mutant allele to its wildtype form in this model, indicating that adenine base editing is a viable method for correcting any ABE-targetable SCN1A mutation in humans, including those listed in Table 1. The SCN1A-p. R613X mutant allele is an example of a nonsense mutation, which leads to premature truncations and exhibits high penetrance. Such mutations result in truncated or misfolded proteins, exacerbated by nonsense-mediated decay of the faulty transcript. According to the ClinVar database, approximately 10% of listed DS-causing nonsense variants involve Arg to stop codon transitions, specifically caused by CGA to TGA mutations.

[0115] Adenine base editing (ABE) is a technique that converts A T base pairs to G C base pairs (or T A base pairs to C G base pairs) at targeted locations in the genome without causing double-stranded DNA breaks. ABEs consist of a nucleic acid-programmable DNA-binding domain, such as a Cas9 nickase, fused to an adenosine deaminase, allowing for modularity that enhances the editor's capabilities. ABEs can be paired with various Cas domains to alter the editing window and PAM preference, which helps accurately position the target base for deamination while minimizing unwanted edits to adjacent bases. The deaminase domain can also be modified to adjust the editing window, improve enzyme efficiency, or reduce unintended deamination events. Since their development, ABEs have been utilized in both ex vivo and in vivo applications, including treatments for sickle cell disease, Hutchinson-Gilford progeria syndrome, acute lymphoblastic leukemia, spinal muscular atrophy, and other genetic disorders. The ABE-mediated correction of the SCN1AR613X / +mutation in mice has shown promise for alleviating DS-related pathology, suggesting that any ABE-targetable SCN1A mutant allele can be effectively corrected, positioning ABE editing as a potential treatment for DS and its symptoms.38 / 224Bl 195.70210WO00#14840465vlNucleic Acid-Programmable DNA Binding Proteins (napDNAbp)

[0116] In various embodiments, the adenine base editors contemplated by the present disclosure comprise a napDNAbp. In some embodiments, the napDNAbp is a Cas protein (e.g., a Cas9 protein, including a Cas9 nickase or a nuclease-inactivated (dCas9) protein). Suitable napDNAbp sequences that can be used in base editors will be apparent to those of skill in the art based on this disclosure, and such proteins include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; which is incorporated herein by reference. Exemplary Cas variants and homologs include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), Cpfl, CasX, CasY, C2cl, C2c2, C2c3, GeoCas9, CjCas9, Cas 12a, Cas 12b, Cas 12g, Casl2h, Casl2i, Cas 13b, Cas 13c, Cas 13d, Cas 14, Csn2, xCas9, SpCas9-NG, Nme2Cas9, circularly permuted Cas9, Argonaute (Ago), Cas9-KKH, SmacCas9, Spy-macCas9, SpCas9-VRQR, SpCas9-NRRH, SpaCas9-NRTH, SpCas9-NRCH, LbCasl2a, AsCasl2a, CeCasl2a, MbCasl2a, Cas3, Cas, and circularly permuted Cas9 domains, such as CP 1012, CP 1028, CP 1041, CP 1249, and CP 1300, and variants and homologs thereof.

[0117] For example, a base editor may include a napDNAbp domain having a wild type Cas9 sequence, including, for example, the canonical Streptococcus pyogenes Cas9 sequence, shown as follows:napDNAbp Sequence SEQ ID NOWild type MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGN 1 Streptococcus TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRR pyogenes KNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHCas9 ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD(SpCas9) LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQL VQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLI AQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAK LQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKAL VRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFI KPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVG PLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS FIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKV KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK39 / 224Bl 195.70210WO00#14840465vlDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGI KELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYV DQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILD SRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFV YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFF KTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKL KSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLII KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEII EQISEF SKRVIL AD ANLDKVLS AYNKHRDKPIREQ AEN IIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0118] In some embodiments, a base editor may include a napDNAbp domain having a modified Cas9 sequence, including, for example, nickase or nuclease-inactivated (dead) variants of Streptococcus pyogenes Cas9, shown as follows:napDNAbp Sequence SEQ ID NOCas9 nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD 2 Streptococcus RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI pyogenes Cas9 CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFwith H840A GNIVDEVAYHEKYPTIYHLRKKLVSTDKADLRLIYLAL AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNRE KIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS40 / 224Bl 195.70210WO00#14840465vlDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEE VVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENII HLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGDCas9 nickase MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTD 3 Streptococcus RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI pyogenes Cas9 CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF with D10A GNIVDEVAYHEKYPTIYHLRKKLVSTDKADLRLIYLAL AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNRE KIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEE VVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENII41 / 224Bl 195.70210WO00#14840465vlHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGDNuclease- MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTD 139 inactivated RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICas9 (dCas9) CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF Streptococcus GNIVDEVAYHEKYPTIYHLRKKLVSTDKADLRLIYLAL pyogenes Cas9 AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE with D10A and NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF H840A GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNRE KIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEE VVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENII HLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0119] In some embodiments, the Cas9 protein included in a base editor can be a wild type Cas9 ortholog from another bacterial species different from the canonical Cas9 from S. pyogenes. For example, modified versions of the following Cas9 orthologs can be used in connection with the base editors described in this specification by making mutations at positions corresponding to D10A and / or H840A or any other amino acids of interest in wild type SpCas9. In addition, any variant Cas9 orthologs having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the below orthologs may also be used with the base editors.42 / 224Bl 195.70210WO00#14840465vlnapDNAbp Sequence SEQ ID NOLfCas9 MKEYHIGLDIGTSSIGWAVTDSQFKLMRIKGKTAIGVRL 4 Lactobacillus FEEGKTAAERRTFRTTRRRLKRRKWRLHYLDEIFAPHL fermentum QEVDENFLRRLKQSNIHPEDPTKNQAFIGKLLFPDLLKKCas9 NERGYPTLIKMRDELPVEQRAHYPVMNIYKLRE AMINE DRQFDLREVYLAVHHIVI< YRGHFLNNASVDI< FI< VGRID FDKSFNVLNEAYEELQNGEGSFTIEPSKVEKIGQLLLDT KMRKLDRQKAVAKLLEVKVADKEETKRNKQIATAMS KLVLGYKADFATVAMANGNEWKIDLSSETSEDEIEKFR EELSDAQNDILTEITSLFSQIMLNEIVPNGMSISESMMDR YWTHERQLAEVKEYLATQPASARKEFDQVYNKYIGQA PKERGFDLEKGLKKILSKKENWKEIDELLKAGDFLPKQ RTSANGVIPHQMHQQELDRIIEKQAKYYPWLATENPAT GERDRHQAKYELDQLVSFRIPYYVGPLVTPEVQKATSG AI< FAWAI< RI< EDGEITPWNLWDI< IDRAESAEAFII< RMT VKDTYLLNEDVLPANSLLYQKYNVLNELNNVRVNGRR LSVGIKQDIYTELFKKKKTVKASDVASLVMAKTRGVNK PSVEGLSDPKKFNSNLATYLDLKSIVGDKVDDNRYQTD LENIIEWRSVFEDGEIFADKLTEVEWLTDEQRSALVKKR YKGWGRLSKKLLTGIVDENGQRIIDLMWNTDQNFKEIV DQPVFKEQIDQLNQKAITNDGMTLRERVESVLDDAYTS PQNKKAIWQVVRVVEDIVKAVGNAPKSISIEFARNEGN KGEITRSRRTQLQKLFEDQAHELVKDTSLTEELEKAPDL SDRYYFYFTQGGKDMYTGDPINFDEISTKYDIDHILPQS FVKDNSLDNRVLTSRKENNKKSDQVPAKLYAAKMKPY WNQLLKQGLITQRKFENLTKDVDQNIKYRSLGFVKRQL VETRQVIKLTANILGSMYQEAGTEIIETRAGLTKQLREEF DLPKVREVNDYHHAVDAYLTTFAGQYLNRRYPKLRSF FVYGEYMKFKHGSDLKLRNFNFFHELMEGDKSQGKVV DQQTGELITTRDEVAKSFDRLLNMKYMLVSKEVHDRS DQLYGATIVTAKESGKLTSPIEIKKNRLVDLYGAYTNGT SAFMTIIKFTGNKPKYKVIGIPTTSAASLKRAGKPGSESY NQELHRIIKSNPKVKKGFEIVVPHVSYGQLIVDGDCKFT LASPTVQHPATQLVLSKKSLETISSGYKILKDKPAIANER LIRVFDEVVGQMNRYFTIFDQRSNRQKVADARDKFLSL PTESKYEGAKKVQVGKTEVITNLLMGLHANATQGDLK VLGLATFGFFQSTTGLSLSEDTMIVYQSPTGLFERRICLK DISaCas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNT 140 Staphylococc DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKN us aureus RICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHCas9 PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIY LALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYN QLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEK KNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTY DDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYK43 / 224Bl 195.70210WO00#14840465vlEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTE ELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQ EDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWM TRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSG EQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEIS GVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTG WGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLI HDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQ KNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLN AKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSD FRI< DFQFYI< VREINNYHHAHDAYLNAVVGTALII< I< YP KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK LIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSK KLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQ ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI TGLYETRIDLSQLGGDSaCas9 MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKE 5 Staphylococc ANVENNEGRRSI< RGARRLI< RRRRHRIQRVI< I< LLFDYN us aureus LLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAK Cas9 RRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAE LQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKA YHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKE WYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNN LVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEIL VNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIEN AELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNL KGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVP KKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIK KYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERI EEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLED LLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKG NRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKE YLLEERDINRF S VQKDFINRNL VDTRYATRGLMNLLRS YFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEK QAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDND 44 / 224Bl 195.70210WO00#14840465vlKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLN AHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKF VTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFI ASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYRE YLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKS KKHPQIIKKStCas9 MLFNKCIIISINLDFSNKEKCMTKPYSIGLDIGTNSVGWA 6 Streptococcu VITDNYKVPSKKMKVLGNTSKKYIKKNLLGVLLFDSGI s T AEGRRLK RT A RRR YTRRRN RI LYLQEI F STEMATLDD A thermophilus FFQRLDDSFLVPDDKRDSKYPIFGNLVEEKVYHDEFPTI Cas9 YHLRI< YLADSTI< I< ADLRLVYLALAHMII< YRGHFLIEGE FNSKNNDIQKNFQDFLDTYNAIFESDLSLENSKQLEEIV KDKISKLEKKDRILKLFPGEKNSGIFSEFLKLIVGNQADF RKCFNLDEKASLHFSKESYDEDLETLLGYIGDDYSDVFL KAKKLYDAILLSGFLTVTDNETEAPLSSAMIKRYNEHKE DLALLKEYIRNISLKTYNEVFKDDTKNGYAGYIDGKTN QEDFYVYLKNLLAEFEGADYFLEKIDREDFLRKQRTFD NGSIPYQIHLQEMRAILDKQAKFYPFLAKNKERIEKILTF RIP YYVGPLARGNSDFAWSIRKRNEKITPWNFED VIDKE SSAEAFINRMTSFDLYLPEEKVLPKHSLLYETFNVYNEL TKVRFIAESMRDYQFLDSKQKKDIVRLYFKDKRKVTDK DIIEYLHAIYGYDGIELKGIEKQFNSSLSTYHDLLNIINDK EFLDDSSNEAIIEEIIHTLTIFEDREMIKQRLSKFENIFDKS VLKKLSRRHYTGWGKLSAKLINGIRDEKSGNTILDYLID DGISNRNFMQLIHDDALSFKKKIQKAQIIGDEDKGNIKE VVKSLPGSPAIKKGILQSIKIVDELVKVMGGRKPESIVVE MARENQYTNQGKSNSQQRLKRLEKSLKELGSKILKENI PAKLSKIDNNALQNDRLYLYYLQNGKDMYTGDDLDID RLSNYDIDHIIPQAFLKDNSIDNKVLVSSASNRGKSDDFP SLEVVKKRKTFWYQLLKSKLISQRKFDNLTKAERGGLL PEDKAGFIQRQLVETRQITKHVARLLDEKFNNKKDENN RAVRTVKIITLKSTLVSQFRKDFELYKVREINDFHHAHD AYLNAVIASALLKKYPKLEPEFVYGDYPKYNSFRERKS ATEKVYFYSNIMNIFKKSISLADGRVIERPLIEVNEETGE SVWNKESDLATVRRVLSYPQVNVVKKVEEQNHGLDRG KPKGLFNANLSSKPKPNSNENLVGAKEYLDPKKYGGY AGISNSFAVLVKGTIEKGAKKKITNVLEFQGISILDRINY RKDKLNFLLEKGYKDIELIIELPKYSLFELSDGSRRMLAS ILSTNNKRGEIHKGNQIFLSQKFVKLLYHAKRISNTINEN HRKYVENHKKEFEELFYYILEFNENYVGAKKNGKLLNS AFQSWQNHSIDELCSSFIGPTGSERKGLFELTSRGSAADF EFLGVKIPRYRDYTPSSLLKDATLIHQSVTGLYETRIDLA KLGEGLcCas9 MKIKNYNLALTPSTSAVGHVEVDDDLNILEPVHHQKAI 7 Lactobacillus GVAKFGEGETAEARRLARSARRTTKRRANRINHYFNEI crispatus MKPEIDKVDPLMFDRIKQAGLSPLDERKEFRTVIFDRPNICas9 ASYYHNQFPTIWHLQKYLMITDEKADIRLIYWALHSLL 45 / 224Bl 195.70210WO00#14840465vlKHRGHFFNTTPMSQFKPGKLNLKDDMLALDDYNDLEG LSFAVANSPEIEKVIKDRSMHKKEKIAELKKLIVNDVPD KDLAKRNNKIITQIVNAIMGNSFHLNFIFDMDLDKLTSK AWSFKLDDPELDTKFDAISGSMTDNQIGIFETLQKIYSAI SLLDILNGSSNVVDAKNALYDKHKRDLNLYFKFLNTLP DEIAKTLKAGYTLYIGNRKKDLLAARKLLKVNVAKNFS QDDFYKLINKELKSIDKQGLQTRFSEKVGELVAQNNFLP VQRSSDNVFIPYQLNAITFNKILENQGKYYDFLVKPNPA KKDRKNAPYELSQLMQFTIPYYVGPLVTPEEQVKSGIPK TSRFAWMVRKDNGAITPWNFYDKVDIEATADKFIKRSI AKDSYLLSELVLPKHSLLYEKYEVFNELSNVSLDGKKL SGGVKQILFNEVFKKTNKVNTSRILKALAKHNIPGSKIT GLSNPEEFTSSLQTYNAWKKYFPNQIDNFAYQQDLEKM IEWSTVFEDHKILAKKLDEIEWLDDDQKKFVANTRLRG WGRLSKRLLTGLKDNYGKSIMQRLETTKANFQQIVYKP EFREQIDKISQAAAKNQSLEDILANSYTSPSNRK AIRKT MSVVDEYIKLNHGKEPDKIFLMFQRSEQEKGKQTEARS KQLNRILSQLKADKS ANKLF SKQLADEF SNAIKKSKYK LNDKQYFYFQQLGRDALTGEVIDYDELYKYTVLHIIPRS KLTDDSQNNKVLTKYKIVDGSVALKFGNSYSDALGMPI KAFWTELNRLKLIPKGKLLNLTTDFSTLNKYQRDGYIA RQLVETQQIVKLLATIMQSRFKHTKIIEVRNSQVANIRY QFDYFRII< NLNEYYRGFDAYLAAVVGTYLYI< VYPI< AR RLFVYGQYLKPKKTNQENQDMHLDSEKKSQGFNFLWN LLYGKQDQIFVNGTDVIAFNRKDLITKMNTVYNYKSQK ISLAIDYHNGAMFKATLFPRNDRDTAKTRKLIPKKKDY DTDIYGGYTSNVDGYMLLAEIIKRDGNKQYGFYGVPSR LVSELDTLKKTRYTEYEEKLKEIIKPELGVDLKKIKKIKI LKNKVPFNQVIIDKGSKFFITSTSYRWNYRQLILSAESQQ TLMDL VVDPDF SNHK ARKD ARKNADERLIK VYEEIL YQ VKNYMPMFVELHRCYEKLVDAQKTFKSLKISDKAMVL NQILILLHSNATSPVLEKLGYHTRFTLGKKHNLISENAV LVTQSITGLKENHVSIKQMLPdCas9 MTNEKYSIGLDIGTS SIGF AVVNDNNRVIRVKGKNAIGV 8 Pedicoccus RLFDEGKAAADRRSFRTTRRSFRTTRRRLSRRRWRLKL damnosus LREIFDAYITPVDEAFFIRLKESNLSPKDSKKQYSGDILF Cas9 NDRSDKDFYEKYPTIYHLRNALMTEHRKFDVREIYLAI HHIMKFRGHFLNATPANNFKVGRLNLEEKFEELNDIYQ RVFPDESIEFRTDNLEQIKEVLLDNKRSRADRQRTLVSDI YQSSEDKDIEKRNKAVATEILKASLGNKAKLNVITNVE VDKEAAKEWSITFDSESIDDDLAKIEGQMTDDGHEIIEV LRSLYSGITLSAIVPENHTLSQSMVAKYDLHKDHLKLFK KLINGMTDTKKAKNLRAAYDGYIDGVKGKVLPQEDFY KQVQVNLDDSAEANEIQTYIDQDIFMPKQRTKANGSIPH QLQQQELDQIIENQKAYYPWLAELNPNPDKKRQQLAK YKLDELVTFRVPYYVGPMITAKDQKNQSGAEFAWMIR KEPGNITPWNFDQKVDRMATANQFIKRMTTTDTYLLGEDVLPAQSLLYQKFEVLNELNKIRIDHKPISIEQKQQIFND 46 / 224Bl 195.70210WO00#14840465vlLFKQFKNVTIKHLQDYLVSQGQYSKRPLIEGLADEKRF NSSLSTYSDLCGIFGAKLVEENDRQEDLEKIIEWSTIFED KKIYRAKLNDLTWLTDDQKEKLATKRYQGWGRLSRKL LVGLKNSEHRNIMDILWITNENFMQIQAEPDFAKLVTD ANKGMLEKTDSQDVINDLYTSPQNKKAIRQILLVVHDI QNAMHGQAPAKIHVEFARGEERNPRRSVQRQRQVEAA YEKVSNELVSAKVRQEFKEAINNKRDFKDRLFLYFMQG GIDIYTGKQLNIDQLSSYQIDHILPQAFVKDDSLTNRVLT NENQVKADSVPIDIFGKKMLSVWGRMKDQGLISKGKY RNLTMNPENISAHTENGFINRQLVETRQVIKLAVNILAD EYGDSTQIISVKADLSHQMREDFELLKNRDVNDYHHAF DAYLAAFIGNYLLKRYPKLESYFVYGDFKKFTQKETKM RRFNFIYDLKHCDQVVNKETGEILWTKDEDIKYIRHLFA YKKILVSHEVREKRGALYNQTIYKAKDDKGSGQESKKL IRIKDDKETKIYGGYSGKSLAYMTIVQITKKNKVSYRVI GIPTLALARLNKLENDSTENNGELYKIIKPQFTHYKVDK KNGEIIETTDDFKIVVSKVRFQQLIDDAGQFFMLASDTY KNNAQQLVISNNALKAINNTNITDCPRDDLERLDNLRL DSAFDEIVKKMDKYFSAYDANNFREKIRNSNLIFYQLPV EDQWENNKITELGKRTVLTRILQGLHANATTTDMSIFKI KTPFGQLRQRSGISLSENAQLIYQSPTGLFERRVQLNKIKFnCas9 MKKQKFSDYYLGFDIGTNSVGWCVTDLDYNVLRFNKK 9 Fusobateriu DMWGSRLFEEAKTAAERRVQRNSRRRLKRRKWRLNLL m nucleatum EEIF SNEILKID SNFFRRLKES SLWLEDKS SKEKFTLFND Cas9 DNYI< DYDFYI< QYPTIFHLRNELII< NPEI< I< DIRLVYLAIH SIFKSRGHFLFEGQNLKEIKNFETLYNNLIAFLEDNGINKI IDKNNIEKLEKIVCDSKKGLKDKEKEFKEIFNSDKQLVAI FKLSVGSSVSLNDLFDTDEYKKGEVEKEKISFREQIYED DKPIYYSILGEKIELLDIAKTFYDFMVLNNILADSQYISE AKVKLYEEHKKDLKNLKYIIRKYNKGNYDKLFKDKNE NNYSAYIGLNKEKSKKEVIEKSRLKIDDLIKNIKGYLPK VEEIEEKDKAIFNKILNKIELKTILPKQRISDNGTLPYQIH EAELEKILENQSKYYDFLNYEENGIITKDKLLMTFKFRIP YYVGPLNSYHKDKGGNSWIVRKEEGKILPWNFEQKVDI EKSAEEFIKRMTNKCTYLNGEDVIPKDTFLYSEYVILNE LNKVQVNDEFLNEENKRKIIDELFKENKKVSEKKFKEY LLVKQIVDGTIELKGVKDSFNSNYISYIRFKDIFGEKLNL DIYKEISEKSILWKCLYGDDKKIFEKKIKNEYGDILTKDE IKKINTFKFNNWGRLSEKLLTGIEFINLETGEC YS S VMD ALRRTNYNLMELLSSKFTLQESINNENKEMNEASYRDLI EESYVSPSLKRAIFQTLKIYEEIRKITGRVPKKVFIEMAR GGDESMKNKKIPARQEQLKKLYDSCGNDIANFSIDIKE MKNSLISYDNNSLRQKKLYLYYLQFGKCMYTGREIDLD RLLQNNDTYDIDHIYPRSI< VII< DDSFDNLVLVLI< NENAE KSNEYPVKKEIQEKMKSFWRFLKEKNFISDEKYKRLTG KDDFELRGFMARQLVNVRQTTKEVGKILQQIEPEIKIVY SK AEI AS S F REM F DF I K VRE LN DTHH A K D A Y LN I VAGNVYNTKFTEKPYRYLQEIKENYDVKKIYNYDIKNAWDKE 47 / 224Bl 195.70210WO00#14840465vlNSLEIVKKNMEKNTVNITRFIKEKKGQLFDLNPIKKGET SNEIISIKPKVYNGKDDKLNEKYGYYKSLNPAYFLYVEH KEKNKRIKSFERVNLVDVNNIKDEKSLVKYLIENKKLV EPRVIKKVYKRQVILINDYPYSIVTLDSNKLMDFENLKP LFLENKYEKILKNVIKFLEDNQGKSEENYKFIYLKKKDR YEKNETLESVKDRYNLEFNEMYDKFLEKLDSKDYKNY MNNKKYQELLDVKEKFIKLNLFDKAFTLKSFLDLFNRK TMADFSKVGLTKYLGKIQKISSNVLSKNELYLLEESVTG LFVI< I< II< LEcCas9 RRKQRIQILQELLGEEVLKTDPGFFHRMKESRYVVEDK 10 Enterococcus RTLDGKQVELPYALFVDKDYTDKEYYKQFPTINHLIVY cecorum LMTTSDTPDIRLVYLALHYYMKNRGNFLHSGDINNVKD Cas9 INDILEQLDNVLETFLDGWNLKLKSYVEDIKNIYNRDLG RGERKKAFVNTLGAKTKAEKAFCSLISGGSTNLAELFD D S SLKEIETPKIEF AS S SLEDKIDGIQE ALEDRF AVIE AAK RL YDWKTLTDILGD S S SL AEARVNS YQMHHEQLLELKS LVKEYLDRKVFQEVFVSLNVANNYPAYIGHTKINGKKK ELEVKRTKRNDFYSYVKKQVIEPIKKKVSDEAVLTKLSE IESLIEVDKYLPLQVNSDNGVIPYQVKLNELTRIFDNLEN RIPVLRENRDKIIKTFKFRIPYYVGSLNGVVKNGKCTNW MVRKEEGKIYPWNFEDKVDLEASAEQFIRRMTNKCTYL VNEDVLPKYSLLYSKYLVLSELNNLRIDGRPLDVKIKQD IYENVFKKNRKVTLKKIKKYLLKEGIITDDDELSGLADD VKSSLTAYRDFKEKLGHLDLSEAQMENIILNITLFGDDK KLLKKRLAALYPFIDDKSLNRIATLNYRDWGRLSERFLS GITSVDQETGELRTIIQCMYETQANLMQLLAEPYHFVEA IEKENPKVDLESISYRIVNDLYVSPAVKRQIWQTLLVIKD IKQVMKHDPERIFIEMAREKQESKKTKSRKQVLSEVYK KAKEYEHLFEKLNSLTEEQLRSKKIYLYFTQLGKCMYS GEPIDFENLVSANSNYDIDHIYPQSKTIDDSFNNIVLVKK SLNAYKSNHYPIDKNIRDNEKVKTLWNTLVSKGLITKE KYERLIRSTPFSDEELAGFIARQLVETRQSTKAVAEILSN WFPESEIVYSKAKNVSNFRQDFEILKVRELNDCHHAHD AYLNIVVGNAYHTKFTNSPYRFIKNKANQEYNLRKLLQ KVNKIESNGVVAWVGQSENNPGTIATVKKVIRRNTVLI SRMVKEVDGQLFDLTLMKKGKGQVPIKSSDERLTDISK YGGYNKATGAYFTFVKSKKRGKVVRSFEYVPLHLSKQ FENNNELLKEYIEKDRGLTDVEILIPKVLINSLFRYNGSL VRITGRGDTRLLLVHEQPLYVSNSFVQQLKSVSSYKLK KSENDNAKLTKTATEKLSNIDELYDGLLRKLDLPIYSY WF S SIKEYLVESRTKYIKLSIEEKAL VIFEILHLFQ SD AQ V PNLKILGLSTKPSRIRIQKNLKDTDKMSIIHQSPSGIFEHEI ELTSLAhCas9 MQNGFLGITVSSEQVGWAVTNPKYELERASRKDLWGV 11 Anaerostipes RLFDKAETAEDRRMFRTNRRLNQRKKNRIHYLRDIFHE hadrus Cas9 EVNQKDPNFFQQLDESNFCEDDRTVEFNFDTNLYKNQF PT VYHLRK YLMETKDKPDIRL VYL A F SKFMKNRGHFL YKGNLGEVMDFENSMKGFCESLEKFNIDFPTLSDEQVKE 48 / 224Bl 195.70210WO00#14840465vlVRDILCDHKIAKTVKKKNIITITKVKSKTAKAWIGLFCG CSVPVKVLFQDIDEEIVTDPEKISFEDASYDDYIANIEKG VGIYYEAIVSAKMLFDWSILNEILGDHQLLSDAMIAEYN KHHDDLKRLQKIIKGTGSRELYQDIFINDVSGNYVCYV GHAKTMS S ADQKQF YTFLKNRLKNVNGIS SED AEWIDT EIKNGTLLPKQTKRDNSVIPHQLQLREFELILDNMQEMY PFLKENREKLLKIFNFVIPYYVGPLKGVVRKGESTNWM VPKKDGVIHPWNFDEMVDKEASAECFISRMTGNCSYLF NEKVLPKNSLLYETFEVLNELNPLKINGEPISVELKQRIY EQLFLTGKKVTKKSLTKYLIKNGYDKDIELSGIDNEFHS NLKSHIDFEDYDNLSDEEVEQIILRITVFEDKQLLKDYLN REFVKLSEDERKQICSLSYKGWGNLSEMLLNGITVTDS NGVEVSVMDMLWNTNLNLMQILSI< I< YGYI< AEIEHYN KEHEKTIYNREDLMDYLNIPPAQRRKVNQLITIVKSLKK TYGVPNKIFFKISREHQDDPKRTSSRKEQLKYLYKSLKS EDEKHLMKELDELNDHELSNDKVYLYFLQKGRCIYSGI< I< LNLSRLRI< SNYQNDIDYIYPLSAVNDRSMNNI< VLTG IQENRADKYTYFPVDSEIQKKMKGFWMELVLQGFMTK EKYFRLSRENDFSKSELVSFIEREISDNQQSGRMIASVLQ YYFPESKIVFVKEKLISSFKRDFHLISSYGHNHLQAAKD AYITIVVGNVYHTKFTMDPAIYFKNHKRKDYDLNRLFL ENISRDGQIAWESGPYGSIQTVRKEYAQNHIAVTKRVVE VKGGLFKQMPLKKGHGEYPLKTNDPRFGNIAQYGGYT NVTGSYFVLVESMEKGKKRISLEYVPVYLHERLEDDPG HKLLKEYLVDHRKLNHPKILLAKVRKNSLLKIDGFYYR LNGRSGNALILTNAVELIMDDWQTKTANKISGYMKRR AIDKKARVYQNEFHIQELEQLYDFYLDKLKNGVYKNR KNNQ AELIHNEKEQFMELKTEDQC VLLTEIKKLF VC SP MQADLTLIGGSKHTGMIAMSSNVTKADFAVIAEDPLGL RNKVIYSHKGEKKvCas9 MSQNNNKIYNIGLDIGDASVGWAVVDEHYNLLKRHGK 12 Kandleria HMWGSRLFTQ ANT AVERRS S RSTRRR YNK RRERI RLLR vitulina Cas9 EIMEDMVLDVDPTFFIRLANVSFLDQEDKKDYLKENYH SNYNLFIDKDFNDKTYYDKYPTIYHLRKHLCESKEKED PRLIYLALHHIVKYRGNFLYEGQKFSMDVSNIEDKMIDV LRQFNEINLFEYVEDRKKIDEVLNVLKEPLSKKHKAEK AFALFDTTKDNKAAYKELCAALAGNKFNVTKMLKEAE LHDEDEKDISFKFSDATFDDAFVEKQPLLGDCVEFIDLL HDIYSWVELQNILGSAHTSEPSISAAMIQRYEDHKNDLK LLKDVIRKYLPKKYFEVFRDEKSKKNNYCNYINHPSKT PVDEFYKYIKKLIEKIDDPDVKTILNKIELESFMLKQNSR TNGAVPYQMQLDELNKILENQSVYYSDLKDNEDKIRSI LTFRIPYYFGPLNITKDRQFDWIIKKEGKENERILPWNAN EIVDVDI< TADEFII< RMRNFCTYFPDEPVMAI< NSLTVSI< YEVLNEINKLRINDHLIKRDMKDKMLHTLFMDHKSISA NAMKKWL VKNQ YF SNTDDIKIEGFQKENAC ST SLTPWI DFTKIFGKINESNYDFIEKIIYDVTVFEDKKILRRRLKKEYDLDEEKIKKILKLKYSGWSRLSKKLLSGIKTKYKDSTR 49 / 224Bl 195.70210WO00#14840465vlTPETVLEVMERTNMNLMQVINDEKLGFKKTIDDANSTS VSGKFSYAEVQELAGSPAIKRGIWQALLIVDEIKKIMKH EPAHVYIEFARNEDEKERKDSFVNQMLKLYKDYDFEDE TEKEANKHLKGEDAKSKIRSERLKLYYTQMGKCMYTG KSLDIDRLDTYQVDHIVPQSLLKDDSIDNKVLVLSSENQ RKLDDLVIPSSIRNKMYGFWEKLFNNKIISPKKFYSLIKT EFNEKDQERFINRQIVETRQITKHVAQIIDNHYENTKVV TVRADLSHQFRERYHIYKNRDINDFHHAHDAYIATILGT YIGHRFESLDAKYIYGEYKRIFRNQKNKGKEMKKNNDG FILNSMRNIYADKDTGEIVWDPNYIDRIKKCFYYKDCFV TKKLEENNGTFFNVTVLPNDTNSDKDNTLATVPVNKYR SNVNKYGGFSGVNSFIVAIKGKKKKGKKVIEVNKLTGIP LMYKNADEEIKINYLKQAEDLEEVQIGKEILKNQLIEKD GGLYYIVAPTEIINAKQLILNESQTKLVCEIYKAMKYKN YDNLDSEKIIDLYRLLINKMELYYPEYRKQLVKKFEDR YEQLKVISIEEKCNIIKQILATLHCNSSIGKIMYSDFKISTT IGRLNGRTISLDDISFIAESPTGMYSKKYKLEfCas9 MRLFEEGHTAEDRRLKRTARRRISRRRNRLRYLQAFFE 13 Enterococcus EAMTDLDENFFARLQESFLVPEDKKWHRHPIFAKLEDE faecalis Cas9 VAYHETYPTIYHLRKKLADSSEQADLRLIYLALAHIVKY RGHFLIEGKLSTENTSVKDQFQQFMVIYNQTFVNGESRL VSAPLPESVLIEEELTEKASRTKKSEKVLQQFPQEKANG LFGQFLKLMVGNKADFKKVFGLEEEAKITYASESYEED LEGILAKVGDEYSDVFLAAKNVYDAVELSTILADSDKK SHAKLSSSMIVRFTEHQEDLKKFKRFIRENCPDEYDNLF KNEQKDGYAGYIAHAGKVSQLKFYQYVKKIIQDIAGAE YFLEKIAQENFLRKQRTFDNGVIPHQIHLAELQAIIHRQA AYYPFLKENQEKIEQLVTFRIPYYVGPLSKGDASTFAWL KRQSEEPIRPWNLQETVDLDQSATAFIERMTNFDTYLPS EKVLPKHSLLYEKFMVFNELTKISYTDDRGIKANFSGKE KEKIFDYLFKTRRKVKKKDIIQFYRNEYNTEIVTLSGLEE DQFNASFSTYQDLLKCGLTRAELDHPDNAEKLEDIIKIL TIFEDRQRIRTQLSTFKGQFSAEVLKKLERKHYTGWGRL SKKLINGIYDKESGKTILDYLVKDDGVSKHYNRNFMQL INDSQLSFKNAIQKAQSSEHEETLSETVNELAGSPAIKKG IYQSLKIVDELVAIMGYAPKRIVVEMARENQTTSTGKRR SIQRLKIVEKAMAEIGSNLLKEQPTTNEQLRDTRLFLYY MQNGKDMYTGDELSLHRLSHYDIDHIIPQSFMKDDSLD NLVLVGSTENRGKSDDVPSKEVVKDMKAYWEKLYAA GLISQRKFQRLTKGEQGGLTLEDKAHFIQRQLVETRQIT KNVAGILDQRYNAKSKEKKVQIITLKASLTSQFRSIFGL YKVREVNDYHHGQDAYLNCVVATTLLKVYPNLAPEFV YGEYPKFQTFKENKATAKAIIYTNLLRFFTEDEPRFTKD GEILWSNSYLKTIKKELNYHQMNIVKKVEVQKGGFSKE SIKPKGPSNKLIPVKNGLDPQKYGGFDSPVVAYTVLFTH EKGKKPLIKQEILGITIMEKTRFEQNPILFLEEKGFLRPRV LMKLPKYTLYEFPEGRRRLLASAKEAQKGNQMVLPEHLLTLLYHAKQCLLPNQSESLAYVEQHQPEFQEILERVVD 50 / 224Bl 195.70210WO00#14840465vlFAEVHTLAKSKVQQIVKLFEANQTADVKEIAASFIQLM QFNAMGAPSTFKFFQKDIERARYTSIKEIFDATIIYQSPT GLYETRRKVVDStaphylococc KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANV 14 us aureus ENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTD Cas9 HSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGV HNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLE RLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQL DQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYE MLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVIT RDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNE EDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAEL LDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKG YTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKK VDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKY GLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEI IRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLL NNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNR TPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYL LEERDINRF S VQKDFINRNL VDTRYATRGLMNLLRS YFR VNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHA EDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAES MPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPN RELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKK LINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYK YYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDIT DDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNN DLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENM NDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI IKKGGeobacillus MKYKIGLDIGITSIGWAVINLDIPRIEDLGVRIFDRAENP 15 thermodenitr KTGESLALPRRLARSARRRLRRRKHRLERIRRLFVREGI ificans Cas9 LTKEELNKLFEKKHEIDVWQLRVEALDRKLNNDELARI (“GeoCas9”) LLHLAKRRGFRSNRKSERTNKENSTMLKHIEENQSILSS YRT VAEMVVKDPKF SLHKRNKEDNYTNT VARDDLERE IKLIFAKQREYGNIVCTEAFEHEYISIWASQRPFASKDDI EKKVGFCTFEPKEKRAPKATYTFQSFTVWEHINKLRLV SPGGIRALTDDERRLIYKQAFHKNKITFHDVRTLLNLPD DTRFKGLLYDRNTTLKENEKVRFLELGAYHKIRKAIDS VYGKGAAKSFRPIDFDTFGYALTMFKDDTDIRSYLRNE YEQNGK RMEN LA DK V YDEELIEELLN LSF SKFGHLSLK ALRNILPYMEQGEVYSTACERAGYTFTGPKKKQKTVLL PNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELA RELSQSFDERRKMQKEQEGNRKKNETAIRQLVEYGLTL NPTGLDIVKFKLWSEQNGKCAYSLQPIEIERLLEPGYTE VDHVIPYSRSLDDSYTNKVLVLTKENREKGNRTPAEYLGLGSERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEE 51 / 224Bl 195.70210WO00#14840465vlNEFKNRNLNDTRYISRFLANFIREHLKFADSDDKQKVY TVNGRITAHLRSRWNFNKNREESNLHHAVDAAIVACTT PSDIARVTAFYQRREQNKELSKKTDPQFPQPWPHFADE LQARLSKNPKESIKALNLGNYDNEKLESLQPVFVSRMP KRSITGAAHQETLRRYIGIDERSGKIQTVVKKKLSEIQLD KTGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEP LYKPKKNGELGPIIRTIKIIDTTNQVIPLNDGKTVAYNSNI VRVDVFEKDGKYYCVPIYTIDMMKGILPNKAIEPNKPY SEWKEMTEDYTFRFSLYPNDLIRIEFPREKTIKTAVGEEI KIKDLF AY YQTID S SNGGLSL VSHDNNF SLRSIGSRTLKR FEKYQVDVLGNIYKVRGEKRVGVASSSHSKAGETIRPLScCas9 MEKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNT 16 S. canis Cas9 NRKSIKKNLMGALLFDSGETAEATRLKRTARRRYTRRK NRIRYLQEIFANEMAKLDDSFFQRLEESFLVEEDKKNER HPIFGNLADEVAYHRNYPTIYHLRKKLADSPEKADLRLI YLALAHIIKFRGHFLIEGKLNAENSDVAKLFYQLIQTYN QLFEESPLDEIEVDAKGILSARLSKSKRLEKLIAVFPNEK KNGLFGNIIALALGLTPNFKSNFDLTEDAKLQLSKDTYD DDLDELLGQIGDQYADLFSAAKNLSDAILLSDILRSNSE VTKAPLSASMVKRYDEHHQDLALLKTLVRQQFPEKYA EIFKDDTKNGYAGYVGIGIKHRKRTTKLATQEEFYKFIK PILEKMDGAEELLAKLNRDDLLRKQRTFDNGSIPHQIHL KELHAILRRQEEFYPFLKENREKIEKILTFRIPYYVGPLA RGNSRFAWLTRKSEEAITPWNFEEVVDKGASAQSFIER MTNFDEQLPNKKVLPKHSLLYEYFTVYNELTKVKYVTE RMRKPEFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK KIECFDSVEIIGVEDRFNASLGTYHDLLKIIKDKDFLDNE ENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK QLKRRHYTGWGRLSRKMINGIRDKQSGKTILDFLKSDG FSNRNFMQLIHDDSLTFKEEIEKAQVSGQGDSLHEQIAD LAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARE NQTTTKGLQQSRERKKRIEEGIKELESQILKENPVENTQ LQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV PQSFIKDDSIDNKVLTRSVENRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAERGGLSEADKAGFIK RQLVETRQITKHVARILDSRMNTKRDKNDKPIREVKVIT LKSKLVSDFRKDFQLYKVRDINNYHHAHDAYLNAVVG TALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA TAKRFFYSNIMNFFKTEVKLANGEIRKRPLIETNGETGE VVWNKEKDF AT VRKVL A M PQ VNIVKKTE VQTGGF SKE SILSKRESAKLIPRKKGWDTRKYGGFGSPTVAYSILVVA KVEKGKAKKLKSVKVLVGITIMEKGSYEKDPIGFLEAK GYKDIKKELIFKLPKYSLFELENGRRRMLASATELQKAN ELVLPQHLVRLLYYTQNISATTGSNNLGYIEQHREEFKE IFEKIIDFSEKYILKNKVNSNLKSSFDEQFAVSDSILLSNS FVSLLKYTSFGASGGFTFLDLDVKQGRLRYQTVTEVLDATLIYQSITGLYETRTDLSQLGGD52 / 224Bl 195.70210WO00#14840465vl

[0120] In some embodiments, a base editor may include a napDNAbp domain having a modified Cas9 sequence, including, for example, Cas9 proteins with alternative PAM specificities, such as the following:napDNAbp Sequence SEQ ID NOSpCas9- MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNT 17 VRQR DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKN RICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP IFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLA LAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKL NREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFL KDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNA SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI QKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIE EGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMY VDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR MNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVRE INNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKV YDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN IVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYG GFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMER SSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRK RMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSP EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDK VLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDSpCas9-NG MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNT 18 DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKN RICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP IFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLA LAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL 53 / 224Bl 195.70210WO00#14840465vlDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKL NREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFL KDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNA SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI QKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIE EGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMY VDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR MNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVRE INNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKV YDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN IVKKTEVQTGGFSKESIRPKRNSDKLIARKKDWDPKKYG GFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMER SSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRK RMLASARFLQKGNELALPSKYVNFLYLASHYEKLKGSP EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDK VLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDSpCas9- MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNT 19 NRCH DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKN RICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP IFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLA LAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMVKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKL NREDLLRKQRTFDNGIIPHQIHLGELHAILRRQGDFYPFL KDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNA SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRLRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI QKAQVSCQGDSLHEHIANLAGSPAIKKGILQTVKVVDELIKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEE 54 / 224Bl 195.70210WO00#14840465vlGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYV DQELDINRLSDYDVDHIVPQSFLKDDSIENKVLTRSDKN RGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLAETRQITKHVAQILDSRMN TKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREIN NYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIV KKTEVQTGGFSKESILPKGNSDKLIARKKDWDPKKYGGF NSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRM LASAGVLQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVL SAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTINRKQYNTTKEVLDATLIRQSITGLYETRIDLSQLGGDSpCas9- MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNT 20 NRRH DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKN RICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP IFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLA LAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMVKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKL NREDLLRKQRTFDNGIIPHQIHLGELHAILRRQGDFYPFL KDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNA SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRLRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI QKAQVSCQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL IKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYV DQELDINRLSDYDVDHIVPQSFLKDDSIENKVLTRSDKN RGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLAETRQITKHVAQILDSRMN TKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREIN NYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIV KKTEVQTGGFSKESILPKGNSDKLIARKKDWDPKKYGGF NSPTAAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIGFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRM LASAGVLHKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVL 55 / 224Bl 195.70210WO00#14840465vlSAYNKHRDKPIREQAENIIHLFTLTNLGVPAAFKYFDTTIDKKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSpCas9- MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNT 21 NRTH DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKN RICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP IFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLA LAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMVKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKL NREDLLRKQRTFDNGIIPHQIHLGELHAILRRQGDFYPFL KDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNA SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRLRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI QKAQVSCQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL IKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYV DQELDINRLSDYDVDHIVPQSFLKDDSIENKVLTRSDKN RGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLAETRQITKHVAQILDSRMN TKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREIN NYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIV KKTEVQTGGFSKESILPKGNSDKLIARKKDWDPKKYGGF NSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIGFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRM LASASVLHKGNELALPSKYVNFLYLASHYEKLKGSSEDN KQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS AYNKHRDKPIREQAENIIHLFTLTNLGASAAFKYFDTTIG RKLYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSpCas9- MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNT 22 SpyMac DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKN RICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP IFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLA LAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFL 56 / 224Bl 195.70210WO00#14840465vlKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNA SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI QKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIE EGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMY VDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR MNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVRE INNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKV YDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN IVKKTEIQTVGQNGGLFDDNPKSPLEVTPSKLVPLKKELN PKKYGGYQKPTTAYPVLLITDTKQLIPISVMNKKQFEQN PVKFLRDRGYQQVGKNDFIKLPKYTLVDIGDGIKRLWAS SKEIHKGNQLVVSKKSQILLYHAHHLDSDLSNDYLQNH NQQFDVLFNEIISFSKKCKLGKEHIQKIENVYSNKKNSASI EELAESFIKLLGFTQLGATSPFNFLGVKLNQKQYKGKKD YILPCTEGTLIRQSITGLYETRVDLSKIGED DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR 23 HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALA HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPL SASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNR EDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP WNFEEVVDKGAS AQ SFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNAS LGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI QKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIE EGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMY VDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR 57 / 224Bl 195.70210WO00#14840465vlMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVRE INNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKV YDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN IVKKTESSpCas9- DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR 244 iSpyMac HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALA HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPL SASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNR EDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP WNFEEVVDKGAS AQ SFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNAS LGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI QKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIE EGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMY VDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR MNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVRE INNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKV YDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE

[0121] Additional suitable napDNAbp sequences that can be used in base editors will be apparent to those of skill in the art based on this disclosure. A person of ordinary skill in the art will also understand that a napDNAbp may be used in a base editor with or without an N-terminal methionine if one is shown in any of the sequences above (i.e., if the napDNAbp is not located at the N-terminus of the base editor, the N-terminal methionine normally present in the napDNAbp sequence (due to the start codon in the nucleotide sequence encoding the napDNAbp) does not need to be present in the base editor).58 / 224Bl 195.70210WO00#14840465vlAdenosine Deaminases

[0122] In various embodiments, the adenine base editors contemplated for use in the present disclosure comprise a deaminase domain. In some embodiments, a base editor converts an A to a G. In some embodiments, the base editor comprises an adenosine deaminase. In some embodiments, the deaminase is an E. coli TadA (ecTadA) deaminase, or a variant thereof. Adenosine deaminases are described, for example, in International PCT Application Publication No. WO 2018 / 027078, which is incorporated herein by reference. In some embodiments, an adenosine deaminase comprises any of the following amino acid sequences, 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 of the following amino acid sequences:Adenosine Deaminase Sequence SEQ ID NOecTadA SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 24VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ GGLVMQNYRLIDATLYVTLEPCVMCAGAMIHS RIGRVVFGARDAKTGAAGSLMDVLHHPGMNH RVEITEGILADECAALLSDFFRMRRQEIKAQKKA QSSTDecTadA (DI 08N) SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 25VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ GGLVMQNYRLIDATLYVTLEPCVMCAGAMIHS RIGRVVFGARNAKTGAAGSLMDVLHHPGMNH RVEITEGILADECAALLSDFFRMRRQEIKAQKKA QSSTDecTadA (DI 08G) SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 26VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ GGLVMQNYRLIDATLYVTLEPCVMCAGAMIHS RIGRVVFGARGAKTGAAGSLMDVLHHPGMNH RVEITEGILADECAALLSDFFRMRRQEIKAQKKA QSSTDecTadA (DI 08 V) SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 27VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ GGLVMQNYRLIDATLYVTLEPCVMCAGAMIHS RIGRVVFGARVAKTGAAGSLMDVLHHPGMNH RVEITEGILADECAALLSDFFRMRRQEIKAQKKA QSSTDecTadA (H8Y, DI 08N, SEVEF S YEYWMRHALTLAKRAWDEREVP VGA 28 N127S) VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ GGLVMQNYRLIDATLYVTLEPCVMCAGAMIHS RIGRVVFGARNAKTGAAGSLMDVLHHPGMSHR VEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD59 / 224Bl 195.70210WO00#14840465vlecTadA (H8Y, DI 08N, SEVEF S YEYWMRHALTLAKRAWDEREVP VGA 29 N127S, E155D) VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ GGLVMQNYRLIDATLYVTLEPCVMCAGAMIHS RIGRVVFGARNAKTGAAGSLMDVLHHPGMSHR VEITEGILADECAALLSDFFRMRRQDIKAQKKA QSSTDecTadA (H8Y, DI 08N, SEVEF S YEYWMRHALTLAKRAWDEREVP VGA 30 N127S, E155G) VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ GGLVMQNYRLIDATLYVTLEPCVMCAGAMIHS RIGRVVFGARNAKTGAAGSLMDVLHHPGMSHR VEITEGILADECAALLSDFFRMRRQGIKAQKKA QSSTDecTadA (H8Y, DI 08N, SEVEF S YEYWMRHALTLAKRAWDEREVP VGA 31 N127S, E155V) VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ GGLVMQNYRLIDATLYVTLEPCVMCAGAMIHS RIGRVVFGARNAKTGAAGSLMDVLHHPGMSHR VEITEGILADECAALLSDFFRMRRQVIKAQKKA QSSTDecTadA (Al 06 V, SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 32 D108N, D147Y, and VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ E155V) GGLVMQNYRLIDATLYVTLEPCVMCAGAMIHS RIGRVVFGVRNAKTGAAGSLMDVLHHPGMNH RVEITEGILADECAALLSYFFRMRRQVIKAQKK AQSSTDecTadA (S2A, I49F, AEVEF SHEYWMRHALTLAKRAWDEREVP VGA 33 Al 06V, D108N, VLVHNNRVIGEGWNRPFGRHDPTAHAEIMALR D147Y, E155V) QGGLVMQNYRLIDATLYVTLEPCVMCAGAMIH SRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNH RVEITEGILADECAALLSYFFRMRRQVIKAQKK AQSSTDecTadA (H8Y, Al 06T, SEVEF S YEYWMRHALTLAKRAWDEREVP VGA 34 D108N, N127S, VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ K160S) GGLVMQNYRLIDATLYVTLEPCVMCAGAMIHS RIGRVVFGTRNAKTGAAGSLMDVLHHPGMSHR VEITEGILADECAALLSDFFRMRRQEIKAQSKAQ SSTDecTadA (R26G, L84F, SEVEF SHEYWMRHALTLAKRAWDEGEVP VGA 35 Al 06V, R107H, VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ D108N, H123Y, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS A142N, A143D, RIGRVVFGVHNAKTGAAGSLMDVLHYPGMNH D147Y, E155V, I156F) RVEITEGILADECNDLLSYFFRMRRQVFKAQKK AQSSTDecTadA (E25G, R26G, SEVEF SHEYWMRHALTLAKRAWDGGEVP VGA 36 L84F, Al 06V, R107H, VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ D108N, H123Y, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS A142N, A143D, RIGRVVFGVHNAKTGAAGSLMDVLHYPGMNH D147Y, E155V, I156F) RVEITEGILADECNDLLSYFFRMRRQVFKAQKKAQSSTD60 / 224Bl 195.70210WO00#14840465vlecTadA (E25D, R26G, SEVEF SHEYWMRHALTLAKRAWDDGEVP VGA 37 L84F, Al 06V, R107K, VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ D108N, H123Y, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS A142N, A143G, RIGRVVFGVKNAKTGAAGSLMDVLHYPGMNH D147Y, E155V, I156F) RVEITEGILADECNGLLSYFFRMRRQVFKAQKK AQSSTDecTadA (R26Q, L84F, SEVEF SHEYWMRHALTLAKRAWDEQEVP VGA 38 Al 06V, D108N, VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ H123Y, A142N, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS D147Y, E155V, I156F RIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECNALLSYFFRMRRQVFKAQKK AQSSTDecTadA (E25M, R26G, SEVEF SHEYWMRHALTLAKRAWDMGEVP VGA 39 L84F, Al 06V, R107P, VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ D108N, H123Y, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS A142N, A143D, RIGRVVFGVPNAKTGAAGSLMDVLHYPGMNHR D147Y, E155V, I156F VEITEGILADECNDLLSYFFRMRRQVFKAQKKA QSSTDecTadA (R26C, L84F, SEVEF SHEYWMRHALTLAKRAWDECEVP VGA 40 Al 06V, R107H, VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ D108N, H123Y, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS A142N, D147Y, RIGRVVFGVHNAKTGAAGSLMDVLHYPGMNH E155V, I156F) RVEITEGILADECNALLSYFFRMRRQVFKAQKK AQSSTDecTadA (L84F, Al 06V SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 41, D108N, H123Y, VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ A142N, A143L, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS D147Y, E155V, I156F) RIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECNLLLSYFFRMRRQVFKAQKK AQSSTDecTadA (R26G, L84F, SEVEF SHEYWMRHALTLAKRAWDEGEVP VGA 42 Al 06V, D108N, VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ H123Y, A142N, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS D147Y, E155V, I156F) RIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECNALLSYFFRMRRQVFKAQKK AQSSTDecTadA (R51H, L84F, SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 43 Al 06V, D108N, VLVHNNRVIGEGWNRPIGHHDPTAHAEIMALR H123Y, D147Y, QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIH E155V, I156F, K157N) SRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLSYFFRMRRQVFNAQKK AQSSTDecTadA (E25A, R26G, SEVEF SHEYWMRHALTLAKRAWD AGE VP VGA 44 L84F, Al 06V, R107N, VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ D108N, H123Y, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS A142N, A143E, RIGRVVFGVNNAKTGAAGSLMDVLHYPGMNH D147Y, E155V, I156F) RVEITEGILADECNELLSYFFRMRRQVFKAQKKAQSSTD61 / 224Bl 195.70210WO00#14840465vlecTadA (N37T, P48T, SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 45 L84F, Al 06V, D108N, VLVHTNRVIGEGWNRTIGRHDPTAHAEIMALR H123Y, D147Y, QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIH E155V, I156F) SRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLSYFFRMRRQVFKAQKK AQSSTDecTadA (N37S, L84F, SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 46 Al 06V, D108N, VLVHSNRVIGEGWNRPIGRHDPTAHAEIMALRQ H123Y, D147Y, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS E155V, I156F) RIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLSYFFRMRRQVFKAQKK AQSSTDecTadA (H36L, L84F, SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 47 Al 06V, D108N, VLVLNNRVIGEGWNRPIGRHDPTAHAEIMALRQ H123Y, D147Y, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS E155V, I156F) RIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLSYFFRMRRQVFKAQKK AQSSTDecTadA (H36L, P48L, SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 48 L84F, Al 06V, D108N, VLVLNNRVIGEGWNRLIGRHDPTAHAEIMALR H123Y, D147Y, QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIH E155V, I156F) SRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLSYFFRMRRQVFKAQKK AQSSTDecTadA (H36L, L84F, SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 49 Al 06V, D108N, VLVLNNRVIGEGWNRPIGRHDPTAHAEIMALRQ H123Y, D147Y, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS El 55V, K57N, I156F) RIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLSYFFRMRRQVFNAQKK AQSSTDecTadA (H36L, L84F, SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 50 Al 06V, D108N, VLVLNNRVIGEGWNRPIGRHDPTAHAEIMALRQ H123Y, S146C, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS D147Y, E155V, I156F) RIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLCYFFRMRRQVFKAQKK AQSSTDecTadA (L84F, SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 51 Al 06V, D108N, VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ H123Y, S146R, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS D147Y, E155V, I156F) RIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLRYFFRMRRQVFKAQKK AQSSTDecTadA (N37S, R51H, SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 52 L84F, Al 06V, D108N, VLVHSNRVIGEGWNRPIGHHDPTAHAEIMALRQ H123Y, D147Y, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS E155V, I156F) RIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLSYFFRMRRQVFKAQKKAQSSTD62 / 224Bl 195.70210WO00#14840465vlecTadA (R51L, L84F, SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 53 Al 06V, D108N, VLVHNNRVIGEGWNRPIGLHDPTAHAEIMALRQ H123Y, D147Y, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS E155V, I156F, K157N) RIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLSYFFRMRRQVFNAQKK AQSSTDecTadA (P48S) SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 54VLVHNNRVIGEGWNRSIGRHDPTAHAEIMALRQ GGLVMQNYRLIDATLYVTLEPCVMCAGAMIHS RIGRVVFGARDAKTGAAGSLMDVLHHPGMNH RVEITEGILADECAALLSDFFRMRRQEIKAQKKA QSSTDecTadA (P48T) SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 55VLVHNNRVIGEGWNRTIGRHDPTAHAEIMALR QGGLVMQNYRLIDATLYVTLEPCVMCAGAMIH SRIGRVVFGARDAKTGAAGSLMDVLHHPGMNH RVEITEGILADECAALLSDFFRMRRQEIKAQKKA QSSTDecTadA (P48A) SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 56VLVHNNRVIGEGWNRAIGRHDPTAHAEIMALR QGGLVMQNYRLIDATLYVTLEPCVMCAGAMIH SRIGRVVFGARDAKTGAAGSLMDVLHHPGMNH RVEITEGILADECAALLSDFFRMRRQEIKAQKKA QSSTDecTadA (A142N) SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 57VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ GGLVMQNYRLIDATLYVTLEPCVMCAGAMIHS RIGRVVFGARDAKTGAAGSLMDVLHHPGMNH RVEITEGILADECNALLSDFFRMRRQEIKAQKKA QSSTDecTadA (W23R) SEVEF SHEYWMRHALTLAKRARDEREVP VGAV 58LVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQG GLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRI GRVVFGARDAKTGAAGSLMDVLHHPGMNHRV EITEGILADECAALLSDFFRMRRQEIKAQKKAQS STDecTadA (W23L) SEVEF SHEYWMRHALTLAKRALDEREVP VGAV 59LVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQG GLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRI GRVVFGARDAKTGAAGSLMDVLHHPGMNHRV EITEGILADECAALLSDFFRMRRQEIKAQKKAQS STDecTadA (R152P) SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 60VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ GGLVMQNYRLIDATLYVTLEPCVMCAGAMIHS RIGRVVFGARDAKTGAAGSLMDVLHHPGMNH RVEITEGILADECAALLSDFFRMPRQEIKAQKKAQSSTD63 / 224Bl 195.70210WO00#14840465vlecTadA (R152H) SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 61VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ GGLVMQNYRLIDATLYVTLEPCVMCAGAMIHS RIGRVVFGARDAKTGAAGSLMDVLHHPGMNH RVEITEGILADECAALLSDFFRMHRQEIKAQKK AQSSTDecTadA (L84F, SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 62 Al 06V, D108N, VLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQ H123Y, D147Y, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS E155V, I156F) RIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLSYFFRMRRQVFKAQKK AQSSTDecTadA (H36L, R51L, SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 63 L84F, Al 06V, D108N, VLVLNNRVIGEGWNRPIGLHDPTAHAEIMALRQ H123Y, S146C, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS D147Y, E155V, I156F, RIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH K157N) RVEITEGILADECAALLCYFFRMRRQVFNAQKK AQSSTDecTadA (H36L, P48S, SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 64 R51L, L84F, A106V, VLVLNNRVIGEGWNRSIGLHDPTAHAEIMALRQ D108N, H123Y, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS S146C, D147Y, RIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH E155V, I156F, RVEITEGILADECAALLCYFFRMRRQVFNAQKK K157N) AQSSTDecTadA (H36L, P48A, SEVEF SHEYWMRHALTLAKRAWDEREVP VGA 65 R51L, L84F, A106V, VLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR D108N, H123Y, QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIH S146C, D147Y, SRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH E155V, I156F, RVEITEGILADECAALLCYFFRMRRQVFNAQKK K157N) AQSSTDecTadA (W23L, H36L, SEVEF SHEYWMRHALTLAKRALDEREVP VGAV 66 P48A, R51L, L84F, LVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQ Al 06V, D108N, GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS H123Y, S146C, RIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH D147Y, R152P, RVEITEGILADECAALLCYFFRMPRQVFNAQKK E155V, I156F, K157N) AQSSTDecTadA (W23R, H36L, SEVEF SHEYWMRHALTLAKRARDEREVP VGAV 67 P48A, R51L, L84F, LVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQG Al 06V, D108N, GLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI H123Y, S146C, GRVVFGVRNAKTGAAGSLMDVLHYPGMNHRV D147Y, R152P, EITEGILADECAALLCYFFRMPRQVFNAQKKAQ E155V, I156F, K157N) SSTD(also known as Tad A7.10)TadA 7.10 (V106W) SEVEF SHEYWMRHALTLAKRARDEREVP VGAV 68 (E. colt) LVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNAKTGAAGSLMDVLHYPGMNHRV 64 / 224Bl 195.70210WO00#14840465vlEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDTadA-8e (E. coll) SEVEF SHEYWMRHALTLAKRARDEREVP VGAV 69LVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQG GLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI GRVVFGVRNSKRGAAGSLMNVLNYPGMNHRV EITEGILADECAALLCDFYRMPRQVFNAQKKAQ SSINTadA-8e(V106W) (E. SEVEF SHEYWMRHALTLAKRARDEREVP VGAV 70 coli) LVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQG GLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI GRVVFGWRNSKRGAAGSLMNVLNYPGMNHRV EITEGILADECAALLCDFYRMPRQVFNAQKKAQSSINAdenine Base Editors

[0123] The present disclosure provides for the use of base editors in treating Dravet syndrome. In some embodiments, the base editor is an adenine base editor. In some embodiments, a base editor comprises at least two adenosine deaminase domains. Without wishing to be bound by any particular theory, dimerization of adenosine deaminases (e.g., in cis or in trans) may improve the ability (e.g., efficiency) of the base editor to modify a nucleic acid base (for example, to deaminate adenosine). In some embodiments, any of the base editors provided herein comprise 2, 3, 4, or 5 adenosine deaminase domains. In some embodiments, any of the base editors provided herein comprise two adenosine deaminases. In certain embodiments, the adenosine deaminases are the same. In some embodiments, the adenosine deaminases are any of the adenosine deaminases provided herein. In certain embodiments, the adenosine deaminases are different. Other adenosine deaminase domains besides those provided herein are known in the art, and a person of ordinary skill in the art would recognize which adenosine deaminase domains could be used in the fusion proteins of the present disclosure.

[0124] In some embodiments, the general architecture of the base editors contemplated by the present disclosure comprises any one of the following structures: NH2-[first adenosine deaminase]-[second adenosine deaminase]-[napDNAbp]-COOH; NH2-[first adenosine deaminase]-[napDNAbp]-[second adenosine deaminase]-COOH; NH2-[napDNAbp]-[first adenosine deaminase]-[second adenosine deaminase]-COOH; NH2-[second adenosine deaminase]-[first adenosine deaminase]-[napDNAbp]-COOH; NH2-[second adenosine deaminase]-[napDNAbp]-[first adenosine deaminase]-COOH; NH2-[napDNAbp]-[second 65 / 224Bl 195.70210WO00#14840465vladenosine deaminase]-[first adenosine deaminase]-COOH. In certain embodiments, the general architecture of the base editor comprises the structure NH₂-[first adenosine deaminase]-[second adenosine deaminase]-[napDNAbp]-COOH.

[0125] In various embodiments, the base editors used in the present disclosure may be fused to one or more nuclear localization sequences (NLS), which help promote translocation of the base editor into the cell nucleus. In some embodiments, the base editors described herein may comprise one or more NLS. Such sequences are well-known in the art and can include the following examples:NLS Sequence SEQ ID NO PKKKRKV 71 MKRTADGSEFESPKKKRKV 72 MDSLLMNRRI< FLYQFI< NVRWAI< GRRETYLC 73 AVKRPAATKKAGQAKKKKLD 74 MSRRRKANPTKLSENAKKLAKEVEN 75PAAKRVKLD 76KLKIKRPVK 77VSRKRPRP 78EGAPPAKRAR 79PPQPKKKPLDGE 80 KRTADGSEFEPKKKRKV 81KRTADGSEFESPKKKRKV 82

[0126] The NLS examples above are non-limiting. The fusion proteins provided herein may comprise any known NLS sequence, including any of those described in Cokol et al., “Finding nuclear localization signals,” EMBO Rep., 2000, 1(5): 411-415; and Freitas et al., “Mechanisms and Signals for the Nuclear Import of Proteins,” Current Genomics, 2009, 10(8): 550-7, each of which are incorporated herein by reference.

[0127] In various embodiments, the base editors and constructs encoding the base editors disclosed herein further comprise one or more, preferably at least two, nuclear localization sequences. In certain embodiments, the base editors comprise at least two NLSs. In embodiments with at least two NLSs, the NLSs can be the same NLSs, or they can be different NLSs. In some embodiments, one or more of the NLSs are bipartite NLSs (“bpNLS”). In certain embodiments, the disclosed base editors comprise two bipartite NLSs. In some embodiments, the disclosed base editors comprise more than two bipartite NLSs. The location of the NLS fusion can be at the N-terminus, the C-terminus, or within a sequence of a base editor66 / 224Bl 195.70210WO00#14840465vl

[0128] In certain embodiments, a base editor comprises an NLS of the amino acid sequence PKKKRKV (SEQ ID NO: 71). In certain embodiments, a base editor comprises an NLS of the amino acid sequence MKRTADGSEFESPKKKRKV (SEQ ID NO: 72). In certain embodiments, a base editor comprises an NLS of the amino acid sequence KRTADGSEFEPKKKRKV (SEQ ID NO: 81).

[0129] Exemplary base editor fusion architectures comprising a first adenosine deaminase, a second adenosine deaminase, a napDNAbp, and an NLS are provided: NH2-[NLS]-[first adenosine deaminase]-[second adenosine deaminase]-[napDNAbp]-COOH; NH₂-[first adenosine deaminase]-[NLS]-[second adenosine deaminase]-[napDNAbp]-COOH; NH₂-[first adenosine deaminase]-[second adenosine deaminase]-[NLS]-[napDNAbp]-COOH; NH₂-[first adenosine deaminase]-[second adenosine deaminase]-[napDNAbp]-[NLS]-COOH; NH₂-[NLS]-[first adenosine deaminase]-[napDNAbp]-[second adenosine deaminase]-COOH; NH2-[first adenosine deaminase]-[NLS]-[napDNAbp]-[second adenosine deaminase]-COOH; NH2-[first adenosine deaminase]-[napDNAbp]-[NLS]-[second adenosine deaminase]-COOH; NH2-[first adenosine deaminase]-[napDNAbp]-[second adenosine deaminase]-[NLS]-COOH; NH2-[NLS]-[napDNAbp]-[first adenosine deaminase]-[second adenosine deaminase]-COOH; NH2-[napDNAbp]-[NLS]-[first adenosine deaminase]-[second adenosine deaminase]-COOH; NH2-[napDNAbp]-[first adenosine deaminase]-[NLS]-[second adenosine deaminase]-COOH; NH2-[napDNAbp]-[first adenosine deaminase]-[second adenosine deaminase]-[NLS]-COOH; NH2-[NLS]-[second adenosine deaminase] -[first adenosine deaminase]-[napDNAbp]-COOH; NH2-[second adenosine deaminase]-[NLS]-[first adenosine deaminase]-[napDNAbp]-COOH; NH2-[second adenosine deaminase]-[first adenosine deaminase]-[NLS]-[napDNAbp]-COOH; NH2-[second adenosine deaminase]-[first adenosine deaminase]-[napDNAbp]-[NLS]-COOH; NH2-[NLS]-[second adenosine deaminase] -[napDNAbp] -[first adenosine deaminase]-COOH; NH2-[second adenosine deaminase]-[NLS]-[napDNAbp]-[first adenosine deaminase]-COOH; NH2-[second adenosine deaminase]-[napDNAbp]-[NLS]-[first adenosine deaminase]-COOH; NH2-[second adenosine deaminase]-[napDNAbp]-[first adenosine deaminase]-[NLS]-COOH; NH2-[NLS]-[napDNAbp]-[second adenosine deaminase]-[first adenosine deaminase]-COOH; NH2-[napDNAbp]-[NLS]-[second adenosine deaminase]-[first adenosine deaminase]-COOH; NH2-[napDNAbp]-[second adenosine deaminase]-[NLS]-[first adenosine deaminase]-COOH; NH2-[napDNAbp]-[second adenosine deaminase] -[first adenosine deaminase]-[NLS]-COOH.67 / 224Bl 195.70210WO00#14840465vl

[0130] In some embodiments, each instance of used in the general architecture above indicates the presence of an optional linker. In some embodiments, a base editor comprises one or more a peptide linkers. Exemplary peptide linkers for use in the base editors contemplated by the present disclosure include, but are not limited to, (GGGGS)n (SEQ ID NO: 83), (G)n (SEQ ID NO: 84), (EAAAK)n(SEQ ID NO: 85), (GGS)n (SEQ ID NO: 86), (SGGS)n (SEQ ID NO: 87), (XP)n(SEQ ID NO: 88), SGSETPGTSESATPES (SEQ ID NO: 89), SGSETPGTSESA (SEQ ID NO: 90), SGSETPGTSESATPEGGSGGS (SEQ ID NO: 97), SGGSSGGSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 91), SGGSGGSGGS (SEQ ID NO: 92), SGGS (SEQ ID NO: 93), SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSSGGS (SEQ ID NO: 94), GGSGGS (SEQ ID NO: 95), GGSGGSGGS (SEQ ID NO: 96), SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 98), or any combination thereof, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid.

[0131] In some embodiments, base editors useful in the present disclosure include base editors comprising any of the following amino acid sequences, or comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the following amino acid sequences:Base Sequence SEQ ID Editor NO ABE8e MKRT ADGSEFESPKKKRK VSEVEF SHEYWMRHALTL AKR 99ARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI GRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILA DECAALLCDFYRMPRQVFNAQKKAQSSINSGGSSGGSSGS ETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVI TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI68 / 224Bl 195.70210WO00#14840465vlVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQK AQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDN VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAY LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI TGLYETRIDLSQLGGDSGGSKRTADGSEFEPKKKRKV ABE8e- MKRT ADGSEFESPKKKRK VSEVEF SHEYWMRHALTL AKR 100 V106W ARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI GRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILA DECAALLCDFYRMPRQVFNAQKKAQSSINSGGSSGGSSGS ETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVI TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQK AQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDN 69 / 224Bl 195.70210WO00#14840465vlVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAY LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI TGLYETRIDLSQLGGDSGGSKRTADGSEFEPKKKRKV ABE8e- MKRT ADGSEFESPKKKRK VSEVEF SHEYWMRHALTL AKR 101 SaCas9 ARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI GRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILA DECAALLCDFYRMPRQVFNAQKKAQSSINSGGSSGGSSGS ETPGTSESATPESSGGSSGGSGKRNYILGLAIGITSVGYGIID YETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRR HRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSE EEF S AALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNS KALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAK QLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPF GWI< DII< EWYEMLMGHCTYFPEELRSVI< YAYNADLYNAL NDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIA KEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEII ENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISN LKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPK KVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYG LPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRT TGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFN YEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLS SSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRF SVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVK SINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIF KEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFI TPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDD KGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDP QTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDN GPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYR FDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKK LKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEV NMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILG NLYEVKSKKHPQIIKKGSGGSKRTADGSEFEPKKKRKV ABE8e- MKRT ADGSEFESPKKKRK VSEVEF SHEYWMRHALTL AKR 102 NG ARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI 70 / 224Bl 195.70210WO00#14840465vlGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILA DECAALLCDFYRMPRQVFNAQKKAQSSINSGGSSGGSSGS ETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVI TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQK AQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDN VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAY LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVA KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYK EVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNELAL PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENII HLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVLDATLIHQS ITGLYETRIDLSQLGGDSGGSKRTADGSEFEPKKKRKV ABE8e- MKRT ADGSEFESPKKKRK VSEVEF SHEYWMRHALTL AKR 103 VRQR ARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI GRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILA DECAALLCDFYRMPRQVFNAQKKAQSSINSGGSSGGSSGS ETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVI TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE 71 / 224Bl 195.70210WO00#14840465vlNLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQK AQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDN VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAY LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSI TGLYETRIDLSQLGGDSGGSKRTADGSEFEPKKKRKV ABE8e- MKRT ADGSEFESPKKKRK VSEVEF SHEYWMRHALTL AKR 104 V106W- ARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI NG MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI GRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILA DECAALLCDFYRMPRQVFNAQKKAQSSINSGGSSGGSSGS ETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVI TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI 72 / 224Bl 195.70210WO00#14840465vlVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQK AQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDN VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAY LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVA KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYK EVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNELAL PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENII HLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVLDATLIHQS ITGLYETRIDLSQLGGDSGGSKRTADGSEFEPKKKRKV ABE8e- MKRT ADGSEFESPKKKRK VSEVEF SHEYWMRHALTL AKR 105 V106W- ARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI VRQR MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI GRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILA DECAALLCDFYRMPRQVFNAQKKAQSSINSGGSSGGSSGS ETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVI TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQK AQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDN 73 / 224Bl 195.70210WO00#14840465vlVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAY LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSI TGLYETRIDLSQLGGDSGGSKRTADGSEFEPKKKRKV ABE8e- MKRT ADGSEFESPKKKRK VSEVEF SHEYWMRHALTL AKR 245 NRRH ARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI GRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILA DECAALLCDFYRMPRQVFNAQKKAQSSINSGGSSGGSSGS ETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVI TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMVKRYDEHHQDLTLLKALVRQQLP EKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLNREDLLRKQRTFDNGIIPHQIHLGELHAILRRQ GDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTR KSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKK AIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRF NASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDR EMIEERLKTYAHLFDDKVMKQLKRLRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ KAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK VMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIK ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEN DKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNG ETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS KESILPKGNSDKLIARKKDWDPKKYGGFNSPTAAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIGFLEAKGY 74 / 224Bl 195.70210WO00#14840465vlKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLHKGNELA LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYL DEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAE NIIHLFTLTNLGVPAAFKYFDTTIDKKRYTSTKEVLDATLIH QSITGLYETRIDLSQLGGDSGGSKRTADGSEFEPKKKRKV ABE8e- MKRT ADGSEFESPKKKRK VSEVEF SHEYWMRHALTL AKR 246 V106W- ARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI NRRH MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI GRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILA DECAALLCDFYRMPRQVFNAQKKAQSSINSGGSSGGSSGS ETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVI TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMVKRYDEHHQDLTLLKALVRQQLP EKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLNREDLLRKQRTFDNGIIPHQIHLGELHAILRRQ GDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTR KSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKK AIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRF NASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDR EMIEERLKTYAHLFDDKVMKQLKRLRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ KAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK VMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIK ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEN DKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNG ETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS KESILPKGNSDKLIARKKDWDPKKYGGFNSPTAAYSVLVV AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIGFLEAKGY KEVKKDLIIKLPKYSLFELENGRKRMLASAGVLHKGNELA LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYL DEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAE NIIHLFTLTNLGVPAAFKYFDTTIDKKRYTSTKEVLDATLIH QSITGLYETRIDLSQLGGDSGGSKRTADGSEFEPKKKRKV ABE8e- MKRT ADGSEFESPKKKRK VSEVEF SHEYWMRHALTL AKR 290 SpyMAC ARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI 75 / 224Bl 195.70210WO00#14840465vlGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILA DECAALLCDFYRMPRQVFNAQKKAQSSINSGGSSGGSSGS ETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVI TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQK AQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDN VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAY LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET GEIVWDKGRDFATVRKVLSMPQVNIVKKTEIQTVGQNGG LFDDNPKSPLEVTPSKLVPLKKELNPKKYGGYQKPTTAYP VLLITDTKQLIPISVMNKKQFEQNPVKFLRDRGYQQVGKN DFIKLPKYTLVDIGDGIKRLWASSKEIHKGNQLVVSKKSQI LLYHAHHLDSDLSNDYLQNHNQQFDVLFNEIISFSKKCKL GKEHIQKIENVYSNKKNSASIEELAESFIKLLGFTQLGATSP FNFLGVKLNQKQYKGKKDYILPCTEGTLIRQSITGLYETRV DLSKIGEDSGGSKRTADGSEFEPKKKRKV _ABE8e- MKRT ADGSEFESPKKKRK VSEVEF SHEYWMRHALTL AKR 247 iSpyMAC ARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI GRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILA DECAALLCDFYRMPRQVFNAQKKAQSSINSGGSSGGSSGS ETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVI TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRKLE 76 / 224Bl 195.70210WO00#14840465vlNLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQK AQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDN VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAY LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET GEIVWDKGRDFATVRKVLSMPQVNIVKKTEIQTVGQNGG LFDDNPKSPLEVTPSKLVPLKKELNPKKYGGYQKPTTAYP VLLITDTKQLIPISVMNKKQFEQNPVKFLRDRGYQQVGKN DFIKLPKYTLVDIGDGIKRLWASSKEIHKGNQLVVSKKSQI LLYHAHHLDSDLSNDYLQNHNQQFDVLFNEIISFSKKCKL GKEHIQKIENVYSNKKNSASIEELAESFIKLLGFTQLGATSP FNFLGVKLNQKQYKGKKDYILPCTEGTLIRQSITGLYETRVDLSKIGEDSGGSKRTADGSEFEPKKKRKVGuide RNAs (gRNAs)

[0132] The present disclosure provides gRNAs for targeting a genome editing agent (e.g., a base editor) to a SCN1A gene (e.g., a human or mouse SCN1A gene). The gRNAs provided herein may be useful for treating Dravet Syndrome.

[0133] In some embodiment, the gRNA is designed to target an ABE to any one of the ABE-targetable SCN1A mutant alleles shown in Table 1, including the SCN1A (R613X) mutant allele.

[0134] In some embodiments, the gRNA targets the SCN1A (R613X) mutant allele. In a further embodiment, the gRNA targeting the SCN1A (R613X) mutant allele is provided in Table 4A, including any one of the guide sequences of SEQ ID NOs: 118-121 full length guide sequences, or a guide comprising any one of the spacer sequences of SEQ ID NOs: 122-125.77 / 224Bl 195.70210WO00#14840465vl

[0135] In another aspect, the present disclosure provides gRNAs for targeting an ABE to introduce an A-to-G mutation into a splice acceptor sequence upstream of exon 20N in SCN1A. In some embodiments, the gRNA comprises a spacer sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence GUAUAGGAUAAUCUUGCUCC (SEQ ID NO: 240), GGUAUAGGAUAAUCUUGCUC (SEQ ID NO: 241), GAUAUAGGAUAAUCUUGCUC (SEQ ID NO: 242), GUAUAGGAUAAUCUUGCUC (SEQ ID NO: 243), or GUAUAGGAUAAUCUUGCUC (SEQ ID NO: 243). In some embodiments, the gRNA comprises a scaffold sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence GUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACUAAAACAAGGCAAAAU GCCGUGUUUAUCUCGUCAACUUGUUGGCGAGA (SEQ ID NO: 126), GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 127), or GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 248). In certain embodiments, the gRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence GUAUAGGAUAAUCUUGCUCCGUUUUAGUACUCUGUAAUGAAAAUUACAGAAU CUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGA(SEQ ID NO: 249), GGUAUAGGAUAAUCUUGCUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 250), GAUAUAGGAUAAUCUUGCUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 251), GUAUAGGAUAAUCUUGCUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 252), or GUAUAGGAUAAUCUUGCUCGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAG78 / 224Bl 195.70210WO00#14840465vlUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG C (SEQ ID NO: 253).

[0136] In other embodiments, the gRNA targets any SCN1A mutant allele of Table 1, or which the guide RNA sequences of Table 4B are provided by way of example. However, the person having ordinary skill in the art will understand and know how to design an appropriate guide sequence to any of the SCN1A mutant alleles of Table 1. Some guiding principles that may be considered when designing a guide RNA to a target site, such as an SCN1A mutant allele of Table 1, include but are not limited to: Target Specificity: Select a target sequence that is unique to the region of interest to minimize off-target effects. Ensure the target sequence is adjacent to a protospacer adjacent motif (PAM) sequence compatible with the Cas9 variant used in the ABE; Length and Structure: Typically, a gRNA should be about 20 nucleotides long. Ensure the gRNA has a stable secondary structure, which can be predicted using RNA folding software to enhance binding efficiency; PAM Compatibility:Ensure that the sequence following the target site contains a PAM that the particular Cas9 variant used in the ABE can recognize. For example, the commonly used SpCas9 requires an NGG PAM; Optimization: Experiment with different gRNA sequences around the target site to identify the most effective one. Utilizing known computational tools and known databases can help predict the efficiency of each gRNA design; Consideration of Editing Window:Understand the editing window of the ABE, as the ABE only modifies adenines within a specific range. This range typically extends from the 5' end of the gRNA target site.Experimental Validation: After designing, validate the gRNA experimentally to confirm its efficiency and specificity in targeting the desired mutation, and evaluate effect on off-target or bystander editing.

[0137] In some embodiments, the guide RNA is designed against a mouse target sequence. In other embodiments, the guide RNA is designed against a human target sequence.

[0138] In some embodiments, the gRNAs target a base editor to a site in the human SCN1A gene of SEQ ID NO: 138. In some embodiments, the gRNAs target a base editor to a site in the human SCN1A gene such that the base editor corrects a mutation in the SCN1A gene, leading to correction of a mutation in the human SCN1A enzyme.

[0139] Additional sequences of suitable gRNAs for targeting a base editor to SCN1A within the scope of the present disclosure will be apparent to those of skill in the art. Such suitable guide RNA sequences typically comprise a spacer sequence that is complementary to a 79 / 224Bl 195.70210WO00#14840465vlnucleic sequence within 50 nucleotides (e.g., within 45, 40, 35, 30, 25, 20, 15, or 10 nucleotides) upstream or downstream of the target nucleotide to be edited (e.g., a target mutation in a SCN1A gene).

[0140] In general, a gRNA is any RNA sequence having sufficient complementarity with a target polynucleotide sequence (e.g., SCN1A) to hybridize with the target sequence and direct sequence-specific binding of a napDNAbp (e.g., Cas9, which may be part of a base editor) to the target sequence. In some embodiments, the degree of complementarity between the spacer of a gRNA and its corresponding target sequence in SCN1A, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more (or the spacer and the corresponding target sequence comprise one, two, three, four, five, six, seven, eight, nine, or ten amino acid differences). In certain embodiments, the spacer of a gRNA is 100% complementary to its corresponding target sequence in SCN1A. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BL AT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0141] The ability of a gRNA to direct sequence-specific binding of a base editor to a target sequence may also be assessed by any suitable assay. For example, a base editor and gRNA may be provided to a host cell (e.g., a cell of the CNS, such as a neuron or a glial cell) having the corresponding target sequence (e.g., SCN1A, or a portion thereof), such as by transfection with vectors encoding the base editor and gRNA or by transfection of a ribonucleoprotein (RNP) complex, followed by an assessment of preferential cleavage, nicking, or editing within the target sequence. Similarly, cleavage or editing of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, base editor, and gRNA to be tested and a control gRNA different from the test gRNA, and comparing binding or rate of cleavage or editing at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will be apparent to those skilled in the art.

[0142] In some embodiments, a gRNA is about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 75, about 100, or more nucleotides in length. In some embodiments, a gRNA is about 50-150, about 60-140, about 70-130, about 80-120, or about 80 / 224Bl 195.70210WO00#14840465vl90-110 nucleotides in length. In some embodiments, the spacer sequence of a gRNA is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides in length.

[0143] In some embodiments, a gRNA comprises the structure 5′-[spacer sequence]-[backbone sequence]-3′. In some embodiments, a gRNA comprises an optional linker sequence. For example, the gRNAs provided herein may comprise an optional linker sequence between the spacer and the backbone sequence of the gRNA. In certain embodiments, the optional linker sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, or at least 50 nucleotides in length.Methods of Use

[0144] In various embodiments, the disclosure relates to methods of using any one of the adenine base editors (ABEs), guide RNAs, complexes, compositions, vectors, cells, and / or kits or combinations thereof disclosed herein for correcting one or more ABE-targetable mutations in the SCN1A gene, including any of the SCN1A mutant alleles of Table 1, including the SCN1A R613X mutant allele.

[0145] In other embodiments, the disclosure relates to methods of treating Dravet Syndrome or one or more symptoms thereof by administering an effective amount of any one of the adenine base editors (ABEs), guide RNAs, complexes, compositions, vectors, cells, and / or kits, or combinations thereof disclosed herein for editing one or more ABE-targetable mutations in the SCN1A gene, including any of the SCN1A mutant alleles of Table 1, including the SCN1A R613X mutant allele.

[0146] In yet other embodiments, the disclosure also relates to methods of using any one of the adenine base editors (ABEs), guide RNAs, complexes, compositions, vectors, cells, and / or kits or combinations thereof disclosed herein for correcting one or more ABE-targetable mutations in the SCN1A gene of Table 1.

[0147] In still other embodiments, the disclosure relates to methods of treating Dravet Syndrome or one or more symptoms thereof by administering an effective amount of any one 81 / 224Bl 195.70210WO00#14840465vlof the adenine base editors (ABEs), guide RNAs, complexes, compositions, vectors, cells, and / or kits, or combinations thereof disclosed herein for editing one or more ABE-targetable mutations in the SCN1A gene of Table 1.

[0148] In yet other embodiments, the disclosure relates to methods of using any one of the adenine base editors (ABEs), guide RNAs, complexes, compositions, vectors, cells, and / or kits or combinations thereof disclosed herein for correcting an SCN1A R613X mutant allele.

[0149] In still other embodiments, the disclosure relates to methods of treating Dravet Syndrome or one or more symptoms thereof by administering an effective amount of any one of the adenine base editors (ABEs), guide RNAs, complexes, compositions, vectors, cells, and / or kits, or combinations thereof disclosed herein for an SCN1A R613X mutant allele.

[0150] In other aspects, the present disclosure provides methods of increasing expression of SCN1A. In some embodiments, the method comprises contacting a nucleic acid sequence comprising SCN1A with an adenine base editor (ABE) and a gRNA targeting the ABE to a splice acceptor sequence upstream of exon 20N in SCN1A. SCN1A exon 20 (also known as the 20N “poison exon”) contains cryptic splice acceptor and splice donor sequences that can contribute to aberrant splicing events, leading to nonproductive SCN1A splicing. The methods described herein use base editing to introduce a mutation (e.g., an A-to-G mutation) into a splice acceptor sequence upstream of exon 20N in the SCN1A coding sequence that can prevent this unproductive splicing, leading to increased expression of SCN1A.Delivery Methods and AAV Particles

[0151] The present disclosure provides, in some aspects, methods comprising delivering any of the gRNAs, complexes, polynucleotides, vectors, and pharmaceutical compositions described herein. In some embodiments, a gRNA is delivered to a cell, e.g., in combination with a base editor. The base editor and / or gRNA can be delivered in any form, e.g., each may independently be delivered in DNA, RNA, or (for the base editor) protein form. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in cells (e.g., mammalian cells) or target tissues. Such methods can be used to administer nucleic acids encoding components of a base editor and gRNA to cells in culture, or in a host organism. Non-viral vector delivery systems include ribonucleoprotein (RNP) complexes, DNA plasmids, RNA, naked nucleic acid, and nucleic acid complexed with, part of, or associated with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to 82 / 224Bl 195.70210WO00#14840465vlthe cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, TIB TECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51 (1):31 -44 (1995); Haddada el al., in Current Topics in Microbiology and Immunology Doerfl er and Bihm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0152] In some embodiments, the gRNA and base editor are delivered or administered as a protein: RNA complex. In certain embodiments, the method of delivery comprises delivering an RNP complex. For example, RNP delivery of base editors markedly increases the DNA specificity of base editing. RNP delivery of base editors leads to fewer off-target effects. RNP delivery ablated off-target editing at non-repetitive sites while maintaining on-target editing comparable to plasmid delivery, and greatly reduced off-target editing even at the highly repetitive VEGFA site 2. See Rees, H. A. et al., Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery, Nat. Commun.8, 15790 (2017), which is incorporated herein by reference.

[0153] Methods of non-viral delivery of nucleic acids include RNP complexes, lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in, e.g., U. S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are sold commercially (e.g., Lipofectamine, Lipofectamine 2000, Lipofectamine 3000, Transfectam™ and Lipofectin™). In certain embodiments of the disclosed methods of editing, a cationic lipid comprising Lipofectamine 2000 is used for delivery of nucleic acids to cells. Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner (see WO 1991 / 17424 and WO 1991 / 16024). Delivery of, e.g., Cas9 proteins and gRNAs using cationic lipids and cationic polymers is also described in International Patent Application Publication Nos. WO 2015 / 035136 and WO 2016 / 070129, each of which is incorporate herein by reference. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).

[0154] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 83 / 224Bl 195.70210WO00#14840465vl270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U. S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,946,787, 9,526,784, and 9,737,604).

[0155] The use of RNA or DNA viral based systems for the delivery of nucleic acids (e.g., nucleic acids encoding a base editor and gRNA as described herein) take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo), or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated, and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

[0156] In some embodiments, an adeno-associated virus (AAV)-based system is used for delivery of nucleic acid molecule(s) encoding a gRNA and base editor. Particularly in applications where transient expression is preferred, adenoviral-based systems may be used. Adenoviral-based vectors are capable of very high transduction efficiency in many different cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. AAV vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West etal., Virology 160:38-47 (1987); U. S. Pat. No.4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors is described in a number of publications, including U. S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol.5:3251-3260 (1985); Tratschin, etal., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); Samulski etal., J. Virol. 63:03822-3828 (1989); and International Patent Application No. PCT / US2023 / 066389, filed April 28, 2023.84 / 224Bl 195.70210WO00#14840465vl

[0157] Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. In some embodiments, the AAV targets the central nervous system (CNS). In some embodiments, the AAV targets neurons. In certain embodiments, the AAV is AAV9.

[0158] In various embodiments, the constructs for expressing a gRNA and base editor described herein may be engineered for delivery in one or more AAV vectors. An AAV as related to any of the methods and compositions provided herein may be of any serotype including any derivative or pseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 2 / 1, 2 / 5, 2 / 8, 2 / 9, 3 / 1, 3 / 5, 3 / 8, or 3 / 9). An AAV may comprise a genetic load (z.e., a recombinant nucleic acid vector that expresses gene products of interest, such as a base editor and / or gRNA that is carried by the AAV into a cell) that is to be delivered to a cell.

[0159] In one aspect, the present disclosure provides one or more AAV particles comprising one or more polynucleotides encoding any of the gRNAs and base editors, or portion(s) thereof, provided herein. In some embodiments, the polynucleotide encoding the base editor is split between a first and a second AAV particle. In certain embodiments, the polynucleotides encoding the split base editor comprise an N-intein and a C-intein. In some embodiments, the first and / or the second AAV particle further comprises the polynucleotide encoding the gRNA. In some embodiments, an AAV vector comprises the sequence of any 85 / 224Bl 195.70210WO00#14840465vlone of SEQ ID NOs: 144-147, 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 of any one of SEQ ID NOs: 144-147.Pharmaceutical Compositions

[0160] Other aspects of the present disclosure relate to pharmaceutical compositions comprising any of the gRNAs, base editors, complexes, AAV particles, polynucleotides, vectors, and / or cells described herein. The term “pharmaceutical composition,” as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g., for specific delivery, increasing half-life, or other therapeutic compounds).

[0161] As used here, the term “pharmaceutically-acceptable carrier” (or “pharmaceutically acceptable excipient”) means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g, lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g, the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.). Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters,86 / 224Bl 195.70210WO00#14840465vlpolycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, and antioxidants can also be present in the formulation. Terms such as “excipient,” “carrier,” “pharmaceutically acceptable carrier,” “pharmaceutically acceptable excipient,” or the like are used interchangeably herein.

[0162] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject for gene editing (e.g., base editing).

[0163] The pharmaceutical compositions described herein may be administered or packaged as a unit dose, for example. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; / .<?., carrier, or vehicle.

[0164] In some embodiments, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierce-able by a hypodermic injection needle. The active agent in the composition is a gRNA and / or base editor described herein, including gRNA-base editor complexes. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer’s solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.87 / 224Bl 195.70210WO00#14840465vlPolynucleotides, Vectors, Cells, and Kits

[0165] The present disclosure provides, in some aspects, polynucleotides and vectors encoding any of the gRNAs, base editors, complexes, and / or AAV particles described herein. In some aspects, the present disclosure provides polynucleotides and vectors encoding a gRNA and a base 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).

[0166] Cells that may contain any of the gRNAs, base editors, complexes, AAV particles, polynucleotides, and / or vectors described herein are also provided by the present disclosure. The methods described herein may be used to deliver a gRNA and base editor into a eukaryotic cell (e.g., a mammalian cell, such as a human cell). In some embodiments, the cell is in vitro (e.g., a cultured cell). In some embodiments, the cell is in vivo (e.g., in a subject, such as a human subject). In some embodiments, the cell is ex vivo e.g, isolated from a subject and may be administered back to the same or a different subject).

[0167] 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 base 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 base editing complex, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence.

[0168] The gRNAs, base editors, complexes, AAV particles, polynucleotides, and / or vectors described herein may also be assembled into kits. In some embodiments, the kit comprises polynucleotides for expression of the gRNAs, base editors, complexes, and / or AAV particles described herein. In some embodiments, the kit comprises appropriate gRNAs or nucleic acid vectors for the expression of such gRNAs to target the Cas9 protein of a base editor to a desired target sequence, e.g, in SCN1A. In some embodiments, the gRNAs in the kit are 88 / 224Bl 195.70210WO00#14840465vluseful for correcting an R208X mutation in a SCN1A enzyme, where X is a premature stop codon.

[0169] 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 base 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.

[0170] 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, efc.), 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.

[0171] 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.89 / 224Bl 195.70210WO00#14840465vl

[0172] 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.EXAMPLESExample 1. Use of mouse model to demonstrate in vivo treatment of Dravet Syndrome by an adenine base editor targeting correction of an exemplary SCN1A mutant allele A. ABE corrects DS mutation in engineered cell lines

[0173] When comparing SCN1AR6I3Xin both human and mouse model contexts, three editing strategies were observed that optimally position the target adenosine in the ABE editing window and use conserved PAM sequences (FIGs. 5A-5B). In an engineered mouse Neuro-2a (N2A) cell line harboring a homozygous SCN1AR6I3Xmutation, these three strategies were assayed, and efficient stop codon correction was observed amongst all of them (FIG. 6). However, ABE8e-VRQR and ABE8e-NG displayed appreciable (>6%) bystander cytosine deamination generating an R612Q missense mutation, while ABE8e-SaCas9 yielded 20.3% bystander adenosine deamination to generate V610A.

[0174] These missense mutations were interrogated in the gnomAD database, and six unaffected individuals were found in the database with SCN1AR612Q, suggesting the mutation is likely benign. SCN1AV6IOAwas not present in the database, but a similar missense mutation, SCN1AV610G, was present in one sequenced individual. AlphaMissense, which predicts the pathogenicity of missense mutations by in silico protein folding18, was used to investigate these V610 mutations further. Both SCN1AV61OAand SCN1AV61OGwere annotated with ambiguous pathogenicity in the database, and ABE8e-SaCas9 was therefore excluded from further studies for biosafety.90 / 224Bl 195.70210WO00#14840465vl

[0175] Despite the likely benign nature of SCN1AR612Qby these in silico methods, it was desirable to reduce the frequency of cytosine deamination at the site. Both ABE8e-V106W-VRQR and ABE8e-V106W-NG were tested to constrict the editing window, and a >4-fold reduction in cytosine deamination and >2-fold reduction in indels was observed for both editors while maintaining high editing efficiency (FIG. 7A). Encouragingly, the lead editing strategies that were observed in N2A cells enabled efficient base editing in a HEK293T cell line harboring the concomitant homozygous SCN1AR613Xmutation (FIG. 7B).B. In vivo delivery of ABE to SCN1AR613X / +mice

[0176] For the efficient delivery of ABE to DS mice, P0 intracerebroventricular (ICV) injections of a previously reported split AAV9 ABE system19were performed. ABE8e-VRQR and -NG were chosen to move forward in vivo as low dose (7.2el0 vg), high efficiency editors, as well as ABE8e-V106W-VRQR as a high dose (1e11 vg), high precision editor. For all injections, lelO vg GFP-KASH AAV9 was co-injected as a transduction control. In both bulk and GFP+ nuclei isolated from tissues, both DNA and RNA were assessed in paired samples to link genomic and transcriptomic editing data.

[0177] At P21 in cortices from VRQR-ABE8e treated mice, only 63.08% of reads were observed to contain the desired R613 codon in bulk nuclei gDNA, which is 26.16% allelic correction above untreated heterozygous mice (FIG. 9A). However, in cDNA isolated from the same bulk nuclei sample, it was found that 81.87% of reads contained R613, equating to 63.74% allelic conversion. These findings suggest that genomic correction was highly enriched in cells which express SCN1A, and this is consistent with previous work establishing that SCN1A is highly expressed throughout interneurons of the cortex and hippocampus20. Additionally, >90% allelic conversion to R613 was seen in GFP+ nuclei for both ABE8e-VRQR and ABE8e-NG, albeit with heightened indels in ABE8e-NG treated samples, indicating high editing efficiency by either enzyme in highly transduced cells.

[0178] At P45, four brain regions (cortex, hippocampus, cerebellum, and rest of brain) were isolated from P0 ABE8e-V106W-VRQR treated mice, and gDNA and cDNA editing efficiencies were assessed again in bulk and GFP+ nuclei. Efficient gDNA stop codon correction was observed in bulk nuclei of both the cortex and hippocampus of treated mice while the cerebellum, which is poorly transduced by AAV9, showed only 3.94% allelic conversion for the correction of R613X (FIG. 9B). In cDNA of the same treated samples,91 / 224Bl 195.70210WO00#14840465vlnear complete correction was observed in all sequenced SCN1A transcripts of the cortex and hippocampus (FIG. 9C). Surprisingly, greater-than-expected frequency of transcripts expressing the desired R613 codon was observed in both the hippocampus and cerebellum of untreated mice.

[0179] As transcripts with premature stop codons are expected to undergo nonsense-mediated decay (NMD) in the cytoplasm, it was sought to assay this phenomenon in whole cell lysates of full brain hemispheres. A two-fold reduction in SCN1A transcripts was noted in P21 vehicle-treated SCN1AR613X / +mice relative to wild-type control mice (FIG. 10A).Encouragingly, a significant increase in SCN1A transcripts was measured following P0 ABE treatment. These findings demonstrate that stop codon correction with ABE ameliorates NMD in DS mice to rescue expression of SCN1A.C. ABE treatment rescues SCN1AR613X / +mice from febrile seizures, SUDEP

[0180] Encouraged by the molecular findings, rescue from two gross phenotypes previously characterized in the SCN1AR613X / +mouse model was quantified next. Previous studies found that P21-24 SCN1AR613X / +mice recapitulate the temperature-sensitive seizures observed in humans with Dravet Syndrome. To test whether the base editing strategy rescued this aspect of the mouse phenotype, the core body temperature of SCN1AR613X / +mice treated with vehicle or ABE was elevated, and seizure activity was monitored up to 42.5°C (~108°F). Vehicle-treated mice exhibited seizures at an average of 40.3°C (FIG. 12A). All mice treated with ABE were seizure-free at temperatures <42.5°C, although two treated mice exhibited seizures when residual heat caused temperatures >42.5°C, temperatures at which WT mice occasionally have seizures. These results demonstrate that ABE enables nearly complete rescue from febrile seizures, a key hallmark of DS.

[0181] This SCN1AR613X / +mouse model also displays a strong SUDEP phenotype, with 60% (9; N=15) of male and 87% (13; N=15) of female mice dying between P20 and P45 (FIG. 12B). ABE treatment drastically ameliorates this phenotype, with only 13% of male mice and 7% of female mice dying by P45. Collectively, these findings represent a 7-fold reduction in SUDEP amongst ABE-treated DS mice relative to vehicle-treated controls. Altogether, these febrile seizure and survival data demonstrate that ABE drastically reduces the risk of epileptic events in DS mice.D. Methods92 / 224Bl 195.70210WO00#14840465vlCell culture

[0182] Neuro-2a (ATCC CCL-131) and HEK293T (ATCC CRL-3216) cells were purchased from American Type Culture Collection and cultured in Dulbecco’s modified Eagle medium with GlutaMax (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) at 37°C with 5% CO2. All cell lines were verified to be free of mycoplasma and were identity-authenticated by their suppliers.Generation of a homozygous SCN1 A R613X N2A cell line using prime editing

[0183] The SCN1AR613X / +mouse model pathogenic allele contains two mutations relative to mmlO ( SCN1A c.1833 and SCN1A c.1837A>T). The SCN1AR613X2A cell line was generated by PE5-style prime editing as previously described21. Briefly, N2A cells were seeded in 48-well poly-D-lysine coated tissue culture plates (Corning) at a density of 25,000 cells per well. After 17-24 hours, cells were transfected with IpL of Lipofectamine 2000 (Thermo Fisher Scientific) following manufacturer’s protocols, as well as 750ng PEmax plasmid, 500 ng murine MLH1dn, 250 ng pegRNA 1, and 83 ng nsgRNA 1. Following a 72-hour incubation, cells were trypsinized, resuspended in DMEM, and plated at a density of 0.5 cells per well in 96-well plates (Corning). Single-cell colonies were evaluated for installation of the desired mutations using high-throughput DNA sequencing.Generation of a homozygous SCN1 A R613X HEK293T cell line using base editing

[0184] The homozygous SCN1AR613XHEK293T cell line was generated by cytosine base editing. HEK293T cells were seeded in 48-well poly-D-lysine coated tissue culture plates (Corning) at a density of 25,000 cells per well. After 17-24 hours, cells were transfected with IpL of Lipofectamine 2000 (Thermo Fisher Scientific) following manufacturer’s protocols, as well as 750 ng TadCBEa-V106W-NRCH and 200 ng sgRNA pDTN1454. Following a 72-hour incubation, cells were trypsinized, resuspended in DMEM, and plated at a density of 0.5 cells per well in 96-well plates (Corning). Single-cell colonies were evaluated for installation of the desired mutations using high-throughput DNA sequencing.N2A and HEK293T ABE transfection

[0185] Cells were seeded in 48-well poly-D-lysine coated tissue culture plates (Corning) at a density of 25,000 cells per well in 0.25 mL media. After 17-24 hours, cells were transfected with 1 pL of Lipofectamine 2000 (Thermo Fisher Scientific) following manufacturer’s 93 / 224Bl 195.70210WO00#14840465vlprotocols, as well as 750 ng editor plasmid and 200 ng sgRNA plasmid. Following 72-hour incubation, cells were lysed for high-throughput sequencing.High-throughput sequencing and data analysis

[0186] Genomic DNA was isolated from cells grown in culture by first removing media, washing with PBS, and adding 100 pL of freshly prepared lysis buffer [10 mM Tris-HCl, pH 8; 0.05% SDS, 20 pg / ml of proteinase K (Qiagen)] directly to each well. Lysates were incubated in the tissue culture plate at 37 °C for 1 hour before transferring to a PCR plate and inactivating the proteinase K by incubation at 80 °C for 30 minutes. DNA sequencing was performed using Illumina MiSeq as previously described21. Briefly, DNA primers containing Illumina forward and reverse adapters were used to amplify the mouse or human SCN1A target site with the following conditions: 0.5 pM each of forward and reverse primer, 1 pl of genomic DNA, and 12.5 pl PhusionU Green Multiplex PCR Master Mix (Thermo Fisher Scientific) in a 25-pl reaction. PCR reactions were as follows: 98 °C for 2 minutes and then 30 cycles of 98 °C for 10 seconds, 61 °C for 20 seconds, 72 °C for 30 seconds, followed by a final 72 °C extension for 2 minutes. Samples were barcoded with Illumina barcode pairs through a second round of PCR (PCR2), with the following conditions: 0.5 pM each of forward and reverse primer, 1 pl of unpurified PCR1, and 12.5 pl PhusionU Green Multiplex PCR Master Mix in a 25-pl reaction. PCR reactions were as follows: 98 °C for 2 minutes and then 10 cycles of 98 °C for 10 seconds, 61 °C for 20 seconds, 72 °C for 30 seconds, followed by a final 72 °C extension for 2 minutes. PCR2 products were pooled and purified by gel electrophoresis on a 1.5% agarose gel using a QIAquick Gel Extraction Kit (Qiagen), eluting with 25 pl water. DNA library concentration was measured by Qubit high-sensitivity fluorometric quantification (Thermo Fisher Scientific) and sequenced on an Illumina MiSeq instrument using Illumina MiSeq control software (version 4.1), according to the manufacturer’s protocols. Sequencing reads were demultiplexed using the onboard MiSeq Analysis Module (Illumina).

[0187] Amplicons were aligned to reference sequences with CRISPResso222, using the following flags: discard indel reads = TRUE, q = 30, and qwc was set to 10 nt upstream and downstream of a given spacer sequence. Indels for samples were calculated as ((‘Discarded’ / ’Reads aligned all amplicons’) x 100). Editing efficiency at a given base was94 / 224Bl 195.70210WO00#14840465vlcalculated as ((frequency of point mutation in the ‘Nucleotide_percentage_summary.txt’ file) x 100).AAV production

[0188] rAAV was produced by transient HEK 293 cell transfection and CsCl sedimentation by the University of Massachusetts Medical School Viral Vector Core, as previously described (Gao GP, Sena-Esteves M. Introducing Genes into Mammalian Cells: Viral Vectors. In: Green MR, Sambrook J, eds. Molecular Cloning, Volume 2: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press; 2012:1209 -1313.). Vector preparations were determined by ddPCR, and purity was assessed by 4%-12% SDS-acrylamide gel electrophoresis and silver staining (Invitrogen).Nuclear isolation for fluorescence-activated sorting

[0189] Mouse brain tissues were homogenized using the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec) instructions for nuclei extraction. Briefly, tissues were disrupted in 4 mL of nuclei extraction buffer (Miltenyi) supplemented with 0.2 U / pL murine RNase inhibitor (NEB). Homogenates were then passed through a 100 pM cell strainer before being pelleted by centrifugation at 500 x g for 5 minutes. Pellets were resuspended in 4 mL cold nuclear suspension buffer (NSB; lx PBS, pH 7.4, 3.33 pM Vybrant DyeCycle Ruby [Thermo Fisher Scientific], 100 pg / pL recombinant albumin [NEB], 0.2 U / pL murine RNase inhibitor [NEB]). Samples were pelleted again by centrifugation at 500 x g for 5 minutes before resuspending in 1 mL of NSB. Samples were passed through a 35 pM cell strainer and sorted using a SH800 Cell Sorter (Sony). Nuclei were sorted into Buffer RLT Plus (Qiagen) supplemented with 40 mM dithiothreitol. Both gDNA and nuclear RNA were isolated using AllPrep DNA / RNA columns (Qiagen) following manufacturer’s instructions. Isolated RNA was reverse transcribed into cDNA using SuperScript IV first-strand synthesis mix with random hexamers (Invitrogen).Western Blot for Nav1.1 expression

[0190] Protein from mouse hippocampi were isolated as previously described1. Whole hippocampi were homogenized in 500 pL 0.32 M sucrose, pH 7.4, supplemented with cOmplete Protease Inhibitor Cocktail (Millipore Sigma). From this, 100 pL of homogenate 95 / 224Bl 195.70210WO00#14840465vlwere diluted into 1 mL of homogenization buffer and disrupted by trituration through a 22 G needle. Membranes were isolated by centrifugation at 24,000 x g for 60 minutes. Pelleted membranes were resuspended in 150mM NaCl, 2% Triton X-100, 25 mM Tris (pH 7.4), supplemented with protease inhibitors. 12 pg aliquots of isolated protein were separated on 4-12% Bis-Tris NuPage protein gels (Life Technologies) and transferred onto a PVDF membrane. Blocked membranes were incubated overnight with either anti-NaVl.l antibody (1:200, Alomone Labs) or anti-calnexin (1:2,000, Stressgen Biotechnologies) followed by incubation with goat anti-rabbit antibody (1:3000, Bio-Rad). Membranes were developed by chemiluminescent visualization using ECL, and densitometry was analyzed in image lab software (version 6.1; Bio-Rad).Mice and Animal Care

[0191] For the generation of testing cohorts, SCN1AR613Xheterozygous animals (JR#34129, also denoted as 129S1 / SvImJ- SCN1Aem1Dsi / J) were bred with C57BL6 / J (JR#00664) to produce B6129F1 offspring. Heterozygous SCN1AR613Xoffspring and their wildtype littermates were sampled via toe clip at Pl-3 to identify phenotype. At P21, animals were weaned and housed with mixed litter sex- and genotype-matched animals. Animals were ear notched for identification and confirmation of genotype. All animals were kept on aspen bedding and fed a 6% fat diet (LabDiet 5K52). Animals were provided a nestlet for enrichment and housed at a density of 3-4 mice per pen. All animals were monitored daily for wellbeing and survival starting at P14. Human euthanasia was applied to any animal that fell to a body condition score of 2 or lower, displayed hydrocephaly, or was determined by veterinary staff to meet humane endpoints.Intracerebroventricular (ICV) Injections

[0192] After P0 genotyping, test article and vehicle were administered via ICV injects at Pl. In brief, pups were anesthetized via hypothermia in chamber set on ice. Once fully anesthetized, pups were injected with either test article or vehicle via dual hemisphere injections using a Hamilton Model 1701 Neuro Syringe with a 33-gauge beveled needle with neuro adaptor. 2 pl per hemisphere of solution were injected at a depth of 2 mm, with a dwell time of 10 seconds per hemisphere. Animals were then placed on a heated recovery pad until96 / 224Bl 195.70210WO00#14840465vlindependently moving and warmed prior to rehousing with their birth dam. Multiple litters were used for each treatment group.Statistics

[0193] All mice were randomly allocated into age-matched and genotype experimental groups. The data were analyzed for statistical significance and graphed using GraphPad Prism 9 (version 10.1.1). It was determined that data followed a normal distribution, and then respected unpaired two-tailed t-test was used for comparison between two groups and oneway analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test for multiple comparison was also used. For survival curves, a Log-rank (Mantel-Cox) test was employed to compare treated vs. vehicle groups. A P value less than 0.05 was considered significant. Investigators were blinded during data collection and analysis.Hyperthermic seizure induction

[0194] Mice were placed in a plexiglass observation chamber and allowed to move freely. Core body temperature was monitored with a rectal probe. Hyperthermia was induced with a heat lamp until core body temperature reached 42.5°C or a seizure occurred. Seizures were identified by visual monitoring by at least two expert observers. The temperature of seizure onset was recorded. Mice were cooled on ice until body temperature returned to baseline.Acute brain slice preparation

[0195] To label parvalbumin interneurons for electrophysiological analysis, SCN1AR613X / +mice on a 129 genetic background were crossed to PV-tdTomato mice (Jax #027395). Mice were treated with ABE + GFP-KASH or GFP-KASH only as described above.

[0196] Coronal brain slices were prepared from Pl 8-21 male and female mice of each genotype and treatment condition. Mice were anesthetized with isofl urane. The brain was dissected and transferred to ice-cold sucrose cutting solution (in mM: 75 sucrose; 10 glucose; 26 NaHCO3; 2.5 KCl; 1.25 NaH2PO4; 87 NaCl; 1 CaCl2; 2 MgSO4) bubbled with 95% O2 / 5% CO2. The cerebellum was removed, and the brain was attached to the specimen holder of a Leica VT1200S vibratome using cyanoacrylate glue. Slices were cut at a thickness of 300 pm in ice-cold sucrose cutting solution and allowed to recover for 30 minutes at 32 °C. Slices97 / 224Bl 195.70210WO00#14840465vlwere then maintained in sucrose cutting solution at room temperature for up to 5 hours before recording.Electrophysiology

[0197] Slices were placed on the recording chamber of a Scientifica SliceScope electrophysiology rig and perfused at 3 mL / min and 30-32 °C with artificial cerebrospinal fluid (in mM: 125 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgSO4).

[0198] Primary somatosensory cortex was identified based on visualization of characteristic “barrels” via infrared differential interference contrast microscopy. PV-INs were identified based on tdTomato reporter expression visualized by epifluorescence microscopy and confirmed by characteristic fast-spiking discharge pattern. In mice treated with ABE + GFP-KASH, both GFP+(edited) and GFP' (unedited) cells were recorded. In mice treated with GFP-KASH only, Only GFP+cells were recorded. All recordings were performed in cortical layer 2 / 3.

[0199] Whole-cell recordings were obtained with borosilicate glass patch pipettes pulled on a P-1000 puller (Sutter Instruments) with a tip resistance of 3-5 MQ. Pipettes were filled with a low-chloride (Cl= -70) internal solution (in mM: 130 K-gluconate, 6.3 KCl, 1 MgCh, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.3 Na-GTP. pH adjusted to 7.3 with KOH and osmolarity adjusted to 285 mOsm with 30% sucrose). All chemicals were purchased from Sigma Aldrich.

[0200] Current-clamp recordings were performed with a MultiClamp 700B amplifier (Molecular Devices) and digitized with an Axon Digidata 1550B digitizer (Molecular Devices). Data were acquired with pCLAMP 10 software, low-pass filtered at 10 kHz, and sampled at 100 kHz. Cells with resting membrane potentials more positive than -55 mV, series resistance greater than 20 MQ, or with changes in access resistance larger than 20% during recording were excluded. Pipette capacitance compensation was applied for all experiments. Liquid-liquid junction potential was not corrected, and was calculated as 15.9 mV at 32°C.

[0201] After breaking in, each cell was recorded two minutes in gap-free current-clamp mode. A ramp protocol (0-400 pA, 2s) was recorded to confirm fast-spiking behavior. Each fast-spiking cell was stimulated with 600 ms current pulses at 25 pA intervals beginning at -98 / 224Bl 195.70210WO00#14840465vl100 pA. When necessary, holding current was applied to maintain membrane potential between -65 and -70 mV. All recordings were performed blind to genotype.Electrophysiology data analysis

[0202] Analysis was performed with custom Matlab (Mathworks) and R (version 4.4.0) scripts and manually confirmed with Clampfit software. Resting membrane potential was measured as the average membrane potential during a 2s sweep with no direct current injection. Input resistance was calculated as the response to a -100 pA direct current injection using Rm = AV / I. Membrane time constant was calculated from a single exponential fit of the hyperpolarizing response to a -100 pA current injection. Membrane sag was measured from the hyperpolarizing response to a -100 pA current injection by calculating the ratio of the steady-state voltage to the maximal hyperpolarization. Rheobase was calculated as the smallest current step capable of inducing the firing of an action potential (AP, defined as meeting having the first derivative of the voltage, dV / dt > 10 mV / ms and a voltage greater than -10 mV).

[0203] AP threshold was defined as the voltage at which dV / dt exceeded 10 mV / ms, calculated from the first AP at rheobase. AP peak was defined as the maximal voltage achieved during the first action potential at rheobase. AP amplitude was calculated as the voltage difference between the AP peak and AP threshold. AP rise time was the time difference between AP peak and AP threshold. The maximal rise slope was defined as the maximal dV / dt achieved between AP threshold and AP peak. AP halfwidth was calculated as the width of the first AP at rheobase at half-maximal amplitude. The after hyperpolarization amplitude was calculated as the absolute minimum voltage achieved during the first AP at rheobase subtracted from the AP threshold.

[0204] The maximal steady-state firing frequency was calculated by dividing the maximum number of APs observed during a single sweep by the sweep duration (600 ms). The maximum instantaneous firing frequency was calculated as the inverse of the smallest inter-AP interval. The spike frequency adaptation was calculated as the ratio of the first and second, tenth, or final inter- AP interval evoked by the first current step to evoke at least 40 APs.99 / 224Bl 195.70210WO00#14840465vlReferences for Example 11. Mavashov, A. et al. Heat-induced seizures, premature mortality, and hyperactivity in a novel SCN1A nonsense model for Dravet syndrome. Front. Cell. Neurosci. 17, 1149391 (2023).2. Ogiwara, I. et al. Nav1.1 Localizes to Axons of Parvalbumin-Positive Inhibitory Interneurons: A Circuit Basis for Epileptic Seizures in Mice Carrying an SCN1A Gene Mutation. J. Neurosci. 27, 5903-5914 (2007).3. Ho, S.-Y. et al. Perampanel Reduces Hyperthermia-Induced Seizures in Dravet Syndrome Mouse Model. Front. Pharmacol. 12, 682767 (2021).4. Valassina, N. et al. SCN1A gene reactivation after symptom onset rescues pathological phenotypes in a mouse model of Dravet syndrome. Nat. Commun. 13, 161 (2022).5. Goff, K. M., Liebergall, S. R., Jiang, E., Somarowthu, A. & Goldberg, E. M. VIP interneuron impairment promotes in vivo circuit dysfunction and autism-related behaviors in Dravet syndrome. Cell Rep. 42, 112628 (2023).6. Kwong, A. K.-Y., Fung, C.-W., Chan, S.-Y. & Wong, V. C.-N. Identification of SCN1A and PCDH19 Mutations in Chinese Children with Dravet Syndrome. PLoS ONE 7, e41802 (2012).7. Schuster, J. et al. Transcriptomes of Dravet syndrome iPSC derived GABAergic cells reveal dysregulated pathways for chromatin remodeling and neurodevelopment.Neurobiol. Dis. 132, 104583 (2019).8. Hammer, M. F. et al. Rare variants of small effect size in neuronal excitability genes influence clinical outcome in Japanese cases of SCN1A truncation-positive Dravet syndrome. PLOS ONE 12, e0180485 (2017).9. Kleinstiver, B. P. et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490-495 (2016).10. Miller, S. M. et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat. Biotechnol. 38, 471-481 (2020).11. Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P.Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368, 290-296 (2020).100 / 224Bl 195.70210WO00#14840465vl12. Huang, T. P. et al. High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs. Nat. Biotechnol. 41, 96-107 (2023).13. Doman, J. L., Raguram, A., Newby, G. A. & Liu, D. R. Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. Nat. Biotechnol. 38, 620-628 (2020).14. Newby, G. A. et al. Base editing of haematopoietic stem cells rescues sickle cell disease in mice. Nature 595, 295-302 (2021).15. Koblan, L. W. et al. In vivo base editing rescues Hutchinson-Gilford progeria syndrome in mice. Nature 589, 608-614 (2021).16. Chiesa, R. et al. Base-Edited CAR7 T Cells for Relapsed T-Cell Acute Lymphoblastic Leukemia. N. Engl. J. Med. 389, 899-910 (2023).17. Arbab, M. et al. Base editing rescue of spinal muscular atrophy in cells and in mice. Science 380, eadg6518 (2023).18. Cheng, J. et al. Accurate proteome-wide missense variant effect prediction with AlphaMissense. Science 381, eadg7492 (2023).19. Levy, J. M. et al. Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Nat. Biomed. Eng. 4, 97-110 (2020).20. Haigh, J. L. et al. Deletion of a non-canonical regulatory sequence causes loss of SCN1A expression and epileptic phenotypes in mice. Genome Med. 13, 69 (2021).21. Doman, J. L., Sousa, A. A., Randolph, P. B., Chen, P. J. & Liu, D. R. Designing and executing prime editing experiments in mammalian cells. Nat. Protoc. 17, 2431-2468 (2022).22. Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224-226 (2019).Example 2. In vivo adenine base editing rescues Dravet syndrome in mice

[0205] Dravet syndrome (DS) is a severe neurodevelopmental disorder characterized by drug-resistant epilepsy, temperature-sensitive seizures, cognitive impairment, and a high incidence of sudden unexpected death in epilepsy (SUDEP). DS is caused by loss-of-function variants in SCN1A, which encodes the voltage-gated sodium channel a subunit Navl.l. Current DS treatments manage symptoms and do not address the root cause of the disease.101 / 224Bl 195.70210WO00#14840465vlThe present disclosure describes the use of adenine base editing (ABE) to directly correct SCN1AR6I3X, a mutation found in DS patients. ABE strategies to efficiently correct R613X in engineered homozygous SCN1AR613X / +HEK293T and Neuro-2a cells were identified (72% and 92% correction efficiencies, respectively). A dual-AAV9 approach was used to deliver an optimized ABE system to Scn1aR613X / +mice, which recapitulated key DS pathologies. AAV9-ABE treatment of Scn1aR613X / +neonates resulted in efficient DNA and mRNA editing (59% and 97%, respectively, in bulk neocortex), restored parvalbumin-expressing inhibitory neuron excitability and function, rescued mice from febrile seizures, and led to a 7.0-fold improvement in 45-day survival over vehicle-treated mice. These findings represent the direct correction of the cause of DS, validate a strategy to correct SCN1A mutations with ABE, and suggest the potential of precision genome editing treatments for DS and other neurodevelopmental disorders.

[0206] Dravet syndrome (DS) is a severe neurodevelopmental disorder defined by treatmentresistant epilepsy, temperature-sensitive seizures, developmental delay, intellectual disability, features of autism spectrum disorder, and high rate of sudden unexpected death in epilepsy (SUDEP)1’2. DS is characterized by symptom onset in the first year of life. The instigating seizure episode is often triggered by a fever, and ensuing epileptic episodes become progressively worse, with many spontaneous events as well as seizures induced by hyperthermic conditions. Approximately 80-90% of DS cases result from de novo loss-of-function mutations in SCN1A3–5, which encodes the voltage-gated sodium channel a subunit Navl.l. This gene is preferentially expressed in GABAergic inhibitory neurons6. Loss of one functional copy of Navl.l impairs the generation and propagation of action potentials in these cells, especially in parvalbumin-expressing inhibitory neurons7. No treatments for DS are available that target the underlying cause of the disease.

[0207] DS is an attractive target for the development of a therapeutic gene editing strategy. Mouse models of Senia haploinsufficiency recapitulate many aspects of the human phenotype, including epilepsy, febrile seizures, SLTDEP, and cognitive deficits7". The human and mouse SCN1A genes share 98.2% amino acid identity and 90.5% coding sequence identity. Symptom onset in DS occurs between 3-6 months of age in humans, coinciding with expression of SCNlA ’n. These observations suggest a window for disease prevention that is also reflected in DS mouse models, in which symptoms do not manifest until 18-21 days after102 / 224Bl 195.70210WO00#14840465vlbirth. Correcting DS-causing mutations prior to the onset of SCN1A expression could offer a one-time treatment of the disease.

[0208] In this study, an adenine base editing strategy was developed to correct a mouse model of the Scn1a-p.R613X mutation8. Unlike Senia deletion or conditional knockout mouse models of DS, Scn1aR613X / +is a recurrent variant identified in DS patients, and -80% of mutations that cause DS are missense, nonsense, or frameshift variants14 l7. TheScn1aR613X / +mouse model closely replicates the key molecular and phenotypic features of the disease. Developing a treatment to correct a nonsense mutation in Senia was of particular interest because variants that cause premature truncation — including nonsense, splice-site, and frameshift mutations — exhibit the highest penetrance among DS patients18,19, and clinical translation might therefore be first applied to presymptomatic patients with nonsense variants predicted to cause severe disease. These variants result in truncated or misfolded proteins, and their pathogenicity is compounded by nonsense-mediated decay of the mutant transcript20. In the ClinVar database, approximately 10% of listed DS-causing nonsense variants are Arg-to-STOP codons, which are caused by CGA> TGA transition mutations.

[0209] Arg-to-STOP mutations can be corrected by adenine base editors (ABEs), which convert A T base pairs to G C at specific locations in the genome in dividing and nondividing cells without requiring double-stranded DNA breaks or donor DNA tempi ate s21 23. An ABE is a programmable DNA-binding protein fused to an laboratory-evolved deoxyadenosine deaminase21,24. ABEs have been used with a variety of Cas domains to change both the editing window and PAM preference25 28. These modifications position the editing window over the target base and reduce unwanted bystander editing of nearby adenines. The deaminase domain can be similarly interchanged to narrow or broaden the editing window, increase editing efficiency, or reduce Cas-independent deamination events29. ABEs have been used in ex vivo and in vivo animal applications including the treatment of sickle cell disease30, Hutchinson-Gilford progeria syndrome31, acute lymphoblastic leukemia32, and spinal muscular atrophy33, with multiple ongoing clinical trials reporting positive patient outcomes34 38.ABE corrects DS mutation in engineered cell lines

[0210] For the correction of Scn1aR613X, ABE treatments that could be translated from mouse model to patients with minimal changes were sought. Three editing strategies were identified,103 / 224Bl 195.70210WO00#14840465vleach consisting of a Cas9-based ABE combined with an sgRNA, that optimally position the target adenosine for correcting SCN1AR6I3Xin the ABE editing window and use PAM sequences that are conserved between mouse and human to enhance translatability (FIG. 13 A). Using prime editing39, a mouse Neuro-2a (N2A) cell line harboring a homozygous 5cw7a-p. R613X mutation was generated. To test the efficiency of the three candidate editing strategies, plasmids encoding each candidate ABE and sgRNA were transfected into N2A 5c«7a-p. R613X cells, and efficient nonsense mutation correction was observed for all three strategies (FIG. 13B). ABE8e-VRQR yielded 82% average A»T-to-G»C editing at the target base As, converting the TGA stop codon to CGA, while ABE8e-NG and ABE8e-SaCas9 showed 76% and 69% average correction, respectively.

[0211] The presence of non-silent bystander editing at nearby bases C7 and A13 across these ABE strategies was also noted. ABE8e-VRQR and ABE8e-NG displayed appreciable (>6.1%) bystander cytosine deamination at C7 that generates an R612Q missense mutation, while ABE8e-SaCas9 yielded 20% bystander adenosine deamination at A13 that results in V610A (FIG. 13B).

[0212] To gain insight into the potential consequence of these non-silent bystander mutations, the variants in the gnomAD database were interrogated, and six unaffected individuals were found in the database with SCN1AR612Q, suggesting this mutation is likely benign40. SCN1AV6IOAwas not present in the database, but a similar missense mutation, SCN1AV610G, is present in one sequenced individual. To investigate these V610 mutations further, AlphaMissense, which predicts the pathogenicity of missense mutations by AlphaF old-mediated computational protein folding41, was also used. Both SCN1AV6IOAand SCN1AV6IOGwere classified as ambiguous pathogenicity by AlphaMissense. ABE8e-SaCas9 was therefore excluded from further studies. To further probe the potential functional consequences of this SCN1AR612Qvariant, whole-cell patch-clamp recording was performed with HEK293T cells transfected with plasmid containing sodium channel P-subunit genes and SCN1AR612Qor wild-type SCN1A. Peak current density and voltage dependence of activation and inactivation were all unchanged compared to WT (FIGs. 22A-22C). These findings, along with database evidence, suggest that the bystander mutation is not likely to have pathogenic effects.

[0213] SCN1AV6IOAwa not present in the gnomAD database, but a similar missense variant, SCN1AV610G, is present in one sequenced individual. To investigate these V610 mutations 104 / 224Bl 195.70210WO00#14840465vlfurther, AlphaMissense was used, which predicts the pathogenicity of missense variants by AlphaF old-mediated computational protein folding41. Both SCN1AV61OAand SCN1AV61OGwere classified as ambiguous pathogenicity by AlphaMissense. ABE8e-SaCas9 was therefore excluded from further studies. While SCN1AR612Qis predicted to be benign by these in silico methods and by human genetic evidence, it was nonetheless desired to minimize the frequency of bystander R612Q editing at the site. It was previously reported that the V106W variant of ABE8e and ABE8e-derived base editors offers reduced aberrant activity that can arise from ABE8e22, 42,43. Both ABE8e-V106W-VRQR and ABE8e-V106W-NG were tested, and a >4.0-fold reduction in the frequency of C7 bystander editing was observed, as well as a >2.0-fold reduction in indels for both base editors while maintaining high on-target editing efficiencies of 92% following ABE8e-V106W-VRQR treatment and 86% from ABE8e-V106W-NG (FIG. 13C). These results demonstrate the utility of ABE8e-V106W variants to enhance base editing precision.

[0214] This optimized treatment yielded similar mutation-correction efficiencies when tested in a human genomic context. The top-performing ABE8e-V106W-VRQR and ABE8e-V106W-NG editing strategies in mouse N2A cells also enabled efficient base editing in a BE-engineered HEK293T cell line harboring a homozygous 5'CMM-p. R613X mutation, with ABE8e-V106W-VRQR enabling 62% correction efficiency and ABE8e-V106W-NG showing 72% efficiency (FIG. 13D). The frequency of undesired editing events in this human cell model was markedly lower compared to the treatment in mouse N2A cells; ABE8e-V106W-VRQR resulted in 0.33% C7 deamination and 0.19% indel formation in HEK293T 5'CMM-p. R613X cells, compared to 1.5% C7 deamination and 0.67% indel formation in N2A 5cw7a-p. R613X cells.

[0215] In addition to pursuing the ABE8e-V106W-VRQR strategy, which is compatible with both human and mouse NGA PAM sequences, albeit with four nucleotide differences in their sgRNA protospacers, one SpCas9-ABE strategy that binds a NGG PAM only found in human SCN1A was also tested. This strategy showed comparable stop codon correction to ABE8e-V106W-VRQR with 52% efficiency but with a higher frequency of indel formation (1.5%) and V610A bystander editing (2.4%; FIGs. 17A-17B). Based on these data, ABE8e-V106W-VRQR was advanced for in vivo experiments. It should be noted that ABE8e-V106W-SpCas9 is likely to also be useful in higher order, humanized 5CA7^4-p. R613X models.105 / 224Bl 195.70210WO00#14840465vl

[0216] CIRCLE-seq44, an unbiased genome-wide off-target site nomination method, was performed to identify Cas-dependent off-target editing events caused by ABE8e-V106W-VRQR treatment in HEK293T SCN1AR613Xcells. CIRCLE-seq nominated 88 candidate off-target genomic loci that are engaged by the DNA-binding domain of the base editor. Editing at each of these candidate sites was assessed by high-throughput sequencing HEK293T SCN1AR613Xcells exposed to ABE8e-V106W-VRQR treatment. In total, four of the 88 nominated candidate off-target loci showed >0.5% A»T-to-G»C editing over untreated controls (FIG. 13E; FIG. 18). Two of these loci, OT9 and OT42 (4.5% and 0.62% average A-to-G reads, respectively), map to intronic regions of long non-coding RNAs, neither of which show detectable expression in human tissues. Additionally, 1.1% average editing was observed in intron 26 oiMTHFDlL (OT2), as well as 1.4% average editing in intron 2 of IGF2BP3 (OT69), neither of which are expressed in the CNS45. An additional time course assay was performed over 9 days to observe off-target editing following extended transfection of HEK293T SCN1AR6I3Xcells, and no increase in on- or off-target editing was observed (FIG. 23). Together, these data suggest that ABE8e-V106W-VRQR efficiently corrects SCN1AR6I3Xin human cells with minimal unintended editing outcomes and no detected off-target edits of anticipated consequence in the CNS.In vivo delivery of ABE to Sen lctR6i3Xmice

[0217] SCN1A is preferentially expressed in inhibitory neurons throughout the cortex and hippocampus in both humans and mice46,47. The therapeutic benefit of a DS treatment therefore is dependent on cerebral cortex biodistribution. To deliver in vivo the optimized ABE strategies to DS mice, a dual-AAV9 ABE system48was used (FIG. 14A). ABE8e-VRQR and ABE8e-NG were chosen to advance into in vivo studies as high-efficiency, low-dose (7.2el0 total vg) treatments, as well as ABE8e-V106W-VRQR as a high-precision, high-dose (lei 1 total vg) treatment. These AAV doses equate to lxl014vg kg'1in typical newborn mice, AAV doses previously established not to cause acute toxicity in vzvo31,66'68. Intracerebroventricular (ICV) injections of the ABE AAV into neonatal (P0) mice were performed. All treatments included coinjection of lelO vg GFP-KASH AAV9 as a transduction control. Following isolation and sorting of bulk and transduced (GFP-positive, GFP+; FIGs. 24A-24B) nuclei populations, both genomic DNA and cellular RNA were isolated in paired samples. This approach allowed genomic and transcriptomic editing data of 106 / 224Bl 195.70210WO00#14840465vlbulk and transduction-enriched tissues to be linked, providing information on editing efficiency in highly transduced cells, as well as in cells that express Senia.

[0218] In bulk cerebral cortex from ABE8e-VRQR-treated mice 21 days after P0 ICV injection, 63% of genomic DNA reads contained the normal R613 codon, which represents 26% correction of the pathogenic allele in these heterozygous mice (FIG. 14B). This editing was accompanied by 0.34% indel formation, which is slightly favorable to the 0.69% indel formation in bulk nuclei by ABE8e-NG. From the same bulk nuclei sample, mRNA analysis revealed 82% of reads containing 5c«7a-p. R613, corresponding to 64% pathogenic allele correction. These findings suggest that AAV9 expression and ABE genomic correction were highly enriched in cells expressing Senia, consistent with previous studies establishing Senia as highly expressed in neocortical and hippocampal interneurons49. This finding was further supported by brain slice immunostaining convergence between the GFP signal and parvalbumin-positive cells (FIG. 25). These data are consistent with previous findings that AAV9 efficiently transduces neurons following ICV injection51. In addition, high allelic conversion was observed in genomic DNA of GFP+nuclei in samples treated with either ABE8e-VRQR (82%) or ABE8e-NG (79%), indicating high editing efficiency by either base editor among transduced cells.

[0219] At P45, four brain samples (neocortex, hippocampus, cerebellum, and rest of brain) were isolated from ABE8e-V106W-VRQR-treated mice, and genomic DNA and cDNA editing efficiencies in bulk and GFP+nuclei were assessed again. As expected, efficient genomic DNA stop codon correction was found in bulk nuclei of both the neocortex (59%) and hippocampus (52%) of treated mice, while the cerebellum, which is poorly transduced by AAV948,50-52, showed only 4.0% allelic conversion for the correction of R613X (FIG. 14C). In cDNA of the same treated samples, near complete correction in bulk sequenced Senia transcripts of the neocortex and hippocampus was observed, with 97% and 93% allelic conversion, respectively, with less than 1% indel formation (FIG. 14D). Similar genomic and cDNA correction efficiencies were found in samples isolated as soon as P21, suggesting that R613X editing is nearly complete (FIGs. 26A-26B). Greater-than-expected frequency of transcripts containing the desired R613 codon was observed in both the hippocampus (75%) and cerebellum (69%) of untreated mice. Because nonsense-mediated decay (NMD) occurs with protein translation, NMD was not expected to de-enrich premature stop codoncontaining transcripts in nuclear isolates. This high degree of wild-type Senia expression 107 / 224Bl 195.70210WO00#14840465vlmay arise from survival bias and transcriptional adaptation amongst untreated mice53,54, as 70% of untreated Scn1aR613X / +mice die by P45. Alternatively, because the nucleus is contiguous with the ER, contaminating mRNAs from the ER could be co-purified during nuclear isolation, skewing observed 5cw7a-p. R613 -containing reads. When analyzing the proportion of 5cw7a-p. R613 reads attributed to the wild-type allele versus the base-edited allele, equal proportions of reads in highly-edited brain regions were observed (FIGs. 27A-27B), demonstrating NMD is not enriching for ABE correction in cDNA. Altogether, these results demonstrate that in vivo ABE treatment efficiently corrects Scn1aR613Xin bulk brain and results in exceptionally high levels of N / r / a-tran script correction.

[0220] Unedited transcripts contain premature stop codons and are expected to undergo nonsense-mediated decay (NMD) in the cytoplasm, resulting in reduced Senia transcript abundance. Senia mRNA levels in whole cell lysates of full brain hemispheres were measured. A 1.8-fold reduction in Senia transcripts in P21 vehicle-treated Sen laR6xXmice was observed, compared to wild-type control mice (FIG. 14E). Encouragingly, a 36% increase in Senia transcripts was measured following P0 ABE treatment compared to untreated DS mice. These findings demonstrate that stop codon correction via ABE ameliorates NMD of Senia transcripts in DS mice to rescue expression in vivo. In addition to measuring expressional changes in Senia, the expression of the other nine voltage-gated sodium ion channel a subunits were measured and observed no significant changes relative to untreated wild-type controls (FIGs. 28A-28J). These findings demonstrated that stop codon correction via ABE ameliorates NMD of Senia transcripts in DS mice to rescue expression in vivo. A western blot analysis was also performed for Navl.l protein expression in isolated P21 mouse neocortices (FIG. 14F; FIG. 29). A 28% reduction in Navl.l expression was observed in untreated Scn1aR613X / +mice relative to wild-type mice. This expression was restored in ABE-treated mice to wild-type levels.ABE rescues electrophysiology of parvalbumin interneurons in DS mice

[0221] Neocortical parvalbumin-positive GABAergic inhibitory interneurons (PVINs) are hypoexcitable in DS mice, exhibiting impaired action potential generation at Pl 8-2155’56. High-frequency PVIN firing is crucial to the regulation of cerebral cortex brain circuits, as PVINs strongly inhibit excitatory pyramidal cells of the neocortex and hippocampus57,58. To assess the functional correction of ABE treatment in PVINs, whole-cell patch clamp 108 / 224Bl 195.70210WO00#14840465vlelectrophysiology of PVINs was performed in acute brain slices prepared from DS mice treated with either GFP-KASH only or with ABE8e-V106W-VRQR + GFP-KASH (FIG. 15 A). The excitability of unedited and edited GFP+PVINs was compared (FIGs. 15A-15B). PVINs from WT and ABE-treated Scnlcfi613X / +animals were substantially more excitable than PVINs from unedited Scnlci1'613-' mice, as measured by input-output curves generated in response to square-wave current injections of ascending amplitude (FIGs. 20A-20C). A 1.3-fold average increase was observed in maximum steady-state firing rate between unedited and edited PVINs (FIG. 20D) and a 1.3-fold average improvement in maximum instantaneous firing rate (FIG. 20E). These findings suggested that correction of the Scn1a-p.R613X mutation rescues the functional deficit of unedited DS PVINs to that of wild-type PVINs. Other electrophysiological abnormalities previously observed in Scnla+l' mice were also corrected in edited PVINs. For example, the input resistance, which is elevated in DS PVINs (86 ± 27 MQ), was normalized to the WT level (73 ± 26) in edited cells (72 ± 20 MO; FIG. 20F). As evidence of improved Na+conductance, edited PVINs display normalized maximum upstroke velocity (306 ± 50 mV / ms in edited cells; 324 ± 50 mV / ms in WT cells) relative to unedited cells (249 ± 48 mV / ms; FIG. 20G). Finally, the action potential halfwidth of WT and ABE-treated cells was narrower (0.36 ± 0.07 ms in WT cells; 0.36 ± 0.04 in edited cells) than unedited cells (0.46 ± 0.11 ms) (FIG. 20H).

[0222] To assess whether the corrected PVIN electrophysiology was due to an increase in sodium current, whole-cell voltage-clamp recordings were performed in nucleated macropatches pulled from PVINs (FIGs. 20I-20K). Compared to WT, unedited DS cells exhibited reduced sodium current (WT peak INa -148 ± 82 pA, n = 25 cells, N= 3 mice; unedited -111 ± 43 pA, n = 12 cells, N= 1 mouse, FIGs. 20J-20K; p = 0.0130 vs. WT). In contrast, edited DS PVIN sodium currents were not significantly different from those of WT PVINs (-153 ± 66 pA, n = 9 cells, N= 1 mouse; p = 0.2722). Edited and unedited DS PVINs also displayed no change in the voltage dependences of activation or inactivation compared to WT PVINs (FIG. 20L; activation p = 0.0833 unedited vs WT, = 0.2445 edited vs WT; inactivation p = 0.1790 unedited vs WT,p = 0.1559 edited vs WT). Taken together, these data demonstrate that ABE treatment leads to restored sodium currents and functional rescue of DS PVINs.

[0223] The effect of base editing on neocortical layer 2 / 3 pyramidal neurons was assessed using whole-cell patch-clamp recordings. No changes in pyramidal cell electrophysiology 109 / 224Bl 195.70210WO00#14840465vlwas identified upon base editing treatment (FIGs. 30A-30J). This result is consistent with previous studies, which have demonstrated that loss of Senia has no or only minor effects on excitatory neuron function (64, 65). We also measured spontaneous inhibitory and excitatory post-synaptic currents in the pyramidal cells to assess the broader function of the cortical microcircuit. Significant differences were not observed in either spontaneous inhibitory or excitatory post-synaptic currents in this microcircuit comparing wild-type, unedited, and edited cells (FIGs. 31 A-31H). Together, these results demonstrated that the most prominent effect of endogenous Senia correction by ABE is normalization of PVIN intrinsic excitability.ABE treatment rescues Scnl(fi6I3X / +mice from febrile seizures and SUDEP

[0224] Following the above molecular and physiological findings, rescue from three hallmark phenotypes of the Scn1aR613X / +mouse model was quantified next (FIG. 16A): temperature-induced seizures, spontaneous seizures, and SUDEP, which are also characteristic features observed in young DS patients. Previous studies found that P21-24 Scn1aR613X / +mice recapitulate the temperature-sensitive seizures observed in humans with DS8. To test whether base editing can rescue this sensitivity, the core body temperature of Pl 8-21 Scn1aR613X / +mice treated with vehicle or ABE8e-V106W-VRQR was elevated, and seizure activity was monitored up to 42.5 °C. While vehicle-treated mice exhibited seizures at an average of 39 °C (FIG. 16B), all mice treated with ABE were seizure-free at temperatures < 42.5 °C. Two of 12 treated mice exhibited seizures when residual heat caused body temperature to rise above 42.5 °C, a temperature at which WT mice occasionally also experience seizures. These results demonstrate that ABE treatment yields near-complete rescue of ScnlaR6I3X / +mice from febrile seizures, a hallmark of DS.

[0225] In addition to induced seizures, rescue of this mouse model from spontaneous seizures was characterized. Scn1aR613X / +mice treated with either ABE or vehicle control at Pl underwent live video recording at P22, which were collected and manually scored for seizure events. 50% of male (2; N=4) and 60% of female (3; N=5) control animals experienced at least one non-terminal seizure during a 24-hour scoring assessment, while all ABE treated animals (N=5 male, N=3 female) remained seizure-free (FIG. 21A). These findings indicate a reduction in overall seizure burden from ABE treatment.110 / 224Bl 195.70210WO00#14840465vl

[0226] This Scn1aR613X / +mouse model also displays a strong SUDEP phenotype, with 60% (9; N=15) of male and 87% (13; N=15) of female mice dying between P20 and P45 (FIG. 15C; FIG. 4D). ABE treatment drastically improves this phenotype, with only 13% (2; N=15) of male mice and 7% (1; N=15) of female mice dying by P45. No additional mortality was observed in either treatment group after P45, the age at which mice reach young adulthood. These data together represent a 7.0-fold reduction in SUDEP amongst ABE-treated DS mice compared to vehicle-treated controls. Collectively, these findings establish that ABE greatly reduces premature death in DS mice.

[0227] Survival rescue in mice treated after the neonatal period was also tested. P12 ScnlaR613X / + mice (developmentally analogous to -3-12 months in a human69'70were treated by ICV injection with 2el 1 total vg of ABE8e-V106W-VRQR, corresponding to a dose of ~3.3el3 vg kg'1in a 6 g mouse. Stark rescue from premature mortality was observed in these mice at P60, with 90% survival (9; N=10) for ABE treated females and 71% survival (5; N=7) for males compared to 0% and 50%, respectively for vehicle controls (FIG. 21C; FIGs. 32A-32C). During a 24-hour period of cage monitoring of these mice between postnatal days P22-26, spontaneous seizures in any P12 ABE-treated ScnlaR613X / +mice was not observed (FIG. 33). Collectively, these results demonstrate that ABE treatment, even after the neonatal period in ScnlaR613X / +mice, confers DS survival benefit.Discussion

[0228] The first direct, permanent genetic correction of Dravet syndrome, a neurodevelopmental disorder characterized by treatment resistant epilepsy, developmental delay / intellectual disability, febrile seizures, and SUDEP, is described herein. Through in vitro editing assays in engineered SCN1AR6I3Xcell lines, multiple base editing strategies capable of efficient stop codon correction with minimal bystander or off-target editing events were identified. ICV injection of AAV9-packaged ABE resulted in robust Scn1aR613Xgenomic correction across multiple brain regions and near complete correction of Scnla-expressing cells in both the cortex and hippocampus. In cellular electrophysiology studies of brain slices from ABE-treated DS mice, greatly improved PVIN excitability was observed. Finally, ABE treatment led to near complete rescue from key DS morbidities, drastically reducing both febrile seizure induction and SUDEP-like phenomenon in mice and also 111 / 224Bl 195.70210WO00#14840465vlgreatly reducing the frequency of premature death in DS mice. These findings suggest the potential of adenine base editing as a transformative therapeutic strategy for DS and related genetic disorders.

[0229] Recently, targeted genetic therapies capable of enhancing SCN1A expression through mutation-agnostic mechanisms have demonstrated promise in both clinical trials and preclinical studies. An antisense oligonucleotide (ASO) that upregulates SCN1A expression by binding pre-mRNA to promote productive splicing of the healthy allele is in phase III clinical trials59. While this strategy yields robust protection from SUDEP and considerable amelioration of seizures in mice when delivered at P0, results in mice at P12 are more modest and the treatment requires repeated dosing in human patients. Study participants receive multiple intrathecal injections per year to maintain expression. An AAV-mediated gene therapy expressing a zinc-finger VP64 transcription factor in GAB Aergic interneurons to upregulate SCN1A transcription has gained FDA clearance to begin phase-I / II trials. This approach may require fewer injections than an ASO but risks long-term off-target transcriptional changes and also does not correct the cause of the disease. An additional, recent gene therapy strategy uses a dual-AAV delivery approach to deliver trans-splicing Navl.l with an IN-specific promoter71. The approach developed in this study directly corrects the root cause of DS using base editing, maintains endogenous regulation of SCN1A expression, and is a permanent one-time treatment, albeit with the need to optimize correction strategies for each targeted mutation.

[0230] This study is limited in its ability to address the therapeutic window for genetic correction of DS. Because disease onset in mice is as soon as P18, determining dose timing capable of rescuing pathology is hindered by the timescale of AAV transgene expression. Importantly, DS onset in humans occurs between 3 and 6 months of age, a timescale much more suitable for AAV delivery and editing than the 18-day window in mice. The P12 ICV injection survival experiment described in this study simulates treatment of a human roughly 3-12 months post-birth69'70, suggesting a therapeutic window. ABE by additional modalities (such as LNPs36or eVLPs60–62) to correct DS can further advance the therapeutic relevance of this approach, as well as further reduce the likelihood of off-target DNA or RNA editing. Previous studies have shown that Cre-mediated reactivation of Senia expression in symptomatic DS mice rescues both spontaneous and temperature-induced seizures10, suggesting that mutation correction could offer similar benefits.112 / 224Bl 195.70210WO00#14840465vl

[0231] The therapeutic threshold of SCN1A correction required for phenotypic rescue is not yet understood. P0 ICV delivery of AAV9-ABE driven by the Cbh promoter conferred almost complete rescue from febrile seizures and SUDEP in mice, an outcome consistent with near complete (>92%) correction of Senia in bulk transcripts from the hippocampus and cortex atP21

[0232] This study highlights the need for early genetic screening and pathogenicity scoring of SCN1A mutations. Currently, 1,421 pathogenic SCN1A variants and an additional 1,465 SCN1A variants of unknown significance, all shorter than 50 bp, are listed in the ClinVar database, suggesting they could be targeted by precision genome editing techniques such as base editing or prime editing. In combination with thorough evaluation of empirical data by neurogeneticists and genetic counselors, tools to score anticipated pathogenicity, such as AlphaMissense and gnomAD used in this study, could enable parents and caregivers of DS patients to make informed decisions on performing interventions before pathology manifests. As suggested by the in vivo outcomes described here, correction prior to DS onset could lead to healthy, neurotypical lives.MethodsStudy Design

[0233] The major goal of this study was to test the rescue of Dravet syndrome in mice by base editing. All mouse experiments were performed in Pl - or P12-treated Scn1aR613X / +mice on a B6 S129 / F1 background. To observe correction in these mice, genomic base editing at the genomic DNA and nuclear mRNA levels was measured in DS mice at days P21 and P45. Additionally, phenotypic readouts such as brain slice electrophysiology, temperature-induced seizure induction, and sporadic seizures were measured at days P18-P24 in a subset of these mice, and a different subset of mice were observed in 45 or 60-day survival study.Cell culture

[0234] Neuro-2a (ATCC CCL-131) and HEK293T (ATCC CRL-3216) cells were purchased from American Type Culture Collection and cultured in Dulbecco’s modified Eagle medium with GlutaMax (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) at 37°C with 5% CO2. All cell lines were verified to be free of mycoplasma and were identity-authenticated by their suppliers.113 / 224Bl 195.70210WO00#14840465vlGeneration of a homozygous Senia R613XN2A cell line using prime editing

[0235] The Scn1aR613X / +mouse model pathogenic allele contains two mutations relative to mmlO Scn1a c.1833 and Scn1a c.1837A>T). The Scn1aR613Xcell line was generated by PE5-style prime editing as previously described63. Briefly, N2A cells were seeded in 48-well poly-D-lysine coated tissue culture plates (Corning) at a density of 25,000 cells per well. After 17-24 hours, cells were transfected with 1 pL of Lipofectamine 2000 (Thermo Fisher Scientific) following manufacturer’s protocols, as well as 750 ng PEmax plasmid, 500 ng murine MLH1dn, 250 ng pegRNA 1, and 83 ng nsgRNA 1. Following a 72-hour incubation, cells were trypsinized, resuspended in DMEM, and plated at a density of 0.5 cells per well in 96-well plates (Coming). Single-cell colonies were evaluated for installation of the desired mutations using high-throughput DNA sequencing.Generation of a homozygous SCN1A R613X HEK293T cell line using base editing

[0236] The homozygous SCN1AR613XHEK293T cell line was generated by cytosine base editing. HEK293T cells were seeded in 48-well poly-D-lysine coated tissue culture plates (Corning) at a density of 25,000 cells per well. After 17-24 hours, cells were transfected with 1 pL of Lipofectamine 2000 (Thermo Fisher Scientific) following manufacturer’s protocols, as well as 750 ng TadCBEa-V106W-NRCH and 200 ng sgRNA 1. Following a 72-hour incubation, cells were trypsinized, resuspended in DMEM, and plated at a density of 0.5 cells per well in 96-well plates (Coming). Single-cell colonies were evaluated for installation of the desired mutations using high-throughput DNA sequencing.N2A and HEK293T ABE transfection

[0237] Cells were seeded in 48-well poly-D-lysine coated tissue culture plates (Corning) at a density of 25,000 cells per well in 0.25 mL media. After 17-24 hours, cells were transfected with 1 pL of Lipofectamine 2000 (Thermo Fisher Scientific) following manufacturer’s protocols, as well as 750 ng editor plasmid and 200 ng sgRNA plasmid. Following 72-hour incubation, cells were lysed for high-throughput sequencing.114 / 224Bl 195.70210WO00#14840465vlHigh-throughput sequencing and data analysis

[0238] Genomic DNA was isolated from cells grown in culture by first removing media, washing with PBS, and adding 100 pL of freshly prepared lysis buffer [10 mM Tris-HCl, pH 8; 0.05% SDS, 20 pg / ml of proteinase K (Qiagen)] directly to each well. Lysates were incubated in the tissue culture plate at 37 °C for 1 hour before transferring to a PCR plate and inactivating the proteinase K by incubation at 80°C for 30 minutes. DNA sequencing was performed using Illumina MiSeq as previously described63. Briefly, DNA primers containing Illumina forward and reverse adapters were used to amplify the mouse or human SCN1A target site with the following conditions: 0.5 pM each of forward and reverse primer, 1 pl of genomic DNA, and 12.5 pl PhusionU Green Multiplex PCR Master Mix (Thermo Fisher Scientific) in a 25-pl reaction. PCR reactions were as follows: 98 °C for 2 minutes and then 30 cycles of 98 °C for 10 seconds, 61 °C for 20 seconds, 72 °C for 30 seconds, followed by a final 72 °C extension for 2 minutes. Samples were barcoded with Illumina barcode pairs through a second round of PCR (PCR2), with the following conditions: 0.5 pM each of forward and reverse primer, 1 pl of unpurified PCR1, and 12.5 pl PhusionU Green Multiplex PCR Master Mix in a 25-pl reaction. PCR reactions were as follows: 98 °C for 2 minutes and then 10 cycles of 98°C for 10 seconds, 61 °C for 20 seconds, 72 °C for 30 seconds, followed by a final 72 °C extension for 2 minutes. PCR2 products were pooled and purified by gel electrophoresis on a 1.5% agarose gel using a QIAquick Gel Extraction Kit (Qiagen), eluting with 25 pl water. DNA library concentration was measured by Qubit high-sensitivity fluorometric quantification (Thermo Fisher Scientific) and sequenced on an Illumina MiSeq instrument using Illumina MiSeq control software (version 4.1), according to the manufacturer’s protocols. Sequencing reads were demultiplexed using the onboard MiSeq Analysis Module (Illumina).

[0239] Amplicons were aligned to reference sequences with CRISPResso264, using the following flags: discard indel reads = TRUE, q = 30, and qwc was set to 10 nt upstream and downstream of a given spacer sequence. Indels for samples were calculated as ((‘Discarded’ / ’Reads aligned all amplicons’) x 100). Editing efficiency at given base was calculated as ((frequency of point mutation in the ‘Nucleotide_percentage_summary.txt’ file) x 100). Descriptions of allelic conversion in genomic DNA were calculated as: allelic conversion = (editing frequency - editing frequency in mock control s) / ( 100 -editing frequency in mock controls).115 / 224Bl 195.70210WO00#14840465vlAA V production

[0240] rAAV was produced by transient HEK293 cell transfection and CsCl sedimentation by the University of Massachusetts Medical School Viral Vector Core, as previously described65. Vector titers were determined by ddPCR, and purity was assessed by 4%-12% SDS-acrylamide gel electrophoresis and silver staining (Invitrogen).CIRCLE-seq sample preparation and off-target analysis

[0241] CIRCLE-seq off-target nomination was performed as previously described44, with the following modifications. Briefly, genomic DNA was extracted from SCN1AR6I3XHEK293T cells and sheared to an average length of 300 bp with a Covaris S2 focused ultrasonicator. Sheared fragments were circularized using NEBnext end-repair, and dA-tailing modules (NEB). For in vitro cleavage assays, 50 ng of circularized DNA (about 8000x coverage) were incubated with purified Cas9-VRQR protein (purified as previously described31) and synthetic 2’-O-methyl-modified (at the first three and final three bases) sgRNA with the following spacer sequence ‘GGUCAUCGAGGAACGAACAG’ (SEQ ID NO: 123). Cleaved products were prepared for HTS using the NEBnext quick ligation module (NEB) and sequenced by a 150-bp / 150-bp paired-end sequencing kit on an Illumina MiSeq instrument. Data analysis was performed using the open-source CHANGE-seq analysis software using GRCh37 / hgl9 as reference.rhAmpSeq off-target site sequencing validation

[0242] rhAmpSeq library design was performed with the IDT rhAmpSeq Design Tool using genomic coordinates from CIRCLE-seq for design input. Samples were prepped for sequencing in a two-step PCR enrichment protocol. Pooled PCR1 library amplification was performed using the rhAmpSeq CRISPR library kit (IDT) in 10 pL reactions as follows: 2.5 pL rhAmpSeq library mix 1, 5-25 ng treated or untreated genomic DNA, 1 pL lOx pooled forward primer library, 1 pL lOx pooled reverse primer library. PCR conditions were as follows: 95°C for 10 minutes, followed by 14 cycles of 95°C for 15 seconds then 61°C for 8 minutes, then enzyme deactivation at 99.5°C for 15 minutes. Samples were diluted 1:20 in nuclease free water before proceeding to PCR2. PCR2 reactions were mixed in 10 pL reactions as follows: 2.5 pL rhAmpSeq library mix 2, 5.5 pL diluted PCR1, 1 pL of 1 pM 116 / 224Bl 195.70210WO00#14840465vlIllumina i5 indexing primer, 1 pL of 1 pM Illumina i7 indexing primer. PCR conditions were as follows: 95°C for 3 minutes, followed by 24 cycles of 95°C for 15 sec, 60°C for 30 sec, and 72°C for 30 sec, followed by a final extension at 72°C for 1 minute. Indexed libraries were pooled and sequenced in 300 cycle single-end reads on an Illumina MiSeq instrument, and sample demultiplexing was performed with on-board software. Amplicon demultiplexing from samples was performed using Samtools with 300 bp amplicons from CIRCLE-seq as indices. Demultiplexed amplicons within each sample were analyzed in CRISPResso2 in NHEJ mode. Reads with at least one A T-to-G C substitution on the edited strand were designated as “edited.” Editing percentages at each amplicon were determined as the number of edited reads divided by the total number of reads for a given amplicon. To remove editing background likely caused by sequencing error, average editing percentage from the untreated group was subtracted from the average editing in the treated group. Significance was determined by student’s T-test, and P values less than 0.01 were determined to be significant.Nuclear isolation for fluorescence-activated sorting

[0243] Mouse brain tissues were homogenized using the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec) instructions for nuclei extraction. Briefly, tissues were disrupted in 4 mL of nuclei extraction buffer (Miltenyi) supplemented with 0.2 U / pL murine RNase inhibitor (NEB). Homogenates were then passed through a 100 pM cell strainer before being, pelleted by centrifugation at 500 x g for 5 minutes. Pellets were resuspended in 4 mL cold nuclear suspension buffer (NSB; lx PBS, pH 7.4, 3.33 pM Vybrant DyeCycle Ruby [Thermo Fisher Scientific], 100 pg / pL recombinant albumin [NEB], 0.2 U / pL murine RNase inhibitor [NEB]). Samples were pelleted again by centrifugation at 500 x g for 5 minutes before resuspending in 1 mL of NSB. Samples were passed through a 35 pM cell strainer and sorted using a SH800 Cell Sorter (Sony). See FIGs. 17A-17B for FACS gating strategy. Nuclei were sorted into Buffer RLT Plus (Qiagen) supplemented with 40 mM dithiothreitol. Both genomic DNA and nuclear RNA were isolated using AllPrep DNA / RNA columns (Qiagen) following manufacturer’s instructions. Isolated RNA was reverse transcribed into cDNA using SuperScript IV first-strand synthesis mix with random hexamers (Invitrogen).117 / 224Bl 195.70210WO00#14840465vlMice and Animal Care

[0244] For the generation of testing cohorts, Scn1aR613Xheterozygous animals (JR#34129, also denoted as 129S1 / SvImJ-Scn1aem1Dsf / J) were bred with C57BL6 / J (JR#00664) to produce B6129F1 offspring. Heterozygous Scn1aR613Xoffspring and their wildtype littermates were sampled via toe clip at Pl -3 to identify phenotype. At P21, animals were weaned and housed with mixed litter sex- and genotype-matched animals. Animals were ear notched for identification and confirmation of genotype. All animals were kept on aspen bedding and fed a 6% fat diet (LabDiet 5K52). Animals were provided a nestlet for enrichment and housed at a density of 3-4 mice per pen. All animals were monitored daily for wellbeing and survival starting at P14. Humane euthanasia was applied to any animal that fell to a body condition score of 2 or lower, displayed hydrocephaly, or was determined by veterinary staff to meet humane endpoints.Intracerebroventricular (ICV) Injections

[0245] After P0 genotyping, test article and vehicle were administered via ICV injects at Pl. In brief, pups were anesthetized via hypothermia in chamber set on ice. Once fully anesthetized, pups were injected with either test article or vehicle via dual hemisphere injections using a Hamilton Model 1701 Neuro Syringe with a 33 gauge beveled needle with neuro adaptor. 2ul per hemisphere of solution were injected at a depth of 2mm, with a dwell time of 10 seconds per hemisphere. Animals were then placed on a heated recovery pad until independently moving and warm prior to rehousing with their birth dam. Multiple litters were used for each treatment group.Hyperthermic seizure induction

[0246] Mice were placed in a plexiglass observation chamber and allowed to move freely. Core body temperature was monitored with a rectal probe (Physitemp, Clifton NJ). Core body temperature was passively elevated with a heat lamp until 42.5 °C or a seizure occurred. Seizures were identified by visual monitoring based on the modified Racine scale by at least two expert observers. The temperature at visually identified seizure onset was recorded. Mice were then cooled on ice until body temperature returned to baseline.

[0247] P12 injections were caried out using anesthesia and stereotaxic surgery table. In brief, mice were anesthetized with 100 mg kg-1ketamine and 10 mg / kg Xylazine. The scalp shaved 118 / 224Bl 195.70210WO00#14840465vland cut back to reveal skull. Pilot holes were drilled to allow Hamilton needle insertion and dosing. For P12 animals, the coordinates for injection were as follows: AP= -0.2mm, ML= -0.8mm, DV= -2.0mm. Post surgery, the animals were sutured and provided carprofen for recovery. Animals were recovered in a warmed cage and provided a warm saline injection for hydration. Animals were returned to their home cage after demonstrating ability to ambulate and move of their own accord.mRNA expression analysis by qPCR

[0248] For expression analysis of Scn4a (Mm. PT.58.8823368, IDT), Scn7a(Mm. PT.58.13836045, IDT), and ScnlOa (Mm. PT.58.9384636, IDT), primer assays were predesigned. qPCR reactions for expression analysis of cDNA transcripts were prepared as follows: 0.5 pM each of forward and reverse primer, lx SYBR Green (1:10,000 dilution, Lonza), 2 pL of cDNA, and 12.5 pL Q5 high-fidelity 2X Master Mix (NEB) in a 25 pl reaction. qPCR reactions were performed using a CFX Opus 96 Real-time PCR system (Biorad). PCR reactions were as follows: 98 °C for 2 minutes and then 35 cycles of 98°C for 10 seconds, 61 °C for 20 seconds, 72 °C for 30 seconds, and plate read for SYBR green signal. Ct values were determined using on-board CFX Maestro analysis software (Biorad). Ct values for target genes were first normalized internally to Gapdh to determine ACt values, and AACt values were found by normalizing ACt values of each sample to that of the average wild-type ACt value. Relative expression is shown as a fold-change in AACt, or 2-(ΔΔCt).Protein expression analysis by western blot

[0249] Brain tissues isolated from mice were lysed in radioimmunoprecipitation assay (RIP A) buffer (20 mM Tris, 140 mMNaCl, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, pH 7.4) supplemented with complete protease inhibitor cocktail, ImM EDTA, and ImM PMSF. Lysis was performed by running samples through a 22-gauge needle until no remaining particulate matter was visible.Solubilized protein concentration was determined by bicinchoninic acid (BCA) quantification, and 20 pg protein aliquots were separated on 4-12% Bolt Bis-Tris Plus mini protein gel (Invitrogen). Gel separations were then transferred to a PVDF membrane by overnight wet transfer at 4°C / 10 V / 40 mA for 15 hours. Membranes were blocked for 2 hours at 4°C with blocking buffer (0.5% Tween-20, 1% bovine serum albumin [BSA], 130119 / 224Bl 195.70210WO00#14840465vlmM NaCl, 2.6 mM KCl, 9.5 mM Na2HPO4, 1.7 mM KH2PO4, pH 7.4). Blocked membranes were incubated overnight at 4°C with blocking buffer supplemented with either 1:200 anti-Navl.l antibody (Alomone Labs, ASC-001) or 1:2000 anti-calnexin (Millipore Sigma, C4731). Membranes were then washed three times with TBST buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% Tween-20, pH 7.4). Membranes were incubated with 1:3000 goat anti-rabbit-HRP (Biorad, #1706515) for two hours at 4°C before washing three times with TBST. Membranes were developed with Clarity western ECL substrate (Biorad, #1705061) and imaged on a ChemiDoc imaging system (Biorad). Densitometry was performed in Image Lab 6.1 software (Biorad). For plotting, total band volume of Nav1.1 was normalized internally to total band volume of calnexin, and these internally controlled values were normalized across samples to the average wild-type normalized value.Temperature-induced seizures

[0250] Mice were placed in a plexiglass observation chamber and allowed to move freely. Core body temperature was monitored with a rectal probe (Physitemp, Clifton NJ). Core body temperature was passively elevated with a heat lamp until 42.5°C or a seizure occurred. Seizures were identified by visual monitoring based on the modified Racine scale by at least two expert observers. The temperature at visually identified seizure onset was recorded. Mice were then cooled on ice until body temperature returned to baseline.Home cage monitoring and seizure burden assessment

[0251] Animals were placed in Discovery Rack with Envision software for 24 / 7 digital video monitoring. Animals were given unique ear identification tags that Envison software uses to identify animals. Diet, water and bedding were as described above in Animal Husbandry. Seizures were scored manually via operator review of video feed. A continuous 24 hour period, including light and dark cycles, was evaluated for each animal. Seizures were identified as episodes of hyperactivity, inability to right self, and clonic-tonic displays lasting 30 seconds -90 seconds. Episodes were not observed to occur back-to-back and animals fully recovered between 30 seconds -90 seconds events.120 / 224Bl 195.70210WO00#14840465vlAcute brain slice preparation

[0252] To label parvalbumin interneurons for electrophysiological analysis, Scn1aR613X / +mice on a 129 genetic background were crossed to PV-tdTomato mice (Jax #027395) on a C57 / B16J background. Mice were treated with ABE + GFP-KASH or GFP-KASH only as described above.

[0253] Coronal brain slices were prepared from Pl 8-21 male and female Scn1aR613X / +mice in either treatment group. Mice were anesthetized with isoflurane. The brain was dissected and transferred to ice-cold sucrose cutting solution (in mM: 75 sucrose; 10 glucose; 26 NaHCO3; 2.5 KCl; 1.25 NaH2PO4; 87 NaCl; 1 CaCl2; 2 MgSO4) bubbled with 95% O2 / 5% CO2. The cerebellum was removed, and the brain was attached to the specimen holder of a Leica VT1200S vibratome using cyanoacrylate glue. Slices were cut at a thickness of 300 pm in ice-cold sucrose cutting solution and allowed to recover for 30 minutes at 32°C. Slices were then maintained in sucrose cutting solution at room temperature for up to 5 hours before recording.Electrophysiology

[0254] Slices were placed on the recording chamber of a Scientifica SliceScope electrophysiology rig and perfused with artificial cerebrospinal fluid (see below) at 3.0 mL / min and 30-32°C. Primary somatosensory cortex was identified based on visualization of characteristic “barrels” via infrared differential interference contrast microscopy. PV-INs were identified based on tdTomato reporter expression visualized by epifluorescence microscopy and confirmed by characteristic fast-spiking discharge pattern. Only GFP+ cells were recorded. All recordings were performed in neocortical layer 2 / 3.

[0255] Whole-cell recordings were obtained with borosilicate glass patch pipettes pulled on a PC-100 puller (Narishige) with a tip resistance of 3-5 MQ. All recordings were performed with a MultiClamp 700B amplifier (Molecular Devices) and digitized with an Axon Digidata 1550B digitizer (Molecular Devices). Data were acquired with pCLAMP 10 software, sampled at 100 kHz, and low-pass filtered at 10 kHz. Pipette capacitance compensation was applied for all experiments. Liquid-liquid junction potential was calculated as 15.9 mV at 32°C and not corrected.

[0256] After break-in, each cell was recorded for two minutes in gap-free current-clamp mode. A ramp protocol (0-400 pA, 2s) was recorded to confirm fast-spiking behavior. Each 121 / 224Bl 195.70210WO00#14840465vlfast-spiking cell was stimulated with 600 ms current pulses at 25 pA intervals beginning at -100 pA. All recordings were performed blind to genotype.

[0257] For current-clamp recordings (FIGs. 15A-15B, FIGs. 20A-20H and FIGs. 30A-30J), pipettes were filled with an intracellular solution containing in mM: 130 K-gluconate, 6.3 KCl, 1 MgCl2, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.3 Na-GTP; pH was adjusted to 7.3 with KOH and osmolarity was adjusted to 285 mOsm with 30% sucrose). The external solution contained in mM: 125 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgSO4. All chemicals were purchased from Sigma Aldrich. Cells were recorded for two minutes after break-in in gap-free mode. For PVIN recordings, a ramp protocol (0-400 pA, 2s) was recorded to confirm fast-spiking behavior. For both PVIN and pyramidal cell recordings, each cell was stimulated with 600 ms current pulses at 25 pA intervals beginning at -100 pA. All recordings were performed blind to genotype. Cells with resting membrane potentials more positive than -55 mV, access resistance greater than 20 MQ, or with changes in access resistance larger than 20% during recording were excluded.

[0258] For nucleated macropatches (FIG. 20I-FIG. 20K), pipettes were filled with an internal solution containing in mM: 140 CsFl, 1.0 EGTA, 2.0 MgCl2, 10 HEPES, 4.0 Na-ATP, 0.3 Na-GTP; pH was adjusted to 7.3 with KOH. External solution was as described above, with the addition of 4 mM 4-AP and 10 mM triethylammonium chloride to block potassium currents. After the cell was patched in the whole-cell configuration, gentle negative pressure was applied to pull the nucleus towards the pipette. The pipette was then slowly retracted from the tissue until the nucleated macropatch was completely removed from the slice. The macropatch was then held at -100 mV in voltage-clamp mode, and 40 ms voltage steps at 5 mV intervals were applied from -80 to +40 mV.

[0259] For spontaneous inhibitory and excitatory post-synaptic current recordings, standard artificial cerebrospinal fluid was used (as described above for current-clamp recordings). Pipettes were filled with Cs-based voltage clamp internal (see previous paragraph) with the addition of 2 mM QX-314 to prevent spiking while holding the cell at 0 mV. After the wholecell configuration was achieved, cells were held at -70 mV and recorded in voltage-clamp gap-free mode for 2 minutes to record spontaneous excitatory post-synaptic currents. The cells were then held at 0 mV and recorded in voltage-clamp gap-free mode for another 2 minutes to record spontaneous inhibitory post-synaptic currents.122 / 224Bl 195.70210WO00#14840465vlElectrophysiology data analysis

[0260] Analysis of current-clamp data was performed with custom Matlab (Mathworks) and R (version 4.4.0) scripts and manually confirmed with Clampfit software. Resting membrane potential was measured as the average membrane potential during a 2s sweep with no direct current injection. Input resistance was calculated as the response to a -100 pA direct current injection using Rm= ΔV / I. Membrane time constant was calculated from a single exponential fit of the hyperpolarizing response to a -100 p A current injection. Membrane sag was measured from the hyperpolarizing response to a -100 pA current injection by calculating the ratio of the steady-state voltage to the maximal hyperpolarization. Rheobase was calculated as the smallest current step capable of inducing the firing of an action potential (AP, defined as meeting having the first derivative of the voltage, dV / dt > 10 mV / ms and a voltage greater than -10 mV).

[0261] AP threshold was defined as the voltage at which dV / dt exceeded 10 mV / ms, calculated from the first AP at rheobase. AP peak was defined as the maximal voltage achieved during the first action potential at rheobase. AP amplitude was calculated as the voltage difference between the AP peak and AP threshold. AP rise time was the time difference between AP peak and AP threshold. The maximal rise slope was defined as the maximal dV / dt achieved between AP threshold and AP peak. AP halfwidth was calculated as the width of the first AP at rheobase at half-maximal amplitude. The after-hyperpolarization amplitude was calculated as the absolute minimum voltage achieved during the first AP at rheobase subtracted from the AP threshold.

[0262] The maximal steady-state firing frequency was calculated by dividing the maximum number of APs observed during a single sweep by the sweep duration (600 ms). The maximum instantaneous firing frequency was calculated as the inverse of the smallest inter-AP interval. The spike frequency adaptation was calculated as the ratio of the first and second, tenth, or final inter- AP interval evoked by the first current step to evoke at least 40 APs.

[0263] Analysis of sodium currents from nucleated macropatches and HEK cells was also performed with a custom Matlab script. For each voltage step, the maximum inward sodium current was recorded to construct a current-voltage plot. For activation and inactivation curves, the maximum inward sodium current density at each voltage step was divided by the123 / 224Bl 195.70210WO00#14840465vloverall maximum sodium current density per cell. Analysis of spontaneous inhibitory and excitatory post-synaptic currents was performed in Clampfit using custom template searches.Statistics and reproducibility

[0264] All mice were randomly allocated into age-matched and genotype experimental groups. The data were analyzed for statistical significance and graphed using GraphPad Prism 9 (version 10.1.1). Whether data followed a normal distribution was determined, and when respected unpaired two-tailed t-test for comparison between two groups and one-way analysis of variance (ANOVA) followed by Dunnett' s multiple comparisons test for multiple comparison were used. For survival curves, a Log-rank (Mantel-Cox) test was employed to compare treated vs vehicle groups. A P value less than 0.05 was considered significant. Statistical tests and number of biological replicates used throughout the manuscript are depicted in figure legends corresponding to each figure. Investigators were blinded during data collection and analysis. Data are presented as mean + / - standard deviation. The sample size and the statistical tests used for each figure are described in the figure legends.124 / 224Bl 195.70210WO00#14840465vlReferences for Example 21. Dravet, C. Dravet syndrome history. Dev. Med. Child Neurol. 53, 1-6 (2011).2. Dravet, C. & Oguni, H. Dravet syndrome (severe myoclonic epilepsy in infancy), in Handbook of Clinical Neurology vol. 111 627–633 (Elsevier, 2013).3. Depienne, C. et al. Spectrum of SCN1 A gene mutations associated with Dravet syndrome: analysis of 333 patients. J. Med. Genet. 46, 183-191 (2008).4. Marini, C. et al. SCN1A duplications and deletions detected in Dravet syndrome: Implications for molecular diagnosis. Epilepsia 50, 1670-1678 (2009).5. Mullen, S. A. & Scheffer, I. E. Translational Research in Epilepsy Genetics: Sodium Channels in Man to Intemeuronopathy in Mouse. Arch. Neurol. 66, (2009).6. Yu, F. H. et al. Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. Nat. Neurosci. 9, 1142-1149 (2006).7. Ogiwara, I. et al. Nav1.1 Localizes to Axons of Parvalbumin-Positive Inhibitory Interneurons: A Circuit Basis for Epileptic Seizures in Mice Carrying an Senia Gene Mutation. J. Neurosci. 27, 5903-5914 (2007).8. Mavashov, A. et al. Heat-induced seizures, premature mortality, and hyperactivity in a novel Senia nonsense model for Dravet syndrome. Front. Cell. Neurosci. 17, 1149391 (2023).9. Ho, S.-Y. et al. Perampanel Reduces Hyperthermia-Induced Seizures in Dravet Syndrome Mouse Model. Front. Pharmacol. 12, 682767 (2021).10. Valassina, N. et al. Senia gene reactivation after symptom onset rescues pathological phenotypes in a mouse model of Dravet syndrome. Nat. Commun. 13, 161 (2022).11. Goff, K. M., Liebergall, S. R., Jiang, E., Somarowthu, A. & Goldberg, E. M. VIP interneuron impairment promotes in vivo circuit dysfunction and autism-related behaviors in Dravet syndrome. Cell Rep. 42, 112628 (2023).12. Voskobiynyk, Y. et al. Aberrant regulation of a poison exon caused by a non-coding variant in a mouse model of Scnla-associated epileptic encephalopathy. PLOS Genet. 17, el009195 (2021).13. Smith, R. S. et al. Sodium Channel SCN3A (NaV1.3) Regulation of Human Cerebral Cortical Folding and Oral Motor Development. Neuron 99, 905-913. e7 (2018).14. Claes, L. R. et al. The SCN1A variant database: a novel research and diagnostic tool. Hum. Mutat. 30, E904-E920 (2009).125 / 224Bl 195.70210WO00#14840465vl15. Meng, H. et al. The SCN1A Mutation Database: Updating Information and Analysis of the Relationships among Genotype, Functional Alteration, and Phenotype. Hum. Mutat.36, 573-580 (2015).16. Xu, X. et al. Amplicon Resequencing Identified Parental Mosaicism for Approximately 10% of “ de novo ” SCN1A Mutations in Children with Dravet Syndrome. Hum. Mutat. 36, 861-872 (2015).17. Liu, Y.-H. et al. Genetics and clinical correlation of Dravet syndrome and its mimics - experience of a tertiary center in Taiwan. Pediatr. Neonatol. 62, 550-558 (2021).18. Kwong, A. K.-Y., Fung, C.-W., Chan, S.-Y. & Wong, V. C.-N. Identification of SCN1A and PCDH19 Mutations in Chinese Children with Dravet Syndrome. PLoS ONE 7, e41802 (2012).19. Schuster, J. et al. Transcriptomes of Dravet syndrome iPSC derived GABAergic cells reveal dysregulated pathways for chromatin remodeling and neurodevelopment. Neurobiol. Dis. 132, 104583 (2019).20. Hammer, M. F. et al. Rare variants of small effect size in neuronal excitability genes influence clinical outcome in Japanese cases of SCN1A truncation-positive Dravet syndrome. PLOS ONE 12, e0180485 (2017).21. Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464-471 (2017).22. Richter, M. F. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat. Biotechnol. 38, 883-891 (2020).23. Gaudelli, N. M. et al. Directed evolution of adenine base editors with increased activity and therapeutic application. Nat. Biotechnol. 38, 892-900 (2020).24. Cho, S.-I. et al. Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases. Cell 185, 1764-1776. el2 (2022).25. KI einstiver, B. P. et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490-495 (2016).26. Miller, S. M. et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat. Biotechnol. 38, 471-481 (2020).27. Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368, 290-296 (2020).28. Huang, T. P. et al. High-throughput continuous evolution of ...

Claims

CLAIMSWhat is claimed is:

1. A method of correcting a SCN1A mutant allele comprising at G> A or OT mutation relative to a wildtype SCN1A nucleotide sequence, said method comprising contacting a nucleic acid sequence comprising the SCN1A mutant allele with an adenine base editor (ABE) and a gRNA targeting the ABE to the SCN1A mutant allele, thereby restoring the G> A or OT mutation to wildtype to produce a corrected SCN1A mutant allele.

2. The method of claim 1, wherein the wildtype SCN1A nucleotide sequence comprises SEQ ID NO: 114 [human] or SEQ ID NO: 116 [mouse],3. The method of claim 2, wherein the wildtype SCN1A nucleotide sequence encodes wildtype voltage-gated sodium channel a subunit (NAV1.1) comprising the amino acid sequence of SEQ ID NO: 106 [human] or SEQ ID NO: 108 [mouse],4. The method of claim 1, wherein the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a G> A or OT mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114 [human],5. The method of claim 1, wherein the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a G> A mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114 [human],6. The method of claim 1, wherein the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a T mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114 [human],7. The method of claim 1, wherein the SCN1A mutant allele comprises a C>T mutation at position 1837 (1837C>T) relative to wildtype SCN1A nucleotide sequence of SEQ ID NO: 114, thereby introducing a stop codon.205 / 224Bl 195.70210WO00#14840465vl8. The method of claim 1, wherein the SCN1A mutant allele encodes a variant of voltage-gated sodium channel a subunit (NAV1.1) having the amino acid change specified in Table 1 relative to wildtype NAV1.1 of SEQ ID NO: 106.

9. The method of claim 1, wherein the SCN1A mutant allele encodes an NAV1.1 variant comprising an R613X substitution relative to NAV1.1 wildtype amino acid sequence of SEQ ID NO: 106, wherein X is a termination codon.

10. The method of claim 1, wherein the gRNA targets a strand at the SCN1A mutant allele.

11. The method of claim 1, wherein the gRNA comprises a spacer having a nucleotide sequence that is the inverse complement of a target strand at the SCN1A mutant allele, wherein the SCN1A mutant allele is any one of the SCN1A mutant alleles identified in Table 1.

12. The method of claim 1, wherein the gRNA comprises a nucleotide sequence of any one of the gRNA sequences of Tables 4A or 4B, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the nucleotide sequences of Tables 4A or 4B.

13. The method of claim 1, wherein the gRNA comprises a spacer comprising a nucleotide sequence of any one of the gRNA spacers of Tables 4A or 4B, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the nucleotide sequences of Tables 4 A or 4B.

14. The method of any one of the above claims, wherein the adenine base editor (ABE) comprises a nucleic acid-programmable DNA-binding protein (napDNAbp) and an adenosine deaminase.

15. The method of claim 14, wherein the nucleic acid-programmable DNA-binding protein (napDNAbp) comprises a Cas9 protein.206 / 224Bl 195.70210WO00#14840465vl16. The method of claim 15, wherein the Cas9 protein is a Cas9 nickase (nCas9).

17. The method of claim 15, wherein the Cas9 protein is a nuclease-inactive Cas9 (dCas9).

18. The method of claim 15, wherein the Cas9 protein is a Streptococcus pyogenes Cas9 protein or a variant thereof.

19. The method of claim 14, wherein the napDNAbp comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1-23 and 139-140.

20. The method of claim 14, wherein the napDNAbp comprises the amino acid sequence of any one of SEQ ID NOs: 1-23 and 139-140.

21. The method of claim 14, wherein the napDNAbp is the napDNAbp of ABE7.10, ABE8e, ABE8e-(V106W)-SaCas9, ABE8e-(V106W)-VRQR / NG, or ABE8e-(V106W)-SpCas9.

22. The method of claim 14, wherein the adenosine deaminase comprises the amino acid sequence of any one of SEQ ID NOs: 24-70.

23. The method of claim 14, wherein the adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 24-70.

24. The method of claim 14, wherein the adenosine deaminase is the adenosine deaminase of ABE7.10, ABE8e, ABE8e-(V106W)-SaCas9, ABE8e-(V106W)-VRQR / NG, or ABE8e-(V106W)-SpCas9.207 / 224Bl 195.70210WO00#14840465vl25. The method of claim 1, wherein the adenine base editor (ABE) is selected from the group consisting of ABE7.10, ABE8e, ABE8e-(V106W)-SaCas9, ABE8e-(V106W)-VRQR / NG, or ABE8e-(V106W)-SpCas9.

26. The method of claim 1, wherein the adenine base editor (ABE) comprises the amino acid sequence of any one of SEQ ID NOs: 99-105, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 99-105.

27. The method of any one of the above claims, wherein the adenine base editor (ABE) further comprises one or more nuclear localization sequences (NLS).

28. The method of claim 27, wherein the one or more NLS comprises the amino acid sequence of any one of SEQ ID NOs: 71-82, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 71-82.

29. The method of claim 14, wherein the adenine base editor (ABE) is a fusion protein further comprising a linker between the napDNAbp and the adenosine deaminase.

30. The method of claim 29, wherein the linker comprises the amino acid sequence of any one of SEQ ID NOs: 83-98, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 83-98.

31. The method of claim 29, wherein the fusion protein comprises in the N-to-C terminal direction the [napDNAbp]-[linker]-[adenosine deaminase],32. The method of claim 29, wherein the fusion protein comprises in the N-to-C terminal direction the [adenosine deaminase]-[linker]-[napDNAbp],33. The method of any one of the above claims, wherein the adenine base editor (ABE) is split fusion protein comprising an N-terminal portion and a C-terminal protein.208 / 224Bl 195.70210WO00#14840465vl34. The method of claim 33 wherein the N-terminal portion comprises an N-intein and the C-terminal portion comprises a C-intein.

35. The method of any one of the above claims, wherein the method results in correction of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the SCN1A mutant allele in a population of cells.

36. The method of claim 35, wherein the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a G> A or OT mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114.

37. The method of claim 35, wherein the SCN1A mutant allele comprises a C>T mutation at position 1837 (1837C>T) relative to wildtype SCN1A nucleotide sequence of SEQ ID NO: 114, thereby introducing a stop codon.

38. The method of claim 35, wherein the SCN1A mutant allele encodes a variant of voltage-gated sodium channel a subunit (NAV1.1) having the amino acid change specified in Table 1 relative to wildtype NAV1.1 of SEQ ID NO: 106.

39. The method of claim 35, wherein the SCN1A mutant allele encodes an NAV1.1 variant comprising an R613X substitution relative to NAV1.1 wildtype amino acid sequence of SEQ ID NO: 106, wherein X is a termination codon.

40. The method of any one of the above claims, wherein one or more polynucleotides encoding the adenine base editor (ABE) and the gRNA are delivered to the SCN1A mutant allele in one or more delivery vehicles.

41. The method of claim 40, wherein the one or more delivery vehicles comprise a viral vector or a non-viral vector, or a combination thereof.

42. The method of claim 41, wherein the viral vector is an AAV vector or lentivirus vector.209 / 224Bl 195.70210WO00#14840465vl43. The method of claim 42, wherein the viral vector is an AAV vector comprising the sequence of any one of SEQ ID NOs: 144-147, 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 of any one of SEQ ID NOs: 144-147.

44. The method of claim 41, wherein the non-viral vector is a virus-like particle (VLP).

45. The method of claim 41, wherein the non-viral vector is a nanoparticle.

46. The method of claim 45, wherein the nanoparticle is a lipid nanoparticle (LNP), polymeric nanoparticle, inorganic nanoparticle, liposome, or a nanostructured lipid carrier.

47. The method of claim 40, wherein the one or more delivery vehicles is a targeted delivery vehicle.

48. The method of claim 47, wherein the targeted delivery vehicle comprises at least one targeting agent capable of targeting the delivery vehicle to a desired cell.

49. The method of claim 48, wherein the desired cell is a GABA interneuron.

50. The method of claim 48, wherein the targeting agent is an antibody or antigen binding domain which is capable of binding to an antigen of the desired cell.

51. The method of any one of the above claims, wherein the adenine base editor (ABE) and the gRNA are encoded on the same polynucleotide.

52. The method of any one of the above claims, wherein the adenine base editor (ABE) and the gRNA are encoded on different polynucleotides.

53. The method of claim 33, wherein the N-terminal portion and C-terminal portion of the split adenine base editor (ABE) are encoded by different polynucleotides.

54. The method of claim 53, wherein the different polynucleotides are encapsulated by the same delivery vehicle.210 / 224Bl 195.70210WO00#14840465vl55. The method of claim 54, wherein the delivery vehicle is an AAV or lentivirus vector.

56. The method of claim 55, wherein the delivery vehicle is an AAV vector comprising the sequence of any one of SEQ ID NOs: 144-147, 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 of any one of SEQ ID NOs: 144-147.

57. The method of claim 53, wherein the different polynucleotides are encapsulated by different delivery vehicles.

58. The method of claim 57, wherein each of the different delivery vehicles comprise an AAV vector or lentivirus vector.

59. The method of claim 58, wherein the delivery vehicle is an AAV vector comprising the sequence of any one of SEQ ID NOs: 144-147, 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 of any one of SEQ ID NOs: 144-147.

60. The method of any one of the above claims, wherein the method results in the treatment, reduction, or prevention of Dravet Syndrome or at least one symptom or condition associated with a SCN1A mutant allele in a subject.

61. The method of claim 60, wherein the at least one symptom or condition associated with the SCN1A mutant allele comprises epilepsy, seizures, developmental delay / intellectual disability, autism spectrum disorder, SUDEP (high rate of sudden unexpected death in epilepsy), and / or severe encephalopathy.

62. The method of any one of the above claims, wherein the step of contacting corrects a G> A transition mutation in a SCN1A mutant allele.

63. The method of claim 62, wherein the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a G> A mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114.211 / 224Bl 195.70210WO00#14840465vl64. The method of any one of the above claims, wherein the step of contacting corrects a C>T transition mutation in a SCN1A mutant allele.

65. The method of claim 64, wherein the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a OT mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114.

66. The method of claim 65, wherein correction of the OT transition mutation in the SCN1A gene results in correction of an R613X mutation.

67. The method of any one of the above claims, wherein the step of contacting a nucleic acid sequence comprising the SCN1A mutant allele is performed in a cell.

68. The method of claim 67, wherein the cell is a neuronal cell.

69. The method of claim 68, wherein the neuronal cell is a GABA (gamma-aminobutyric acid) interneuron.

70. The method of any one of the above claims, wherein the step of contacting a nucleic acid sequence comprising the SCN1A mutant allele is performed in vivo.

71. The method of any one of the above claims, wherein the step of contacting a nucleic acid sequence comprising the SCN1A mutant allele is performed in vitro.

72. The method of any one of the above claims, wherein the step of contacting a nucleic acid sequence comprising the SCN1A mutant allele is performed ex vivo.

73. The method of any one of the above claims, wherein the step of contacting a nucleic acid sequence comprising the SCN1A mutant allele is performed in a subject.

74. The method of claim 73, wherein the subject is a human.

75. The method of claim 73, wherein the subject is a mouse.212 / 224Bl 195.70210WO00#14840465vl76. A complex for correcting a SCN1A mutant allele having a G>A or C>T mutation in a SCN1A gene, said complex comprising an adenine base editor (ABE) and a gRNA targeting the ABE to the SCN1A mutant allele, thereby restoring the G>A or C>T mutation to wildtype to produce a corrected SCN1A mutant allele.

77. The complex of claim 76, wherein the wildtype SCN1A nucleotide sequence comprises SEQ ID NO: 114 [human] or SEQ ID NO: 116 [mouse],78. The complex of claim 76, wherein the wildtype SCN1A nucleotide sequence encodes wildtype voltage-gated sodium channel a subunit (NAV1.1) comprising the amino acid sequence of SEQ ID NO: 106 [human] or SEQ ID NO: 108 [mouse],79. The complex of claim 76, wherein the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a G> A or OT mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114.

80. The complex of claim 76, wherein the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a G> A mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114.

81. The complex of claim 76, wherein the SCN1A mutant allele is one of the SCN1A mutant alleles identified in Table 1, wherein said mutant allele comprises a T mutation relative to the wildtype SCN1A nucleotide sequence of SEQ ID NO: 114.

82. The complex of claim 76, wherein the SCN1A mutant allele comprises a C>T mutation at position 1837 (1837C>T) relative to wildtype SCN1A nucleotide sequence of SEQ ID NO: 114, thereby introducing a stop codon.

83. The complex of claim 76, wherein the SCN1A mutant allele encodes a variant of voltage-gated sodium channel a subunit (NAV1.1) having the amino acid change specified in Table 1 relative to wildtype NAV1.1 of SEQ ID NO: 106.213 / 224Bl 195.70210WO00#14840465vl84. The complex of claim 76, wherein the SCN1A mutant allele encodes an NAV 1.1 variant comprising an R613X substitution relative to NAV1.1 wildtype amino acid sequence of SEQ ID NO: 106, wherein X is a termination codon.

85. The complex of claim 76, wherein the gRNA targets a strand at the SCN1A mutant allele.

86. The complex of claim 85, wherein the SCN1A mutant allele encodes an NAV 1.1 variant comprising an R613X substitution relative to NAV1.1 wildtype amino acid sequence of SEQ ID NO: 106, wherein X is a termination codon.

87. The complex of claim 76, wherein the gRNA comprises a spacer having a nucleotide sequence that is the inverse complement of a target strand at the SCN1A mutant allele, wherein the SCN1A mutant allele is any one of the SCN1A mutant alleles identified in Table 1.

88. The complex of claim 76, wherein the gRNA comprises a nucleotide sequence of any one of the gRNA sequences of Tables 4A or 4B, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the nucleotide sequences of Tables 4A or 4B.

89. The complex of claim 76, wherein the gRNA comprises a spacer comprising a nucleotide sequence of any one of the gRNA spacers of Tables 4A or 4B, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the nucleotide sequences of Tables 4 A or 4B.

90. The complex of claim 76, wherein the adenine base editor (ABE) comprises a nucleic acid-programmable DNA-binding protein (napDNAbp) and an adenosine deaminase.

91. The complex of claim 90, wherein the nucleic acid-programmable DNA-binding protein (napDNAbp) comprises a Cas9 protein.214 / 224Bl 195.70210WO00#14840465vl92. The complex of claim 91, wherein the Cas9 protein is a Cas9 nickase (nCas9) or a nuclease-inactive Cas9 (dCas).

93. The complex of claim 90, wherein the napDNAbp comprises 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: 1-23 and 139-140.

94. The complex of claim 90, wherein the napDNAbp is the napDNAbp of ABE7.10, ABE8e, ABE8e-(V106W)-SaCas9, ABE8e-(V106W)-VRQR / NG, or ABE8e-(V106W)-SpCas9.

95. The complex of claim 90, wherein the adenosine deaminase comprises 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: 24-70.

96. The complex of claim 90, wherein the adenosine deaminase is the adenosine deaminase of ABE7.10, ABE8e, ABE8e-(V106W)-SaCas9, ABE8e-(V106W)-VRQR / NG, or ABE8e-(V106W)-SpCas9.

97. The complex of claim 76, wherein the adenine base editor (ABE) is selected from the group consisting of ABE7.10, ABE8e, ABE8e-(V106W)-SaCas9, ABE8e-(V106W)-VRQR / NG, and ABE8e-(V106W)-SpCas9.

98. The complex of claim 76, wherein the adenine base editor (ABE) comprises the sequence of any one of SEQ ID NOs: 99-105, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of any one of SEQ ID NOs: 99-105.

99. The complex of claim 76, wherein the adenine base editor (ABE) comprises the sequence of SEQ ID NO: 99-105.

100. The complex of claim 76, wherein the adenine base editor (ABE) further comprises one or more nuclear localization sequences (NLS).215 / 224Bl 195.70210WO00#14840465vl101. The complex of claim 100, wherein the one or more NLS comprise the sequence of any one of SEQ ID NOs: 71-82, 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 of any one of SEQ ID NOs: 71-82.

102. The complex of claim 76, wherein the adenine base editor (ABE) is a fusion protein further comprising a linker between the napDNAbp and the adenosine deaminase.

103. The complex of claim 102, wherein the linker comprises the sequence of any one of SEQ ID NOs: 83-98, 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 of any one of SEQ ID NOs: 83-98.

104. The complex of claim 102, wherein the fusion protein comprises in the N-to-C terminal direction the [napDNAbp]-[linker]-[adenosine deaminase],105. The complex of claim 102, wherein the fusion protein comprises in the N-to-C terminal direction the [adenosine deaminase]-[linker]-[napDNAbp],106. The complex of claim 76, wherein the adenine base editor (ABE) is split fusion protein comprising an N-terminal portion and a C-terminal protein.

107. The complex of claim 106, wherein the N-terminal portion comprises an N-intein and the C-terminal portion comprises a C-intein.

108. A guide RNA (gRNA) comprising (a) a spacer sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence GGUCAUCGGGGCACAAACAA (SEQ ID NO: 124) [human], or a spacer sequence comprising one, two, three, four, or five nucleotide substitutions relative to the sequence GGUCAUCGGGGCACAAACAA (SEQ ID NO: 124) [human] or (b) a spacer sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence GCGUGUCAUCGAGGAACGAACA (SEQ ID NO: 122) [mouse] or a spacer sequence216 / 224Bl 195.70210WO00#14840465vlcomprising one, two, three, four, or five nucleotide substitutions relative to the sequence GCGUGUCAUCGAGGAACGAACA (SEQ ID NO: 122) [mouse].

109. A polynucleotide molecule comprising a nucleotide sequence encoding a gRNA of claim 108.

110. The polynucleotide molecule of claim 109, further comprising a nucleotide sequence encoding an adenine base editor (ABE).

111. The polynucleotide molecule of claim 110, wherein the adenine base editor is a split adenine base editor.

112. A composition comprising a first polynucleotide molecule encoding a gRNA of claim 108 and one or more additional polynucleotide molecules encoding an adenine base editor (ABE).

113. The composition of claim 112, wherein the adenine base editor (ABE) is a split adenine base editor (ABE).

114. One or more vectors comprising a polynucleotide molecule of any of claims 109-111 or a composition of claims 112-113.

115. The one or more vectors of claim 114, wherein each of the vectors is a viral or non-viral vector.

116. The one or more vectors of claim 115, wherein the viral vector is an AAV or a lentivirus vector.

117. The one or more vectors of claim 116, wherein the viral vector is an AAV vector comprising the sequence of any one of SEQ ID NOs: 144-147, 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 of any one of SEQ ID NOs: 144-147.

118. The one or more vectors of claim 115, wherein the non-viral vector is a nanoparticle.217 / 224Bl 195.70210WO00#14840465vl119. The one or more vectors of claim 118, wherein the nanoparticle is a lipid nanoparticle (LNP), polymeric nanoparticle, inorganic nanoparticle, liposome, or a nanostructured lipid carrier.

120. The one or more vectors of claim 115, wherein the non-viral particle is a virus-like particle (VLP).

121. A pharmaceutical composition comprising the complex of claims 76-107, the gRNA of claim 108, the polynucleotide molecule of claims 109-111, the composition of claims 112-113, or the one or more vectors of claims 114-120, or any combinations thereof, and at least one pharmaceutically acceptable excipient.

122. A cell comprising the complex of claims 76-107, the gRNA of claim 108, the polynucleotide molecule of claims 109-111, the composition of claims 112-113, or the one or more vectors of claims 114-120, or any combinations thereof, and at least one pharmaceutically acceptable excipient.

123. A tissue comprising the complex of claims 76-107, the gRNA of claim 108, the polynucleotide molecule of claims 109-111, the composition of claims 112-113, or the one or more vectors of claims 114-120, or any combinations thereof, and at least one pharmaceutically acceptable excipient.

124. The cell of claim 122, wherein the cell is a GABA interneuron.

125. The cell of claim 124, wherein the tissue is a neuronal or CNS tissue.

126. A kit comprising the complex of claims 76-107, the gRNA of claim 108, the polynucleotide molecule of claims 109-111, the composition of claims 112-113, or the one or more vectors of claims 114-120, or any combinations thereof, and at least one pharmaceutically acceptable excipient, optionally an administration device, and optionally a set of instructions for use.218 / 224Bl 195.70210WO00#14840465vl127. Use of the complex of claims 76-107, the gRNA of claim 108, the polynucleotide molecule of claims 109-111, the composition of claims 112-113, or the one or more vectors of claims 114-120, or any combinations thereof in the manufacture of a medicament for the treatment of Dravet Syndrome or at least one symptom or condition associated with a SCN1A mutant allele in a subject.

128. The use of claim 127, wherein the at least one symptom or condition associated with the SCN1A mutant allele comprises epilepsy, seizure, developmental delay / intellectual disability, autism spectrum disorder, SUDEP (high rate of sudden unexpected death in epilepsy), severe encephalopathy.

129. The complex of claims 76-107, the gRNA of claim 108, the polynucleotide molecule of claims 109-111, the composition of claims 112-113, or the one or more vectors of claims 114-120, or any combinations thereof for use in the treatment of Dravet Syndrome or at least one symptom or condition associated with a SCN1A mutant allele in a subject.

130. The use of claim 129, wherein the at least one symptom or condition associated with the SCN1A mutant allele comprises epilepsy, seizure, developmental delay / intellectual disability, autism spectrum disorder, SUDEP (high rate of sudden unexpected death in epilepsy), severe encephalopathy.

131. A method of increasing expression of SCN1A, said method comprising contacting a nucleic acid sequence comprising SCN1A with an adenine base editor (ABE) and a gRNA targeting the ABE to a splice acceptor sequence upstream of exon 20N in SCN1A.

132. The method of claim 131, wherein the gRNA comprises a spacer sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence: GUAUAGGAUAAUCUUGCUCC (SEQ ID NO: 240), GGUAUAGGAUAAUCUUGCUC (SEQ ID NO: 241), GAUAUAGGAUAAUCUUGCUC (SEQ ID NO: 242), GUAUAGGAUAAUCUUGCUC (SEQ ID NO: 243), or GUAUAGGAUAAUCUUGCUC (SEQ ID NO: 243).

133. The method of claim 131 or 132, wherein the gRNA comprises a scaffold sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at 219 / 224Bl 195.70210WO00#14840465vlleast 99%, or 100% identical to the sequence:GUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACUAAAACAAGGCAAAAU GCCGUGUUUAUCUCGUCAACUUGUUGGCGAGA (SEQ ID NO: 126), GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 127), or GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 248).

134. The method of any one of claims 131-133, wherein the gRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence:GUAUAGGAUAAUCUUGCUCCGUUUUAGUACUCUGUAAUGAAAAUUACAGAAU CUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGA(SEQ ID NO: 249), GGUAUAGGAUAAUCUUGCUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 250), GAUAUAGGAUAAUCUUGCUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 251), GUAUAGGAUAAUCUUGCUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 252), or GUAUAGGAUAAUCUUGCUCGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAG UUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 253).

135. The method of any one of claims 131-134, wherein the ABE comprises SpCas9-NRRH, SpCas9-SpyMAC, or SpCas9-iSpyMAC.

136. The method of any one of claims 131-135, wherein the ABE is ABE8e-NRRH, ABE8e-V106W-NRRH, ABE8e-SpyMAC, or ABE8e-iSpyMAC.220 / 224Bl 195.70210WO00#14840465vl137. The method of any one of claims 131-136, wherein introduction of an A-to-G mutation in the splice acceptor sequence by the ABE prevents nonproductive splicing of SCN1A.

138. The method of any one of claims 131-137, wherein the method is performed in vivo.

139. The method of any one of claims 131-138, wherein the method is performed in a subject.

140. The method of claim 139, wherein the subject is a mouse.

141. The method of claim 139, wherein the subject is a human.

142. The method of any one of claims 131-137, wherein the method is performed in vitro or ex vivo.

143. The method of any one of claims 131-141, wherein the method is a method of treating Dravet syndrome.

144. A gRNA comprising a spacer sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence: GUAUAGGAUAAUCUUGCUCC (SEQ ID NO: 240), GGUAUAGGAUAAUCUUGCUC (SEQ ID NO: 241), GAUAUAGGAUAAUCUUGCUC (SEQ ID NO: 242), GUAUAGGAUAAUCUUGCUC (SEQ ID NO: 243), or GUAUAGGAUAAUCUUGCUC (SEQ ID NO: 243).

145. The gRNA of claim 144, wherein the gRNA comprises a scaffold sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence:GUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACUAAAACAAGGCAAAAU GCCGUGUUUAUCUCGUCAACUUGUUGGCGAGA (SEQ ID NO: 126), GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 127), or221 / 224Bl 195.70210WO00#14840465vlGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 248).

146. The gRNA of claim 144 or 145, wherein the gRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence:GUAUAGGAUAAUCUUGCUCCGUUUUAGUACUCUGUAAUGAAAAUUACAGAAU CUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGA(SEQ ID NO: 249), GGUAUAGGAUAAUCUUGCUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 250), GAUAUAGGAUAAUCUUGCUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 251), GUAUAGGAUAAUCUUGCUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 252), or GUAUAGGAUAAUCUUGCUCGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAG UUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 253).

147. A complex comprising a gRNA of any one of claims 144-146 and an adenine base editor (ABE).

148. The complex of claim 147, wherein the ABE comprises SpCas9-NRRH, SpCas9-SpyMAC, or SpCas9-iSpyMAC.

149. The complex of claim 147 or 148, wherein the ABE is ABE8e-NRRH, ABE8e-V106W-NRRH, ABE8e-SpyMAC, or ABE8e-iSpyMAC.

150. One or more polynucleotides encoding the gRNA of any one of claims 144-146 or the gRNA and the ABE of the complex of any one of claims 147-149.222 / 224Bl 195.70210WO00#14840465vl151. One or more vectors comprising the one or more polynucleotides of claim 150.

152. A pharmaceutical composition comprising the gRNA of any one of claims 144-146, the gRNA and the ABE of the complex of any one of claims 147-149, the one or more polynucleotides of claim 150, or the one or more vectors of claim 151.

153. A kit comprising the gRNA of any one of claims 144-146, the gRNA and the ABE of the complex of any one of claims 147-149, the one or more polynucleotides of claim 150, or the one or more vectors of claim 151.

154. Use of the gRNA of any one of claims 144-146, the gRNA and the ABE of the complex of any one of claims 147-149, the one or more polynucleotides of claim 150, the one or more vectors of claim 151, the pharmaceutical composition of claim 152, or the kit of claim 153, in the manufacture of a medicament for treating Dravet syndrome.

155. The gRNA of any one of claims 144-146, the gRNA and the ABE of the complex of any one of claims 147-149, the one or more polynucleotides of claim 150, the one or more vectors of claim 151, the pharmaceutical composition of claim 152, or the kit of claim 153, for use in treating Dravet syndrome.223 / 224Bl 195.70210WO00#14840465vl

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