Disruption of splice acceptor site of disease-associated gene using adenosine deaminase base editor, including treatment of genetic disease

Abstract

To provide methods for treating, reducing, or ameliorating the debilitating effects of neurological disorders, particularly Amyotrophic Lateral Sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA).SOLUTION: Provided is a method for treating neurological disorders in a subject in need thereof comprising administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding it; and (ii) a guide polynucleotide or a nucleic acid sequence encoding it, where the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, and the guide polynucleotide directs the adenosine base editor to effect a single nucleobase modification at a splice site of a target gene associated with the neurological disorder in the subject, thereby treating the neurological disorder in the subject.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application No. 62 / 805,271, filed February 13, 2019, and U.S. Provisional Application No. 62 / 805,271, filed May 23, 2019. U.S. Provisional Application No. 62 / 852,228 filed May 23, 2019, No. 224, filed July 11, 2019; U.S. Provisional Application No. 62 / 873,140, ​​filed July 11, 2019; U.S. Provisional Application No. 62 / 873,144, filed November 6, 2019; U.S. Provisional Application No. 62 / 931,722, filed November 6, 2019 , U.S. Provisional Application No. 62 / 941,569, filed November 27, 2019, and U.S. Provisional Application No. 62 / 941,569, filed January 27, 2020 The benefit of U.S. Provisional Application No. 62 / 966,526 is claimed, the disclosure of which is incorporated herein by reference in its entirety. be incorporated into the book.

[0002] Incorporation by Reference All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety. The patent or patent application is specifically and individually indicated to be incorporated by reference; To the same extent, the invention is incorporated herein by reference. All cited publications, patents, and patent applications are incorporated herein by reference in their entirety. can be. [Background technology]

[0003] Targeted editing of nucleic acid sequences, such as targeted cleavage or targeted editing of genomic DNA. These modifications are a very promising approach for studying gene function and may be useful in the treatment of human genetic diseases. Currently available base editors include target C·G Cytidine base editors (e.g., BE4) convert base pairs to T·A and A·T to G·C Includes adenine base editors (e.g., ABE7.10) that target targets with higher specificity and efficiency. There is a need in the art for improved base editors that can introduce modifications into sequences. It is being done. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention provides compositions comprising novel adenine base editors (e.g., ABE8) with increased efficiency. and a base enzyme containing adenosine deaminase variants for editing target sequences. A method for using the editor is provided. [Means for solving the problem]

[0005] In some embodiments, provided herein are methods for treating a neurological disorder in a subject in need thereof. (i) an adenosine base editor or a nucleic acid sequence encoding the same; and (ii) a guide and administering to said subject a nucleic acid sequence encoding the polynucleotide. the adenosine base editor comprises a programmable DNA binding domain and an adenosine a deaminase domain, and the guide polynucleotide comprises the adenosine base edta and inducing a single gene encoding a target gene associated with a neurological disorder in said subject. and a method for treating said neurological disorder in a subject, comprising: will be done.

[0006] In one embodiment, the adenosine deaminase is adenosine deaminase as numbered in SEQ ID NO:2. It contains an amino acid substitution at amino acid position 82 or 166 or a corresponding substitution. In embodiments, the single nucleobase modification is In one embodiment, the alternative splicing results in alternative splicing of A truncated or non-functional protein encoded by said target gene In one embodiment, the single nucleobase modification results in the production of a protein in the subject. In one embodiment, the target gene is a superoxide anion. The gene responsible for this is the SOD1 gene, and the neurological disease is amyotrophic lateral sclerosis (ALS). In one embodiment, the target gene is the androgen receptor (AR) gene, and the target gene is used to treat a neurological disorder. is spinal and bulbar muscular atrophy (SBMA).

[0007] In one aspect, provided herein is a method for treating amyotrophic lateral sclerosis (ALS) in a subject in need thereof. 1. A method of treating a subject having a leukemia, comprising administering to the subject (i) a base editor or a nucleic acid encoding the same; and (ii) a guide polynucleotide or a nucleic acid sequence encoding the same. and wherein the base editor comprises a programmable DNA binding domain and a deaminator. a guide polynucleotide comprising a nucleotide domain, and wherein the guide polynucleotide induces the adenosine base editor. This leads to a splice site in the superoxide dismutase 1 (SOD1) gene of the subject. and a method for treating ALS in said subject. It is served.

[0008] In one aspect, provided herein is a method for treating amyotrophic lateral sclerosis (ALS) in a subject in need thereof. 1. A method of treating a subject with a steroid hormone, comprising administering to the subject (i) a base editor or a nucleic acid encoding same; and (ii) a guide polynucleotide or a nucleic acid sequence encoding the same. and wherein the base editor comprises a programmable DNA binding domain and a deaminator. a guide polynucleotide comprising a nucleotide domain, and wherein the guide polynucleotide induces the adenosine base editor. A single nucleobase in the superoxide dismutase 1 (SOD1) gene of the subject The single nucleic acid base modification results in a premature stop codon in the SOD1 gene. and thereby treating ALS in said subject.

[0009] In one embodiment, the deaminase is selected from the group consisting of amino acid sequence 8 and 9, as numbered in SEQ ID NO:2. or an adenosine deaminase containing an amino acid substitution at 166 or a corresponding substitution In one embodiment, the single nucleobase modification is an A to G modification. In embodiments, the single nucleobase modification is at the splice acceptor site of the SOD1 gene. In one embodiment, the splice site is located at the exon of the SOD1 gene. In one embodiment, the splice acceptor site at the 5' end of the SOD1 gene is The exon is exon 3 corresponding to SEQ ID NO: 3 or a variant thereof. In the example, the exon 3 of the SOD1 gene is the SOD1 polynucleotide according to the numbering in SEQ ID NO: 3. splice acceptor AG at nucleotide position 6828 of the nucleotide sequence or its variants In one embodiment, the exon of the SOD1 gene is adjacent to In one embodiment, the single nucleobase modification is exon 4 or a variant thereof. However, a transcript lacking exons 3 to 5 of the human SOD1 gene corresponding to SEQ ID NO: 3 or In one embodiment, after said administration, a variant of the SOD1 gene is generated in said subject. expression is reduced by at least 40%.

[0010] In one embodiment, the guide polynucleotide is In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence complementary to a target nucleic acid sequence or a splice donor nucleic acid sequence. wherein the guide polynucleotide is any nucleic acid sequence selected from Table 19 or Table 23. In one embodiment, the guide polynucleotide comprises a sequence of 5'-UUAAAGGAAAGUAAUGGA CCAGU-3′, 5′-UAAAUAGGCUGUACCAGUGCAGG-3′, 5′-UUCAUUAUUAGGCAUGUUGGAGA-3′, 5′ -AAAUAGGCUGUACCAGUGCAGGU-3′, 5′-UAUUAGGCAUGUUGGAGACUUGG-3′ The nucleic acid sequence includes a nucleic acid sequence that is

[0011] In some embodiments, the present invention provides a method for treating spinal and bulbar muscular atrophy (SBMA) in a subject. A method comprising: (i) a base editor or a nucleic acid sequence encoding the same; and (ii) a guidepo administering to said subject a nucleic acid sequence encoding the same; The base editor contains a programmable DNA binding domain and a deaminase domain. wherein the guide polynucleotide guides the adenosine base editor to A single nucleobase alteration at a splice site in the elephant androgen receptor (AR) gene and thereby treating SBMA in said subject.

[0012] In some embodiments, the present invention provides a method for treating spinal and bulbar muscular atrophy (SBMA) in a subject. A method comprising: (i) a base editor or a nucleic acid sequence encoding the same; and (ii) a guidepo administering to said subject a nucleic acid sequence encoding the same; The adenosine base editor comprises a programmable DNA binding domain and a deaminase a domain, wherein the guide polynucleotide guides the adenosine base editor. resulting in a single nucleobase alteration in the androgen receptor (AR) gene of said subject. wherein the single nucleobase modification results in a premature stop codon in the AR gene, A method is provided for treating SBMA in the subject by

[0013] In one embodiment, the nucleobase modification results in a CAG-TAG codon change in the AR gene. In one embodiment, the codon change is in exon 1 or exon 2 of the AR gene. In one embodiment, the deaminase is Adenosine deaminase containing an amino acid substitution at amino acid position 82 or 166. In one embodiment, the single nucleobase modification is an A to G modification. The present invention relates to a method for identifying a nucleic acid sequence in which the A to G modification occurs in the splice acceptor site of the AR gene. In one embodiment, the splice site is located 5' of an exon of the AR gene. In one embodiment, the exon of the AR gene is a splice acceptor site. exon 2 or a variant thereof, which corresponds to sequence number 4. The splice site is a splice donor site 3' of an exon of the AR gene. In this embodiment, the exon of the AR gene is exon 1 corresponding to SEQ ID NO: 4 or a variant thereof. In one embodiment, the expression of the AR gene in the subject after said administration is at least Decreases by 40%.

[0014] In one embodiment, the guide polynucleotide is a polynucleotide that encodes a splice acceptor of the AR gene. In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence complementary to the splice donor nucleic acid sequence. wherein the guide polynucleotide comprises a nucleic acid sequence selected from Table 41 A or 41 B. In one embodiment, the guide polynucleotide has the sequence 5′-ACUUACCGCAUGUCCCCGUAAGG-3′ , 5′-AGUGCAGUUAGGGCUGGGAAGGG-3′, 5′-AAGUGCAGUUAGGGCUGGGAAGG-3′ The nucleic acid sequence includes a nucleic acid sequence selected from the group consisting of:

[0015] In one embodiment, the subject is a mammal or a human. In one embodiment, administration is via delivery to cells of the subject's central nervous system (CNS). wherein the cell is a motor neuron.

[0016] In one embodiment, the present invention relates to a target gene or its regulatory element associated with a neurological disorder. A method for modifying a target gene or its regulatory element, comprising: (i) (ii) a nucleic acid sequence encoding a nucleotide sequence containing a nucleotide sequence encoding a nucleotide sequence; and contacting said adenosine base with a nucleic acid sequence encoding the same; The editors contain programmable DNA-binding and adenosine deaminase domains. wherein the guide polynucleotide guides the adenosine base editor to Methods are provided that result in the modification of a single nucleobase at a splice site of the target gene. do.

[0017] In one embodiment, the adenosine deaminase is adenosine deaminase as numbered in SEQ ID NO:2. In one embodiment, the single nucleic acid comprises an amino acid substitution at amino acid position 82 or 166. The base modification induces alternative splicing of the transcript encoded by the target gene, truncated and / or non-functional proteins encoded by the or results in decreased expression of the target gene when expressed in a cell. The target gene is the superoxide dismutase 1 (SOD1) gene, and the neurological disease is muscle atrophy. In one embodiment, the target gene is androgen receptor agonist (ARG). The body (AR) gene and the neurological disease is spinal and bulbar muscular atrophy (SBMA).

[0018] In one aspect, the present invention relates to a method for the expression of the superoxide dismutase (SOD1) gene. A method for regulating expression of the SOD1 gene or a regulatory element thereof, comprising: (i) modifying the base edition of the SOD1 gene; (ii) a target or a nucleic acid sequence encoding the target; and (iii) a guide polynucleotide or a nucleic acid sequence encoding the target. wherein the base editor is programmable. a DNA binding domain and a deaminase domain, and the guide polynucleotide The adenosine base editor is then induced to transduce the superoxide dismutase 1 (SOD1) gene. Methods are provided that result in the modification of a single nucleobase at a splice site of a child.

[0019] In one embodiment, the superoxide dismutase (SOD1) gene is modified as described herein. A method for modifying an SOD1 gene or a regulatory element thereof, comprising: (i) using a base editor or or a nucleic acid sequence encoding the same, and (ii) a guide polynucleotide or a nucleic acid sequence encoding the same. and contacting the guide polynucleotide with a nucleic acid sequence that encodes the adenosine monophosphate (ADP), and inducing a nucleotide editor to modify the target superoxide dismutase 1 (SOD1) gene. and the single nucleobase modification results in a single nucleobase modification in the SOD1 gene. Methods are provided for translating a nucleotide sequence into a premature stop codon.

[0020] In one embodiment, the single nucleobase modification is an A to G modification. In the example, the modification of the nucleic acid base occurs in the splice acceptor site of the SOD1 gene. In one embodiment, the splice site is a splice site located 5' of an exon of the SOD1 gene. In one embodiment, the exon of the SOD1 gene has the sequence In one embodiment, the SOD1 gene is exon 3 corresponding to number 3 or a variant thereof. wherein said exon 3 is a nucleotide of the SOD1 polynucleotide sequence as numbered in SEQ ID NO: 3 In one embodiment, the SOD1 Said exon of the gene is exon 4 corresponding to SEQ ID NO: 3 or a variant thereof. In one embodiment, the single nucleobase modification is in the exon 1 of the SOD1 gene corresponding to SEQ ID NO: 3. Generate transcripts lacking sequences 3-5 or variants thereof.

[0021] In one embodiment, the guide polynucleotide is In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence complementary to a target nucleic acid sequence or a splice donor nucleic acid sequence. wherein the guide polynucleotide comprises a nucleic acid sequence selected from Table 19 or Table 23. In one embodiment, the guide polynucleotide has the sequence 5'-UUAAAGGAAAGUAAUGGACCAGU-3' , 5′-UAAAUAGGCUGUACCAGUGCAGG-3′, 5′-UUCAUUAUUAGGCAUGUUGGAGA-3′, 5′-AAAUAGGC a nucleic acid selected from the group consisting of UGUACCAGUGCAGGU-3′, 5′-UAUUAGGCAUGUUGGAGACUUGG-3′ Contains arrays.

[0022] In one embodiment, the compounds described herein regulate expression of the androgen receptor (AR) gene. The method comprises: (i) converting an AR gene or a regulatory element thereof into a base editor or a gene encoding the base editor; and (ii) a guide polynucleotide or a nucleic acid sequence encoding the same. and contacting the base editor with a programmable DNA binding domain. and a deaminase domain, and the guide polynucleotide comprises the adenosine A base editor was induced to insert a single fragment at a splice site in the androgen receptor (AR) gene. Methods are provided that result in the modification of a single nucleobase.

[0023] In one aspect, methods for modifying the androgen receptor (AR) gene are described herein. The AR gene or its regulatory elements are modified by: (i) a base editor or a gene encoding the same; and (ii) a guide polynucleotide or a nucleic acid sequence encoding the same. and wherein the base editor comprises a programmable DNA-binding domain. and a deaminase domain, wherein the guide polynucleotide comprises the adenosine base Inducing an editor in a single nucleus in the androgen receptor (AR) gene in said subject. resulting in an acid-base modification, the single nucleobase modification causing a premature termination in the AR gene. A method is provided for generating codons.

[0024] In one embodiment, the deaminase is selected from the group consisting of amino acid sequence 8 and 9, as numbered in SEQ ID NO:2. In one embodiment, the adenosine deaminase comprises an amino acid substitution at position 2 or 166. In one embodiment, the deaminase is a cytidine deaminase. The base modification is a C to T modification. In one embodiment, the nucleobase modification is a C to T modification at the AR gene. In one embodiment, the codon change results in a CAG-TAG codon change in the AR gene. In one embodiment, the single nucleobase modification is in exon 1 or exon 2 of the gene. In one embodiment, the A to G nucleobase modification is an A to G nucleobase modification in the AR gene. at the rice acceptor site.

[0025] In one embodiment, the splice site is a splice site 5' of an exon of the AR gene. In one embodiment, the exon of the AR gene is a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ In one embodiment, the splice region is exon 2 or a variant thereof. In one embodiment, the site is a splice donor site 3' of an exon of the AR gene. and wherein the exon of the AR gene is exon 1 corresponding to SEQ ID NO: 4 or a variant thereof. do.

[0026] In one embodiment, the guide polynucleotide is selected from Table 41 A or 41 B. In one embodiment, the guide polynucleotide comprises a splice of the AR gene. The splice acceptor nucleic acid sequence or the splice donor nucleic acid sequence may comprise a nucleic acid sequence complementary to the splice acceptor nucleic acid sequence or the splice donor nucleic acid sequence. In one embodiment, the guide polynucleotide is a nucleic acid selected from Table 41A or 41B. In one embodiment, the guide polynucleotide comprises the sequence 5'-ACUUACCGCAUGUC CCCGUAAGG-3′, 5′- AGUGCAGUUAGGCUGGGAAGGG-3′, 5′- AAGUGCAGUUAGGGCUGGGAAGG-3 ', and its complementary strand.

[0027] In some embodiments, the contacting is intracellular. The alterations result in fewer than 15% indels in the genome of the cell. The modification of a single nucleobase results in less than 5% indels in the genome of the cell. In some embodiments, the single nucleobase modification results in less than 2% indels in the genome of the cell. In some embodiments, the cell is a mammalian cell or a human cell. In one embodiment, the cell is a central nervous system cell. In another embodiment, the cell is a motor neuron. do.

[0028] In one embodiment, the contact is in a population of cells. Following the contacting, at least 40% of the cell population comprises the single nucleobase modification.

[0029] In one embodiment, after said contacting, at least 50% of the cell population have a single nucleobase modification. In one embodiment, after said contacting, at least 60% of the cell population comprises a single nucleobase modification. In one embodiment, at least 85% of the cell population is viable after said contacting. In some embodiments, the population of cells is mammalian or human cells. In one embodiment, the population of cells is a central nervous system cell. are motor neurons.

[0030] In one embodiment, the adenosine deaminase comprises TadA deaminase. In one embodiment, the adenosine deaminase is TadA7.10.

[0031] In one embodiment, the adenosine deaminase is V28 in the numbering in SEQ ID NO:2. In one embodiment, the TadA comprises the S mutation or the T166R mutation or a corresponding mutation. wherein the adenosine deaminase is selected from the group consisting of Y147T, Y147R, Q154S, as numbered in SEQ ID NO: 2; Y123H, and Q154R, or one or more of the mutations corresponding thereto. In embodiments, the adenosine deaminase has the sequence Y147T + Q according to the numbering in SEQ ID NO:2. 154R; Y147T + Q154S; Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V8 2S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123 H + Q154R; Y147R + Q154R +Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y1 23H + Y147R + Q154R + I76Y; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y1 A combination of mutations selected from the group consisting of 23H + Y147R + Q154R or their corresponding mutations Includes differences.

[0032] In one embodiment, the adenosine deaminase is selected from the group consisting of 149, 150, 151, 152, 153, 154, The deletions include C-terminal deletions beginning at residues selected from the group consisting of 155, 156, and 157. In some embodiments, the adenosine deaminase comprises a TadA dimer. The adenosine deaminase comprises an adenosine deaminase monomer.

[0033] In the various aspects and embodiments described above, the polynucleotide-programmable In one embodiment, the DNA binding domain is a Cas9 domain. In one embodiment, the Cas9 domain comprises a nickase domain. In one embodiment, the SpCas9 domain is selected from the group consisting of D10A and D10B, as numbered in SEQ ID NO: 1. and / or H840A amino acid substitution or a corresponding amino acid substitution. In one embodiment, the Cas9 domain comprises a SaCas9 domain. In one embodiment, the Cas9 domain has specificity for the engineered PAM. , NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, and NGC where N is A, G, C, or T and R is A or G. is.

[0034] In one embodiment, provided herein are cells produced by the methods described herein. A population of 100 is provided.

[0035] In some embodiments, provided herein are (i) base editors or nucleic acid sequences encoding same. and (ii) a guide polynucleotide or a nucleic acid sequence encoding the same. a base editor system, wherein the base editor comprises a programmable DNA binding domain and a deaminase domain, and the guide polynucleotide comprises the adenosine salt Induction of a base editor at the splice site of superoxide dismutase 1 (SOD1)

[0006] Base editor systems are provided that result in the modification of single nucleobases in a nucleic acid.

[0036] In some embodiments, provided herein are (i) base editors or nucleic acid sequences encoding same. and (ii) a guide polynucleotide or a nucleic acid sequence encoding the same. a base editor system, wherein the base editor comprises a programmable DNA binding domain and a deaminase domain, wherein the guide polynucleotide comprises the adenosine base Inducing editors to create a single nucleic acid in the superoxide dismutase 1 (SOD1) gene resulting in a base modification, and the single nucleic acid base modification results in a premature stop codon in the SOD1 gene. A base editor system is provided, which results in:

[0037] In one embodiment, the deaminase is selected from the group consisting of amino acid sequence 8 and 9, as numbered in SEQ ID NO:2. In one embodiment, the adenosine deaminase comprises an amino acid substitution at position 2 or 166. In one embodiment, the deaminase is a cytidine deaminase. The base modification is an A to G modification. In one embodiment, the A to G nucleobase modification is In one embodiment, the mutation is in the splice acceptor site of the SOD1 gene. The splice site is a splice acceptor site located 5' of an exon of the SOD1 gene. In one embodiment, the exon of the SOD1 gene is the exon corresponding to SEQ ID NO: 3. In one embodiment, the exon 3 of the SOD1 gene is At nucleotide position 6828 of the SOD1 polynucleotide sequence according to the numbering in SEQ ID NO:3 In one embodiment, the sequence is flanked by a splice acceptor AG or a variant thereof. Alternative splicing of the SOD1 transcript results in exons 3-5 of the SOD1 gene corresponding to SEQ ID NO:3. In one embodiment, the SOD1 gene is expressed as a transcript lacking the SOD1 gene or a variant thereof. The exon is exon 4 corresponding to SEQ ID NO: 3 or a variant thereof.

[0038] In one embodiment, the guide polynucleotide is In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence complementary to a target nucleic acid sequence or a splice donor nucleic acid sequence. wherein the guide polynucleotide comprises a nucleic acid sequence selected from Table 19 or Table 23. In one embodiment, the guide polynucleotide has the sequence 5'-UUAAAGGAAAGUAAUGGACCAGU-3' , 5′-UAAAUAGGCUGUACCAGUGCAGG-3′, 5′-UUCAUUAUUAGGCAUGUUGGAGA-3′, 5′-AAAUAGGC UGUACCAGUGCAGGU-3′, 5′-UAUUAGGCAUGUUGGAGACUUGG-3′, and their complementary sequences The nucleic acid sequence comprises a nucleic acid sequence selected from the group:

[0039] In some embodiments, provided herein are (i) base editors or nucleic acid sequences encoding same. and (ii) a guide polynucleotide or a nucleic acid sequence encoding the same. a base editor system, wherein the base editor comprises a programmable DNA binding domain and a deaminase domain, wherein the guide polynucleotide comprises the adenosine base Inducing editors at splice sites in the androgen receptor (AR) gene A base editor system is provided that provides acid-base modifications.

[0040] In some embodiments, provided herein are (i) base editors or nucleic acid sequences encoding same. and (ii) a guide polynucleotide or a nucleic acid sequence encoding the same. adenosine base editor system, wherein the adenosine base editor is a programmable DNA binding a deaminase domain and a binding domain of the guide polynucleotide, A single nucleotide editor was introduced into the androgen receptor (AR) gene in a subject. The single nucleobase modification results in a premature termination codon in the AR gene. A base editor system is provided that generates the don.

[0041] In one embodiment, the deaminase is selected from the group consisting of amino acid sequence 8 and 9, as numbered in SEQ ID NO:2. In one embodiment, the adenosine deaminase comprises an amino acid substitution at position 2 or 166. In one embodiment, the deaminase is a cytidine deaminase. The base modification is a C to T modification. In one embodiment, the single nucleobase modification is This results in a CAG-TAG codon change in the AR gene. In one embodiment, the codon change is , exon 1 or exon 2 of the AR gene corresponding to SEQ ID NO: 4, or a variant thereof In one embodiment, the single nucleobase modification is an A to G modification. In this embodiment, the A to G nucleobase modification is In one embodiment, the splice site is located 5' of an exon of the AR gene. In one embodiment, the exon is exon 2 corresponding to SEQ ID NO: 4 or a variant thereof. The splice site is the splice donor site located 3' of the exon of the AR gene. In some embodiments, the exon of the AR gene is exon 1, which corresponds to SEQ ID NO: 4, or It is a variant.

[0042] In one embodiment, the guide polynucleotide is a polynucleotide that encodes a splice acceptor of the AR gene. In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence complementary to the splice donor nucleic acid sequence. wherein the guide polynucleotide comprises a nucleic acid sequence selected from Table 41 A or 41 B. In one embodiment, the guide polynucleotide has the sequence 5′-ACUUACCGCAUGUCCCCGUAAGG-3′ , 5′- AGUGCAGUUAGGGCUGGGAAGGG-3′, 5′- AAGUGCAGUUAGGGCUGGGAAGG-3′, and and the complementary strand of the nucleic acid sequence selected from the group consisting of:

[0043] In one embodiment, the adenosine deaminase comprises TadA deaminase. In one embodiment, the adenosine deaminase is TadA7.10. The nosine deaminase has a V28S mutation or a T166R mutation according to the numbering in SEQ ID NO: 2 or TadA containing a corresponding mutation. The enzyme is Y147T, Y147R, Q154S, Y123H, and Q154R as numbered in SEQ ID NO:2. In one embodiment, the adenosine dehydrogenase (ADD) comprises one or more of the following mutations: The aminase may be selected from the group consisting of Y147T + Q154R; Y147T + Q154S; Y147R + Q 154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; Y147R + Q154R +Y123 H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y123H + Y147R + Q154R + I76Y; V8 2S + Y123H + Y147R + Q154R; and the group consisting of I76Y + V82S + Y123H + Y147R + Q154R or a combination of mutations selected from the above or their corresponding mutations.

[0044] In one embodiment, the adenosine deaminase is selected from the group consisting of 149, 150, 151, 152, 153, 154, and C-terminal deletions beginning at residues selected from the group consisting of 155, 156, and 157. In one embodiment, the adenosine deaminase comprises a TadA dimer. The adenosine deaminase comprises an adenosine deaminase monomer.

[0045] In one embodiment, the polynucleotide-programmable DNA binding domain is C In one embodiment, the Cas9 domain is a Cas9 nickase domain. In some embodiments, the Cas9 domain comprises an SpCas9 domain. wherein the SpCas9 domain is selected from the group consisting of D10A and / or H840A amino acids according to the numbering in SEQ ID NO: 1. In one embodiment, the Cas9 domain comprises a nucleotide substitution or a corresponding amino acid substitution. In one embodiment, the Cas9 domain comprises a SaCas9 domain. In one embodiment, the Cas9 domain is linked to an engineered PAM. In one embodiment, the Cas9 domain has specificity for NGG, NGA, NGCG, NGN , NNGRRT, NNNRRT, NGCG, NGCN, NGTN, and NGC. where N is A, G, C, or T and R is A or G.

[0046] In some embodiments, the base editor systems described herein include A nucleic acid sequence encoding a DNA-binding domain programmable by said polynucleotide in and a nucleic acid sequence encoding the adenosine deaminase domain. In one embodiment, the vector comprises a nucleic acid encoding the guide polynucleotide. In one embodiment, the vector is a viral vector.

[0047] In some embodiments, the base editor systems described herein or In one embodiment, the cell is a mammalian cell, such as a human cell. In one embodiment, the cells are in vivo, ex vivo, or motor neurons. In one embodiment, the cells are autologous cells isolated from a subject. In one embodiment, the cells are allogeneic cells.

[0048] In some embodiments, the base editor systems described herein or In one embodiment, the population of cells is a mammalian cell. In some embodiments, the population of cells is an in vitro cell, a human cell, or a motor neuron. In some embodiments, the cells are isolated from a subject. These are the patient's own cells.

[0049] In some embodiments, the present disclosure provides a method for the production of a base editor, vector, or In one embodiment, a pharmaceutical composition is provided comprising the cells and a pharmaceutically acceptable carrier. The pharmaceutical compositions described herein further comprise a lipid. The pharmaceutical composition further comprises a virus.

[0050] In some embodiments, the base feeder system or vector described herein Kits containing the target are provided.

[0051] In various embodiments of the methods described herein, at least one of the guide polynucleotides Another nucleotide comprises a non-natural modification. wherein at least one nucleotide of the nucleic acid sequence contains a non-natural modification. In various embodiments of the method described above, at least one of the nucleic acid sequences of the base editor system Another nucleotide comprises a non-natural modification. The modification is a chemical modification. In some embodiments, the chemical modification is 2'-O-methylation. In some embodiments, the nucleic acid sequence comprises a phosphorothioate.

[0052] The description and examples herein particularly illustrate embodiments of the present disclosure. It is understood that the present invention is not limited to the particular embodiments described in the specification, as such may vary. Those skilled in the art will recognize that this disclosure contains numerous variations and modifications that are encompassed within its scope. You will realize there are fixes.

[0053] Implementation of the embodiments disclosed herein is within the skill of those of ordinary skill in the art, unless otherwise indicated. Immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and and conventional techniques of recombinant DNA. See, e.g., Sambrook and Green, Molecular Cloning : A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molec ular Biology (FM Ausubel, et al. eds.); the series Methods In Enzymology (Aca (demic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialize See Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0054] The section headings used herein are for organizational purposes only. and should not be construed as limiting the subject matter described.

[0055] Various features of the disclosure may be described in the context of a single embodiment, but these features may also be combined in a single embodiment. They may also be provided separately or in any suitable combination. Although for clarity, may be described in the context of separate embodiments, the present disclosure also provides a single embodiment. The section headings used herein are for organizational purposes only. The information is for illustrative purposes only and should not be construed as limiting the subject matter described.

[0056] The features of the present disclosure are set forth with particularity in the appended claims. , which presents an illustrative embodiment in which the principles of the present disclosure are utilized. A better understanding of the features and advantages of the present disclosure will be provided in light of the accompanying drawings, which are described below. is obtained.

[0057] definition The following definitions supplement those in the art and are intended for this application: Related or unrelated matters, such as those resulting from commonly owned patents or applications Any methods and materials similar or equivalent to those described herein are not intended to be limiting. Although the materials and methods described herein may be used in carrying out the tests shown, preferred materials and methods are described herein. Therefore, the terminology used herein is for the purpose of describing particular embodiments only. It is for illustrative purposes only and is not intended to be limiting.

[0058] Unless otherwise defined, all technical and scientific terms used herein are defined by the It has the meaning commonly understood by one of ordinary skill in the art to which the invention pertains. , provides those skilled in the art with general definitions of many of the terms used in this invention: n et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The C ambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary o f Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0059] In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms "a," "an," and "the" are used unless the context clearly indicates otherwise. It should be noted that unless specifically indicated, plural referents are included. In this context, the use of "or" means and includes "and / or" unless otherwise stated. It will be understood that the terms "including," "include," "includes," and "i The use of "includes" and other forms such as "included" is non-limiting.

[0060] As used in this specification and claims, the terms "comprising" (and " "comprise" and "comprises" and all its forms), "having" g) (any of its forms, such as "have" and "has"), "include "including" ("include" and "includes" and any of its forms) or "including containing (any of its forms, such as "contains" and "contain") ) is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. Any embodiment discussed herein may be used with respect to any method or composition of the present disclosure. It is believed that the same can be done, and vice versa. The method of the present disclosure can be achieved by

[0061] The terms "about" or "approximately" refer to a range of values ​​as determined by one of ordinary skill in the art. This means that the value is within an acceptable margin of error for a particular value, which indicates how How it is measured or determined depends in part on the limitations of the measurement system. For example, "about" means, according to practice in the art, within 1 or more than 1 standard deviation. Alternatively, "about" can mean up to 20%, up to 10%, up to 5%, or Alternatively, it may refer to a range of up to 1% of the total mass of a biological system or process. Therefore, the term can mean values ​​within the same order of magnitude, e.g., within 5 times, or within 2 times. Where specific values ​​are described in the application and claims, unless otherwise stated, The term "about" means within an acceptable range of error for that particular value. It should be estimated.

[0062] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, the range 1 to 50 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 , 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 It is understood to include any number, combination of numbers, or subrange.

[0063] In the specification, "some embodiments," "an embodiment," "one embodiment," or Reference to "another embodiment" may include any particular features, structures, or feature is included in at least some embodiments of the present disclosure, but not necessarily all. This means that the embodiments are not necessarily included.

[0064] "Abasic base editor" is a tool that extracts nucleobases and converts them into DNA nucleobases (A, T, Abasic base editors refer to agents that can insert nucleic acid groups (C, G, or G). In one embodiment, the nucleic acid glycosylase comprises a glycosylase polypeptide or a fragment thereof. at amino acid 204 of the following sequence or the corresponding position in uracil DNA glycosylase: containing Asp (e.g., substituting Asn at amino acid 204) and cytosine-DNA glycosylation A mutant human uracil DNA glycosylase or an active fragment thereof having enzyme activity. In one embodiment, the nucleic acid glycosylase comprises amino acid 147 or uracil of the following sequence: containing Ala, Gly, Cys, or Ser at the corresponding positions of a DNA glycosylase (e.g., a mutation in which Tyr is substituted at amino acid 147, and which has thymine-DNA glycosylase activity An exemplary human uracil-DNA glycosylase is a human uracil-DNA glycosylase or an active fragment thereof. The sequence of A glycosylase, isoform 1 is as follows: 1 mgvfclgpwg lgrklrtpgk gplqllsrlc gdhlqaipak kapagqeepg tppssplsae 61 qldriqrnka aallrlaarn vpvgfgeswk khlsgefgkp yfiklmgfva eerkhytvyp 121 pphqvftwtq mcdikdvkvv ilgqdp y hgp nqahglcfsv qrpvppppsl eniykelstd 181 iedfvhpghg dlsgwakqgv lll n avltvr ahqanshker gweqftdavv swlnqnsngl 241 vfllwgsyaq kkgsaidrkr hhvlqtahps p l svy r gffg crhfsktnel lqksgkkpid 301 wkel

[0065] The sequence of human uracil-DNA glycosylase, isoform 2 is as follows: 1 migqktlysf fspsparkrh apspepavqg tgvagvpees gdaaaipakk apagqeepgt 61 ppssplsaeq ldriqrnkaa allrlaarnv pvgfgeswkk hlsgefgkpy fiklmgfvae 121 erkhytvypp phqvftwtqm cdikdvkvvi lgqdp y hgpn qahglcfsvq rpvppppsle 181 niykelstdi edfvhpghgd lsgwakqgvl ll n avltvra hqanshkerg weqftdavvs 241 wlnqnsnglv fllwgsyaqk kgsaidrkrh hvlqtahpsp l svy r gffgc rhfsktnell 301 qksgkkpidw kel

[0066] In other embodiments, the abasic editor is a base editor as described in PCT / JP205 / 080958 and US20170321210. The base editors may be any of those listed in the literature, which are incorporated herein by reference. In certain embodiments, the abasic editor is shown in bold and underlined in the sequence above. or any other abasic editor or uracil degumming known in the art. In one embodiment, the abasic enzyme comprises a mutation at the corresponding amino acid in the glycosylase. The diter contains mutations at Y147, N204, L272, and / or R276 or corresponding positions. In another embodiment, the abasic editor is a Y147A or Y147G mutation or the corresponding In another embodiment, the abasic editor comprises a N204D mutation or a corresponding In another embodiment, the abasic editor comprises a L272A mutation or a corresponding In another embodiment, the abasic editor comprises a R276E or R276C mutation. or corresponding mutations.

[0067] "Adenosine deaminase" refers to the enzyme that hydrolyzes the deamination of adenine or adenosine. In one embodiment, the term "de" refers to a polypeptide or fragment thereof that is capable of catalyzing the deactivation of a protein. The aminase or deaminase domain converts adenosine to inosine or deoxyribonucleic acid. Adenosine deamina catalyzes the hydrolytic deamination of adenosine to deoxyinosine. In some embodiments, the adenosine deaminase is a deoxyribonucleic acid (DNA) The compounds provided herein catalyze the hydrolytic deamination of adenine or adenosine in the ribozyme. Adenosine deaminases (e.g., engineered adenosine deaminases, evolved The adenosine deaminase (enzyme-activated adenosine deaminase) can be from any organism, such as a bacterium.

[0068] In one embodiment, the adenosine deaminase is TadA deaminase. In some embodiments, the TadA deaminase is a TadA variant. The dA variant is TadA*8. In one embodiment, the deaminase or deaminase The domains may be, for example, human, chimpanzee, gorilla, monkey, cow, dog, rat, or mammal. In some embodiments, the deaminase is a variant of a naturally occurring deaminase from an organism such as a mouse. The enzyme or deaminase domain is non-naturally occurring. For example, in some embodiments In this embodiment, the deaminase or deaminase domain is At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least At least 92%, at least 93%, at least, at least 94%, at least 95%, at least 96%, At least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, At least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99 0.7%, at least 99.8%, or at least 99.9% identity. For example, Zedaima is a registered trademark of International PCT Application Nos. PCT / 2007 / 045381 (WO2018 / 027078) and PCT / US201 6 / 058344 (WO 2017 / 070632), each of which is incorporated by reference in its entirety. See also Komor, AC, et al., “Programmable editing of fa target base in genomic DNA without double-stranded DNA cleavage” Nature 533 , 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, AC, et al., “Improved base excision repair inhibition and bacteriophage Mu Ga m protein yields C:G-to-T:A base editors with higher efficiency and product puri ty” Science Advances 3:eaao4774 (2017) ), and Rees, HA, et al., “Base edit ing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 Dec;19(12):770-788. See also doi: 10.1038 / s41576-018-0059-1 (see also the entire contents of which are incorporated herein by reference).

[0069] The wild-type TadA (wt) adenosine deaminase has the following sequence (also known as the TadA reference sequence): called): MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHEIMALRQGGLVMQNYRLIDATL YVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQK KAQSSTD (SEQ ID NO:2).

[0070] In some embodiments, the adenosine deaminase has a modification in the following sequence: include: MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV IGEGWNRAIG LHDPTAHAEI MALRQGGLVM QNY RLIDATL YVTFEPCVMC AGAMIHSRIG RVVFGVRNAK TGAAGSLMDV LHYPGMNHRV EITEGILADE CAALLC YFFR MPRQVFNAQK KAQSSTD (also known as TadA*7.10).

[0071] In some embodiments, TadA*7.10 comprises at least one modification. In embodiments, TadA*7.10 contains modifications at amino acids 82 and / or 166. In embodiments, variants of the reference sequence include one or more of the following modifications: Y1 47T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. The modification Y123H is It is also called H123H in the literature (the modification H123Y in TadA*7.10 has been reverted to Y123H(wt)). In other embodiments, the variant of the TadA*7.10 sequence is selected from the group consisting of: Includes combinations of changes: Y147T + Q154R; Y147T + Q154S; Y147R + Q154S; V82S + Q1 54S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T ; V82S + Y123H + Y147R; V82S + Y123H + Q154R; Y147R + Q154R +Y123H; Y147R + Q154 R + I76Y; Y147R + Q154R + T166R; Y123H + Y147R + Q154R + I76Y; V82S + Y123H + Y1 47R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R.

[0072] In other embodiments, the present invention provides a method for the preparation of a nucleic acid comprising a nucleic acid sequence selected from residues 149, 150, 151, 152, 153, 154, 155, 156, or or adenosine deaminase variants containing deletions, including C-terminal deletions beginning at 157 In another embodiment, adenosine deaminase variants, such as TadA*8, are provided. The target is a TadA (e.g., TadA*8) monomer containing one or more of the following modifications: Y147T , Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. The adenosine deaminase variants are those that contain a combination of modifications selected from the group consisting of: TadA monomers containing the following combinations (e.g., TadA*8): Y147T + Q154R; Y147T + Q154S; Y147 R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; Y147R + Q154R +Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y123H + Y147R + Q154R + I76 Y; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R.

[0073] In yet other embodiments, the adenosine deaminase variants each have the following modifications: One or more of the following variants: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R It is a homodimer containing two adenosine deaminase domains (e.g., TadA*8) In other embodiments, the adenosine deaminase variants are each selected from the following: two adenosine deaminase domains having a combination of modifications selected from the group consisting of: (e.g., TadA*8) are homodimers containing: Y147T + Q154R; Y147T + Q154S; Y147R + Q1 54S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; Y147R + Q154R +Y123H ; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y123H + Y147R + Q154R + I76Y; V82 S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R.

[0074] In other embodiments, the adenosine deaminase variant is a wild-type TadA adenosine deaminase variant. The deaminase domain and the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and adenosine deaminase variant domains containing one or more of Q154R and / or Q154R (e.g. In another embodiment, the adenosine deaminase The variant comprises a wild-type TadA adenosine deaminase domain and a variant selected from the group consisting of: adenosine deaminase variant domains (e.g., TadA) containing a combination of modifications to be *8) and heterodimers containing: Y147T + Q154R; Y147T + Q154S; Y147R + Q154S; V 82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; Y147R + Q154R +Y123H; Y147 R + Q154R + I76Y; Y147R + Q154R + T166R; Y123H + Y147R + Q154R + I76Y; V82S + Y1 23H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R.

[0075] In another embodiment, the adenosine deaminase variant comprises a TadA*7.10 domain and: one of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R adenosine deaminase variant domains containing TadA*8 or more (e.g., TadA*8) and heterodimers containing TadA*8 or more In another embodiment, the adenosine deaminase variant is TadA*7.1 0 domain and adenosine deaminase variant domains containing a combination of the following modifications: and heterodimers containing the following amino acids (e.g., TadA*8): Y147T + Q154R; Y147T + Q154S; Y147 R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; Y147R + Q154R +Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y123H + Y147R + Q154R + I76 Y; V82S + Y123H + Y147R + Q154R; or I76Y + V82S + Y123H + Y147R + Q154R.

[0076] In one embodiment, the adenosine deaminase has the following sequence or adenosine deaminase TadA*8 comprises or consists essentially of a fragment thereof having enzyme activity: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATL YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQK KAQSSTD.

[0077] In some embodiments, TadA*8 is truncated. , the truncated TadA*8 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 Lacking 4, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues. In terms of morphology, the truncated TadA*8 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , lacking 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues. In some embodiments, the adenosine deaminase variant is full-length TadA*8. be.

[0078] In certain embodiments, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from one of the following:

[0079] In certain embodiments, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from one of the following:

[0080] Escherichia coli TadA: MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLV MQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFF RMRRQEIKAQKKAQSSTD

[0081] E. coli TadA (N-terminal truncated): MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHEIMALRQGGLVMQNYRLIDATL YVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQK KAQSSTD

[0082] Staphylococcus aureus (S. aureus) TadA: MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTL YVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN

[0083] Bacillus subtilis (B. subtilis) TadA: MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTL EPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLS E

[0084] Salmonella typhimurium (S. typhimurium) TadA: MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLV LQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFF RMRRQEIKALKKADRAEGAGPAV

[0085] Shewanella putrefaciens (S. putrefaciens) TadA: MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEP CAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQ QGIE

[0086] Haemophilus influenzae F3031 (H. influenzae) TadA: MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTΑΗAEIIALRNGAKNIQNYR LNLSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRRE EKKIEKALLKSLSDK

[0087] Caulobacter crescentus (C. crescentus) TadA: MRTDESEDQDHRMMRLALDAARAAAAEGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIIAAMRAAAAKLGNYRLT DLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAK IN

[0088] Geobacter sulfurreducens (G. sulfurreducens) TadA: MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLT GATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRK KAKATPALFIDERKVPPEP

[0089] TadA*7.10 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQK KAQSSTD

[0090] An additional TadA7.10 or TadA7.10 is proposed as a component of the heterodimer with TadA*8. TadA7.10 variants include:

[0091] GSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIM ALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADEC AALLCYFFRMPRQVFNAQKKAQSSTD

[0092] TadA7.10 CP65 TAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITE GILADECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREV PVGAVLVLNNRVIGEGWNRAIGLHDP

[0093] TadA7.10 CP83 YRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMP RQVFNAQKKAQSSTDGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWN RAIGLHDPTAHAEIMALRQGGLVMQN

[0094] TadA7.10 CP136 MNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLA KRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI GRVVFGVRNAKTGAAGSLMDVLHYPG

[0095] TadA7.10 C-truncated GSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIM ALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADEC AALLCYFFRMPRQVFN

[0096] TadA7.10 C-truncated 2 GSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIM ALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADEC AALLCYFFRMPRQ

[0097] TadA7.10 delta59-66+C-truncated GSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNNRVIGEGWNRAHAEIMALRQGGLV MQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFF RMPRQVFN

[0098] TadA7.10 delta 59-66 GSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNNRVIGEGWNRAHAEIMALRQGGLV MQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFF RMPRQVFNAQKKAQSSTD.

[0099] In some embodiments, the adenosine deaminase variant is in TadA7.10. In some embodiments, TadA7.10 comprises a modification at amino acid 82 or 166. In certain embodiments, the variant of the reference sequence includes any of the following modifications: Includes one or more of: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R. In this state, the adenosine deaminase variants are Y147R + Q154R + Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y147T + Q154R; Y147T + Q154S; and Y123H + Y147R + Q154R + I76Y.

[0100] In other embodiments, the present invention provides adenosine deaminase variants comprising deletions, e.g. For example, C-terminal deletions beginning at residues 149, 150, 151, 152, 153, 154, 155, 156, or 157 In another embodiment, the adenosine deaminase variant TadA7.10 is provided. is a TadA monomer containing one or more of the following modifications: Y147T, Y147R, Q154S, Y12 3H, V82S, T166R, Q154R. In other embodiments, the adenosine deaminase variant is Monomers containing the following modifications: Y147R + Q154R + Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y147T + Q154R; Y147T + Q154S; and Y123H + Y147R + Q154R + I7 6Y. In yet other embodiments, the adenosine deaminase variants are each Two with one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, Q154R In another embodiment, the adenosine deaminase domain is a homodimer containing the adenosine deaminase domain. The adenosine deaminase variants are expressed in either the wild-type adenosine deaminase domain or the TadA7.10 domain. and an adenosine deaminase variant domain comprising one or more of the following modifications: It is a heterodimer containing: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R In another embodiment, the adenosine deaminase variant comprises a TadA7.10 domain and and a heterodimer containing a TadA7.10 adenosine deaminase variant containing the following modifications: Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y147T + Q154R; Y147T + Q154S; and Y123H + Y147R + Q154R + I76Y.

[0101] "Administering" refers to providing one or more compositions described herein to a patient or subject. By way of example and not limitation, administration of a composition For example, injections can be intravenous (iv), subcutaneous (sc), intradermal (id), or intraperitoneal (i. p.) injection or intramuscular (i.m.) injection. Using more than one such route. Parenteral administration can be, for example, by bolus injection or by gradual infusion over time. In some embodiments, parenteral administration can be achieved by infusing the Intraductal, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, intradermal, intraperitoneal, transtracheal, subcutaneous, subcorneal , including intra-articular, intracapsular, intrathecal and intrasternal infusion or injection; or Concurrently, administration can be by the oral route.

[0102] An "agent" is any small molecule compound, antibody, nucleic acid molecule, or polypeptide. , or fragments thereof.

[0103] "Alteration" refers to any alteration that can be detected by standard art known methods such as those described herein. Such a change in the structure, expression level or activity of a gene or polypeptide (e.g., an increase or As used herein, modification means a change in a polynucleotide or A change in the sequence of a polypeptide or a change in expression level, for example, a 10% change, a 25% change, This includes a 40% change, a 50% change, or a greater change in expression level.

[0104] "Ameliorate" means to reduce, inhibit, or attenuate the occurrence or progression of a disease. "To cause, reduce, stop, or stabilize" means to cause, reduce, stop, or stabilize.

[0105] "Analog" means a molecule that is not identical but has similar functional or structural characteristics. For example, a polynucleotide or polypeptide analog is a polynucleotide or polypeptide analog that is similar to the corresponding naturally occurring polynucleotide. A naturally occurring polynucleotide or polypeptide can be synthesized while retaining the biological activity of the nucleotide or polypeptide. or polypeptides, and have specific modifications that enhance the function of the analogs. Such modifications can improve the DNA affinity, efficiency, etc. of the analogs without altering, for example, ligand binding. affinity, specificity, protease or nuclease resistance, membrane permeability, and / or half-life Analogs can be used to increase the frequency of non-naturally occurring polynucleotides or amino acids. It may include.

[0106] A "base editor (BE)" or "nucleobase editor (NBE)" is a In various embodiments, the term "agent" refers to an agent that binds to an oxidase and has nucleobase modifying activity. Base editors include nucleobase-modifying polypeptides (e.g., deaminases) and nucleic acid promoters. The ramifiable nucleotide-binding domain is attached to a guide polynucleotide (e.g., a guide RNase A). In various embodiments, the agent comprises a protein having base editing activity. domains, i.e., bases (e.g., A, T, C, G, U) within a nucleic acid molecule (e.g., DNA), In some embodiments, the biomolecular complex comprises a domain capable of binding to a polypeptide. A nucleotide-programmable DNA-binding domain fused to a deaminase domain In one embodiment, the agent is a domain having base editing activity. In another embodiment, the fusion protein comprises a protein domain having base editing activity. The deaminase is ligated to the guide RNA (e.g., by ligating the RNA-binding motif and deaminase on the guide RNA). In some embodiments, the nucleic acid molecule has base editing activity (e.g., via an RNA-binding domain fused to an enzyme). In one embodiment, the domain is capable of deaminating a base within a nucleic acid molecule. The base editor can deaminate one or more bases in a DNA molecule. In , the base editor can deaminate adenosines (A) in DNA. In some embodiments, the base editor is an adenosine base editor (ABE).

[0107] "Cytidine deaminase" is a enzyme that converts amino groups into carbonyl groups through a deamination reaction. In some embodiments, the term "catalyzed polypeptide" refers to a polypeptide or fragment thereof that is capable of catalyzing In this case, the cytidine deaminase has at least about 85% identity to APOBEC or AID. In one embodiment, the cytidine deaminase converts cytosine to uracil and converts 5-methylcytosine to thymine. PmCDA1 from Petromyzon marinus (Petromyzon marinus cytosine deaminase 1, “PmCDA1”), or mammalian (e.g., human, porcine) AID (activation-induced cytidine deaminase; AICDA) derived from mammals such as cattle, horses, and monkeys, and APOBECs are exemplary cytidine deaminases.

[0108] In some embodiments, the base editor is a deaminase (e.g., adenosine Reprogrammable base editor fused to deaminase or cytidine deaminase In some embodiments, the base editor is a deaminase (e.g., Cas9 fused to adenosine deaminase or cytidine deaminase. In embodiments, the base editor is a deaminase (e.g., adenosine deaminase or nuclease-inactivated Cas9 (dCas9) fused to a cytidine deaminase (CdCas9). In some embodiments, the Cas9 is a circular permutant Cas9 (e.g., spCas9 or saCas9). Circularly permuted Cas9s are known in the art and are described, for example, in Oakes et al. et al., Cell 176, 254-267, 2019. In some embodiments, the salt Base editors are fused to inhibitors of base excision repair, e.g., UGI domains or dISN domains. In one embodiment, the fusion protein comprises a deaminase and a UGI domain or or a Cas9 nickase fused to an inhibitor of base excision repair, such as a dISN domain. In other embodiments, the base editor is an abasic base editor.

[0109] In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the adenosine deaminase is evolved from TadA. In some embodiments, the base editors of the invention are internally fused It contains a napDNAbp domain with a catalytic (e.g., deaminase) domain. In embodiments, napDNAbp comprises a Cas12a deaminase domain fused internally. In some embodiments, the napDNAbp is an internally fused deamidation In some embodiments, the napDNAbp is a Cas12b(c2c1) having a cleavage domain. is Cas12c(c2c3) with an internally fused deaminase domain. In one embodiment, napDNAbp comprises a Ca 2+ deaminase domain fused internally to the napDNAbp. In some embodiments, the napDNAbp is an internally fused deoxyribonucleotide. In some embodiments, napD is Cas12e (CasY) with an aminase domain. NAbp is a Cas12g with an internally fused deaminase domain. In an embodiment, napDNAbp is a Cas12h gene having an internally fused deaminase domain. In some embodiments, the napDNAbp is an internally fused deaminase In some embodiments, the base editor is a Cas12i having a deamidating domain. A catalytically dead Cas12 (dCas12) fused to a nase domain. In this study, the base editor is a Cas12 nickase (nCa) fused to a deaminase domain. s12).

[0110] In some embodiments, the base editor is an adenosine deaminase variant (e.g., TadA*8) with a circularly permuted Cas9 (e.g., spCAS9 or saCAS9) and a bipartite nuclear-localized Circular permutation is generated by cloning into a scaffold containing the targeting sequence (e.g., ABE8). The Cas9 enzyme is known in the art and is described, for example, in Oakes et al., Cell 176, 254-267, 2019. Exemplary circular permutations are described below, where the bolded sequences are derived from Cas9. The italicized sequences indicate linker sequences, and the underlined sequences indicate bipartite nuclear localization. The sequence is shown.

[0111] CP5 (with MSP “NGC=Pam Variant with mutations Regular Cas9 likes NGG” PID=Prot ein Interacting Domain and “D10A” nickase): JPEG2025143260000002.jpg170164

[0112] In some embodiments, ABE8 is a base editor from Tables 6-9, 13, or 14 below. In some embodiments, ABE8 is an adenosine triphosphate (ABT)-dependent ATPase (ATA ... In some embodiments, the adenosine deaminase variant of ABE8 is The amino acid sequence variant is a TadA*8 variant as set forth in Tables 7, 9, 13, or 14 below. In some embodiments, the adenosine deaminase variant is Y147T, Y14 7R, Q154S, Y123H, V82S, T166R, and / or Q154R. In various embodiments, the ABE is a TadA*7.10 variant (e.g., TadA*8) that includes the mutation. 8 is Y147T + Q154R; Y147T + Q154S; Y147R + Q154S; V82S + Q154S; V82S + Y147R; V8 2S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147 R; V82S + Y123H + Q154R; Y147R + Q154R +Y123H; Y147R + Q154R + I76Y; Y147R + Q15 4R + T166R; Y123H + Y147R + Q154R + I76Y; V82S + Y123H + Y147R + Q154R; and I7 6Y + V82S + Y123H + Y147R + Q154R In some embodiments, ABE8 is a monomeric construct and includes TadA*7.10 (e.g., TadA*8). In some embodiments, ABE8 is a heterodimeric construct. In the ABE8 base editor, the sequence includes: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATL YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQK KAQSSTD.

[0113] In some embodiments, the polynucleotide programmable DNA binding domain is CRISP In some embodiments, the base editor is a deamidating enzyme. In one embodiment, the Cas9 domain is a catalytically dead Cas9 (dCas9) fused to a cleavage domain. The base editor is a Cas9 nickase (nCas9) fused to a deaminase domain. In some embodiments, the base editor is fused to an inhibitor of base excision repair (BER). In some embodiments, the inhibitor of base excision repair is a uracil DNA glycosylase inhibitor. In some embodiments, the inhibitor of base excision repair is an inosine base excision repair inhibitor. It is a reversible inhibitor.

[0114] For more information on the base editor, see International PCT Application No. PCT / 2017 / 045381 (International Publication No. WO 2018 / 027078). ) and PCT / US 2016 / 058344 (International Publication No. 2017 / 070632), Komor, AC, et al., “Programmable editing ing of a target base in genomic DNA without double-stranded DNA cleavage” Natur e 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A ·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); K omor, AC, et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and produc t purity” Science Advances 3:eaao4774 (2017), and Rees, HA, et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 Dec;19(12):770-788. See also doi: 10.1038 / s41576-018-0059-1 (the entire contents of which are incorporated herein by reference).

[0115] By way of example, base editing compositions, systems, and methods described herein may be used. The cytidine base editor used is the following nucleic acid sequence (8877 base pairs) (Addgene, Watertown, MA) .; Komor AC, et al., 2017, Sci Adv., 30;3(8):eaao4774. doi: 10.1126 / sciadv.aao47 74) A polynucleotide having at least 95% identity to the BE4 nucleic acid sequence. Arrangements of the amino acid sequence are also included.

[0116] 1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gagctcagag 421 actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 481 gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 541 gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 601 aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 661 acctggttc tcagctggag cccatgcggc gaatgtagta gggccatcac tgaattcctg 721 tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 781 ccccgcaatc gacaaggcct gcgggatttg atctcttcag gtgtgactat ccaaattatg 841 actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 901 gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 961 atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 1021 ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 1081 gccaccgggt tgaaatctgg tggttcttct ggtggttcta gcggcagcga gactcccggg 1141 acctcagagt ccgccacacc cgaaagttct ggtggttctt ctggtggttc tgataaaaag 1201 tattctattg gtttagccat cggcactaat tccgttggat gggctgtcat aaccgatgaa 1261 tacaaagtac cttcaaagaa atttaaggtg ttgggggaaca cagaccgtca ttcgattaaa 1321 aagaatctta tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg 1381 aaacgaaccg ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa 1441 atttttagca atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc 1501 ttccttgtcg aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat 1561 gaggtggcat atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac 1621 tcaactgata aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc 1681 cgtgggcact ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg 1741 ttcatccagt tagtacaac ctataatcag ttgtttgaag agaaccctat aaatgcaagt 1801 ggcgtggatg cgaaggctat tcttagccc cgcctctcta atcccgacg gctagaaac 1861 ctgatcgcac aattacccgg agagagaaa aatggttgt tcggtaacct tatagcgctc 1921 tcactaggcc tgacaccaaa tttagtcg aacttcgact tagctgaga tgccaattg 1981 cagcttagta aggacacgta cgatgacgat ctcgacaatc tactggcaca attggagat 2041 cagtagcgg acttatttt ggctgccaa aaccttagcg atgcaatcct cctactcgac 2101 atactgagag ttatactga gattaccaag gcgccgtta ccgctcaat gatcaaagg 2161 tacgatgaac atcaccaga cttgacactt ctcaagccc tagtccgtca gcaactgcct 2221 gagaaata aggaaatatt cttgatcag tcgaaaaacg ggtacgcagg ttatattgac 2281 ggcggagcga gtcagagga attctacaag tttatcaac ccatattaga gagatggat 2341 gggacggaag agttgcttgt aaaaccaat cgcgagatc tactgcgaaa gcagcggact 2401 ttcgacacg gtagcattcc acatcaatc cacttaggcg aattgcatgc tatacttaga 2461 aggcaggagg atttttacc gttcccaaa gatacgtg aaagattga gaaaatccta 2521 acctttcgca taccttacta tgtgggaccc ctggcccgag ggaactcg gttcgcatgg 2581 atgacagaa agtccgaaga aacgattact ccatggaatt tgaggaagt tgtcgataaa 2641 gtgcgtcag ctcaatcgtt catcgagagg atgaccact ttgacagaa tttaccgaac 2701 gaaaagtat tgcctaagca cagtttactt tacgagtatt tcacagtgta caatgaactc 2761 acgaaagtta agtatgtcac tgaggcatg cgtaaacccg cctttctaag cggagacag 2821 aagaaagcaa tagtagatct gttattcaag accaccgca aagtgacagt tagcattg 2881 aagaggact actttaagaa attgaatgc tcgattctg tcgagatc cggggtagaa 2941 gatcgattta atgcgtcact tgtacgtat catgacctcc taagataat taagataag 3001 gacttcctgg ataacgaaga gatgagat atcttagaag atagtgtt gactcttacc 3061 ctctttgaag atcgggaat gattgaggaa agactaaaa catacgctca cctgttcgac 3121 gataagtta tgaacagtt aaagaggcgt cgctatacgg gctgggacg attgtcgcgg 3181 3241 agcgacggct tcgccaatag gaactttatg cagctgatcc atgatgactc tttaaccttc 3301 aaagaggata tacaaaaggc acaggtttcc ggacaagggg actcattgca cgaacatatt 3361 gcgaatcttg ctggttcgcc agccatcaaa aagggcatac tccagacagt caaagtagtg 3421 gatgagctag ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca 3481 cgcgaaaatc aaacgactca gaagggcaa aaaaacagtc gagagcggat gaagagaata 3541 gaagggta ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc 3601 3661 3721 tccttttga aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga 3781 gggaaaagtg acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg 3841 3901 aggggtggct tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc 3961 cgccaaatca caaagcatgt tgcacagata ctagattccc gaatgaatac gaaatacgac 4021 gagaacgata agctgattcg ggaagtcaaa gtaatcactt taaagtcaaa attggtgtcg 4081 gacttcagaa aggattttca attctataaa gttagggaga taaataacta ccaccatgcg 4141 cacgacgctt atcttaatgc cgtcgtaggg accgcactca ttaagaaata cccgaagcta 4201 gaaagtgagt ttgtgtatgg tgattacaaa gtttatgacg tccgtaagat gatcgcgaaa 4261 agcgaacagg agataggcaa ggctacagcc aatacttct tttattctaa cattatgaat 4321 ttctttaaga cggaaatcac tctggcaaac ggagagatac gcaaacgacc tttaattgaa 4381 accaatgggg agacaggtga aatcgtatgg gataagggcc gggacttcgc gacggtgaga 4441 aaagttttgt ccatgcccca agtcaacata gtaaagaaaa ctgaggtgca gaccggaggg 4501 tttcaaagg aatcgattct tccaaaagg aatagtgata agctcatcgc tcgtaaaaag 4561 gactgggacc cgaaaaagta cggtggcttc gatagcccta cagttgccta ttctgccta 4621 gtagtggcaa aagttgaga gggaaaatcc aagaaactga agtcagtcaa agaattattg 4681 gggataacga ttatggagcg ctcgtcttttt gaaaagaacc ccatcgactt ccttgaggcg 4741 aaaggttaca aggagtaa aaaggatctc aatattaac taccaaagta tagtctgttt 4801 gagttagaaa atggccgaaa acggatgttg gctagcgccg gagagcttca aaaggggaac 4861 gaactcgcac taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag 4921 ttgaaaggtt cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat 4981 tatctcgacg aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat 5041 gccaatctgg acaaagtatt aagcgcatac aacaagcaca gggataacc catacgtgag 5101 caggcggaa atattatcca tttgtttact cttaccaacc tcggcgctcc agccgcattc 5161 aagtattttg acacaacgat agatcgcaaa cgatacactt ctaccaagga ggtgctagac 5221 gcgacactga ttcaccaatc catcacggga ttattgaaa ctcggataga tttgtcacag 5281 cttggggggtg actctggtgg ttctggagga tctggtggtt ctactaatct gtcagatatt 5341 attgaaaagg agaccggtaa gcaactggtt atccaggaat ccatcctcat gctcccagag 5401 gaggtggaag aagtcattgg gaacaagccg gaaagcgata tactcgtgca caccgcctac 5461 gacgagagca ccgacgagaa tgtcatgctt ctgactagcg acgcccctga atacaagcct 5521 tgggctctgg tcatacagga tagcaacggt gagaacaaga ttaagatgct ctctggtggt 5581 tctggaggat ctggtggttc tactaatctg tcagatatta ttgaaaagga gaccggtaag 5641 caactggtta tccaggaatc catcctcatg ctcccagagg aggtggaaga agtcattggg 5701 aacaagccgg aaagcgatat actcgtgcac accgcctacg acgagagcac cgacgagaat 5761 gtcatgcttc tgactagcga cgcccctgaa tacaagcctt gggctctggt catacaggat 5821 agcaacggtg agaacaagat taagatgctc tctggtggtt ctcccaagaa gaagaggaaa 5881 gtctaaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg 5941 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 6001 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 6061 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 6121 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 6181 ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc 6241 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 6301 taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 6361 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 6421 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 6481 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 6541 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 6601 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 6661 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 6721 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 6781 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 6841 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 6901 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 6961 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 7021 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 7081 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 7141 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 7201 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 7261 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 7321 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 7381 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 7441 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 7501 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 7561 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 7621 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 7681 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 7741 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 7801 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 7861 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 7921 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 7981 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 8041 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 8101 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 8161 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 8221 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 8281 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 8341 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg 8401 gagatcgatc tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat 8461 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca 8521 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg 8581 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat 8641 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt 8701 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 8761 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 8821 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatc

[0117] BE4 amino acid sequence: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNT RCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYS PSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPPHILWATGLKSGGSSGGSSGS ETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE ATRLKRTARRRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRK KLVDSTDKADLRIYLALAMHIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSR RLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDLNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPIL EKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRER MKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMN TKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPID FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTK EVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILV HTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRK

[0118] By way of example, adenine dinucleotides used in the base editing compositions, systems, and methods described herein can be used. The Additive Base Editor (ABE) has the nucleic acid sequence (8877 base pairs) provided below (Addge ne, Watertown, MA.; Gaudelli NM, et al., Nature. 2017 Nov 23;551(7681):464-471. doi: 10.1038 / nature24644; Koblan LW, et al., Nat Biotechnol. 2018 Oct;36(9):8 43-846. doi: 10.1038 / nbt.4172.) At least 95% identity to the ABE nucleic acid sequence Also encompassed are polynucleotide sequences having the following structure:

[0119] ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT GACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGATTTCCAAGTCTCCACCCCATTG ACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCC ATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGT CAGATCCGCTAGAGATCCGCGGCCGCTAATACGACTCACTATAGGGAGAGCCGCCACCATGAAACGGACA GCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTCTCTGAAGTCGAGTTTAGCCACGAGT ATTGGATGAGGCACGCACTGACCCTGGCAAAGCGAGCATGGGATGAAAGAGAAGTCCCCGTGGGCGCCGT GCTGGTGCACAACAATAGAGTGATCGGAGAGGGATGGAACAGGCCAATCGGCCGCCACGACCCTACCGCA CACGCAGAGATCATGGCACTGAGGCAGGGAGGCCTGGTCATGCAGAATTACCGCCTGATCGATGCCACCC TGTATGTGACACTGGAGCCATGCGTGATGTGCGCAGGAGCAATGATCCACAGCAGGATCGGAAGAGTGGT GTTCGGAGCACGGGACGCCAAGACCGGCGCAGCAGGCTCCCTGATGGATGTGCTGCACCACCCCGGCATG AACCACCGGGTGGAGATCACAGAGGGAATCCTGGCAGACGAGTGCGCCGCCCTGCTGAGCGATTTCTTTA GAATGCGGAGACAGGAGATCAAGGCCCAGAAGAAGGCACAGAGCTCCACCGACTCTGGAGGATCTAGCGG AGGATCCTCTGGAAGCGAGACACCAGGCACAAGCGAGTCCGCCACACCAGAGAGCTCCGGCGGCTCCTCC GGAGGATCCTCTGAGGTGGAGTTTTCCCACGAGTACTGGATGAGACATGCCCTGACCCTGGCCAAGAGGG CACGCGATGAGAGGGAGGTGCCTGTGGGAGCCGTGCTGGTGCTGAACAATAGAGTGATCGGCGAGGGCTG GAACAGAGCCATCGGCCTGCACGACCCAACAGCCCATGCCGAAATTATGGCCCTGAGACAGGGCGGCCTG GTCATGCAGAACTACAGACTGATTGACGCCACCCTGTACGTGACATTCGAGCCTTGCGTGATGTGCGCCG GCGCCATGATCCACTCTAGGATCGGCCGCGTGGTGTTTGGCGTGAGGAACGCAAAAACCGGCGCCGCAGG CTCCCTGATGGACGTGCTGCACTACCCCGGCATGAATCACCGCGTCGAAATTACCGAGGGAATCCTGGCA GATGAATGTGCCGCCCTGCTGTGCTATTTCTTTCGGATGCCTAGACAGGTGTTCAATGCTCAGAAGAAGG CCCAGAGCTCCACCGACTCCGGAGGATCTAGCGGAGGCTCCTCTGGCTCTGAGACACCTGGCACAAGCGA GAGCGCAACACCTGAAAGCAGCGGGGGCAGCAGCGGGGGGTCAGACAAGAAGTACAGCATCGGCCTGGCC ATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGG TGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGA AACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGC TATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGT CCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGC CTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGAC CTGCGGCTGATCTATCTGGCGCTGGCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACC TGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGA GGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGA CGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGGAGAAAGAATGGCCTGTTCGGAAACCTGATTGCCC TGAGCCTGGGCCTGACCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAG CAAGGACACCTACGACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGGCCGACCTGTTTT CTGGCCGCCAAGAACCTGCTCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCA AGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGC TCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCC GGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGG ACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAA CGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTAC CCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCC CTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAA CTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAG AACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGC TGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGC CATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAG AAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACAT ACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGA AGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCC CACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCC GGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGG CTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAA GCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTA AGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGA GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGA ATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACA CCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGA ACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGAC TCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAG AGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTT CGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAG CTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACG ACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCG GAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAAC GCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACA AGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTT CTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGG CCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGC GGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAA AGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAG TACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGT CCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAA TCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAG TACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAA ACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGG CTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATC GAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCT ACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAA TCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAA GAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTC AGCTGGGAGGTGACTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAG GAAAGTCTAACCGGTCATCATCACCATCACCATTGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTT CTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGT GGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCT CTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGATACCGTCGACCTCTAGCTAGAGCTTGGCGTA ATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA AGCATAAAGTGTAAAGCCTAGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGC CCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGG TTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGA GCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCT CCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGAT ACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTC GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTA TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTA ACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTA CACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGC TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACACTCAGTGGAACGAAAACTC ACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTAC GATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCT CCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGT TGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCC CAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGA TCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTAC TGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGT ATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAA AAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGA GCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATAC TCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATG TATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGA TCGGGAGATCGATCTCCCGATCCCCTAGGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAA GCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAAC AAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGAT GTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCAT TAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCAT TGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC

[0120] "Base editing activity" refers to the ability to chemically modify bases within a polynucleotide. In one embodiment, the first base is converted to the second base. base editing activity is cytidine deaminase activity, e.g., the activity of converting the target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity. In another embodiment, the base editing activity is a cytogenetic activity, for example, the activity of converting A·T to G·C. Deaminase activity, e.g., converting the target C·G to T·A, resulting in adenosine or Adenine deaminase activity, for example, converting A·T to G·C. In this embodiment, base editing activity is evaluated by editing efficiency. Base editing efficiency can be measured using any suitable method. by suitable means, e.g., Sanger sequencing or next-generation sequencing. In some embodiments, the base editing efficiency can be measured by the base editor. The percentage of all sequencing reads with nucleobase conversions resulting from, e.g., G. by the percentage of all sequencing reads with the target AT base pair converted to a C base pair. In some embodiments, base editing efficiency is measured by determining the number of bases in a population of cells. Whole cells with nucleobase changes effected by base editors when editing occurs It is measured by the percentage of

[0121] The term "base editor system" refers to a system that edits nucleic acid bases in a target nucleotide sequence. In various embodiments, the base editor system comprises: (1) (2) a polynucleotide-programmable nucleotide-binding domain (e.g., Cas9); a deaminase domain for deaminating the nucleic acid base (e.g., adenosine deaminase) and (3) one or more guide polynucleases. In some embodiments, the polynucleotide primer comprises a nucleic acid sequence (e.g., a guide RNA). The programmable nucleotide binding domain is a polynucleotide programmable DNA In some embodiments, the base editor is an adenine or In some embodiments, the base editor system is an adenosine base editor (ABE). The stem is ABE8.

[0122] In some embodiments, the base editor system comprises two or more base editing components. For example, a base editor system can include multiple deaminases. In some embodiments, the base editor system comprises one or more adenosine deoxyribonucleotides. In some embodiments, a single guide polynucleotide may be utilized. can be used to target different deaminases to a target nucleic acid sequence. In some embodiments, a single pair of guide polynucleotides is utilized to identify different determinants. The enzyme can be targeted to a target nucleic acid sequence.

[0123] Deaminase domains of base editor systems and polynucleotide programmability The nucleotide-binding moieties may be covalently or non-covalently bound to one another or by any of the bonds. The molecules may be linked by any combination of binding and interaction. In the present invention, the deaminase domain is capable of forming programmable nucleotide bonds in polynucleotides. The target nucleotide sequence can be targeted by the binding domain. In this case, the polynucleotide programmable nucleotide binding domain is a deaminator. In some embodiments, the polynucleotide may be fused or linked to a nucleotide domain. The programmable nucleotide-binding domain binds non-covalently to the deaminase domain By selectively interacting or binding to the deaminase domain, the deaminase domain is directed to the target nucleotide sequence. For example, in some embodiments, Deamina The enzyme domain is one of the polynucleotide-programmable nucleotide binding domains. interacting with, associating with, or forming a complex with a further heterologous moiety or domain that is part of the In some embodiments, the polypeptide may contain additional heterologous moieties or domains. In the form, the additional heterologous moiety binds to, interacts with, associates with, or In some embodiments, the additional heterologous moiety can form a complex with a poly It is capable of binding to, interacting with, associating with, or forming a complex with a nucleotide. In some embodiments, the additional heterologous moiety is capable of binding to the guide polynucleotide. In some embodiments, the additional heterologous moiety is attached to the polypeptide linker. In some embodiments, the additional heterologous moiety can be a polynucleotide. The additional heterologous moiety can be a protein domain or a In some embodiments, the additional heterologous moiety is a K homology (KH) domain, an MS2 domain, or a nucleotide sequence. coat protein domain, PP7 coat protein domain, SfMu Com coat protein domain In, steryl α motif, telomerase Ku binding motif and Ku protein, telomerase The motif may be a Sm7-binding motif and an Sm7 protein, or an RNA recognition motif.

[0124] The base editor system can further comprise a guide polynucleotide component. The components of the base editor system may be linked by covalent bonds, non-covalent interactions, or both. It should be understood that the compounds may be coupled to one another via any combination of bonds and interactions. In some embodiments, the deaminase domain is selected from the group consisting of: It can be targeted to a target nucleotide sequence. For example, in some embodiments The deaminase domain may be a portion or segment of the guide polynucleotide (e.g., a polynucleotide Further variants that can interact, bind, or form complexes with the nucleotide motif A species moiety or domain (e.g., a polynucleotide binding protein, such as an RNA or DNA binding protein) In some embodiments, additional heterologous moieties or domains ( Polynucleotide-binding domains, e.g., RNA- or DNA-binding proteins, are deaminants of In some embodiments, the additional heterologous moiety may be fused or linked to the enzyme domain. The polypeptide binds to, interacts with, associates with, or forms a complex with the polypeptide. In some embodiments, the additional heterologous moiety can be a polynucleotide. capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide In some embodiments, the additional heterologous moiety is attached to the guide polynucleotide. In some embodiments, the additional heterologous moiety can be a polypeptide linker. In some embodiments, the additional heterologous moiety can be linked to a polynucleotide. The additional heterologous moiety can be a protein domain or a nucleotide linker. In some embodiments, the additional heterologous moiety may be a K homology (KH) domain, MS2 Coat protein domain, PP7 coat protein domain, SfMu Com coat protein domain, sterile alpha motif, telomerase Ku binding motif and Ku protein, Te It may be a chromosome enzyme Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0125] In one embodiment, the base editor system comprises an inhibitor of base excision repair (BER). The components of the base editor system can further include a shared Covalent bonds, non-covalent interactions, or any combination of these bonds and interactions. It should be understood that the BER components can be linked to each other via a BER inhibitor. In one embodiment, the inhibitor of BER is uracil DNA glycosylase In one embodiment, the inhibitor of BER can be an inosine BER inhibitor (UGI). In one embodiment, the inhibitor of BER is a polynucleotide protease inhibitor. Targeting to target nucleotide sequences via a rammable nucleotide-binding domain In some embodiments, the polynucleotide may be a programmable nucleotide. The binding domain may be fused or linked to an inhibitor of BER. The programmable nucleotide-binding domain is a deaminase domain. In some embodiments, the polynucleotides may be fused or linked to an inhibitor of BER. Nucleotide-programmable nucleotide-binding domains are not covalent inhibitors of BER. BER inhibitors by covalently interacting with or associating with BER inhibitors. The molecule can be targeted to a target nucleotide sequence. In embodiments, the inhibitor of the BER component is a polynucleotide programmable nucleotide. interacting with, associating with, or having associated with further heterologous moieties or domains that are part of the octide-binding domain or may contain further heterologous moieties or domains capable of complexing.

[0126] In one embodiment, the inhibitor of BER is a nucleotide sequence that is mediated by a guide polynucleotide to target nucleotides. For example, in some embodiments, the inhibitor may be targeted to a nucleotide sequence that inhibits BER. The deleterious agent may be a portion or segment of a guide polynucleotide (e.g., a polynucleotide Further heterologous moieties or domains (e.g., nucleotides) that can interact with, associate with, or complex with the nucleotide motif (e.g., nucleotides) For example, a polynucleotide binding domain such as an RNA or DNA binding protein. In some embodiments, the guide polynucleotide may further comprise a heterologous moiety or a The main (e.g., polynucleotide-binding domain, such as an RNA- or DNA-binding protein) In some embodiments, the additional heterologous nucleotide may be fused or linked to an inhibitor of BER. The moiety is capable of binding, interacting, associating, or complexing with a polynucleotide In some embodiments, the additional heterologous moiety is attached to the guide polynucleotide. In some embodiments, the additional heterologous moiety can be a polypeptide linker. In some embodiments, the additional heterologous moiety can be linked to a polynucleotide. The additional heterologous moiety can be a protein domain or a nucleotide linker. In some embodiments, the additional heterologous moiety may be a K homology (KH) domain, MS2 Coat protein domain, PP7 coat protein domain, SfMu Com coat protein domain, sterile alpha motif, telomerase Ku binding motif and Ku protein, Te It may be a chromosome enzyme Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0127] The term "Cas9" or "Cas9 domain" refers to a Cas9 protein or a fragment thereof (e.g., Cas9 Active, inactive, or partially active DNA cleavage domains of Cas9 and / or gRNA binding of Cas9 Cas9 nuclease refers to an RNA-guided nuclease containing a c domain. asnl nuclease or CRISPR (clustered regularly interspaced short palindromic CRISPR is also known as a repeat-associated nuclease. The adaptive immune system provides defense against transposable elements (transposable elements, conjugative plasmids). CRISPR cluster CRIS comprises a spacer, a sequence complementary to the preceding mobile element, and a target invader nucleic acid. The PR cluster is transcribed and processed into CRISPR RNA (crRNA). In the endothelial cell line, the correct processing of pre-crRNA is mediated by a small trans-coding RNA (tracrRNA), an endogenous RNA. tracrRNA requires ribonuclease 3 (rnc) and Cas9 protein. This guides the processing of pre-crRNA by Cas9 / crRNA / tracrRNA. A endonucleolytically cleaves a linear or circular dsDNA target complementary to the spacer. The target strand that is not complementary to the crRNA is first endonucleolytically cleaved and then exonucleolytically cleaved. In nature, DNA binding and cleavage are carried out by proteins. However, both crRNA and tracrRNA aspects are typically required. A single guide RNA ("sgRNA," or simply "gRNA") is engineered to incorporate into a single RNA species. See, for example, Jinek M. et al., Science 337:816-821 (2012). Cas9 is a CRISPR repeat sequence. It recognizes a short motif (PAM or protospacer adjacent motif) in the spacer to distinguish self from non-self. The sequence and structure of Cas9 nuclease are well known to those skilled in the art. (e.g., "Complete genome sequence of an M1 strain of Streptococcus pyog" Ferretti et al., Proc. Natl. Acad. Sci. USA 98:4658-4663(2001); SPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltc heva E. et al., Nature 471:602-607(2011); and “A programmable dual-RNA-guide d DNA endonuclease in adaptive bacterial immunity.” Jinek M. et al., Science 33 7:816-821 (2012), the entire contents of which are incorporated herein by reference. Orthologs include, but are not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences are described in the present disclosure. Such Cas9 nucleases and sequences will be apparent to those skilled in the art based on the disclosures of Chylinsk i, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas Organisms and genes disclosed in “immunity systems” (2013) RNA Biology 10:5, 726-737 The Cas9 sequences from the locus are included, the entire contents of which are incorporated herein by reference.

[0128] An exemplary Cas9 is Streptococcus pyogenes Cas9 (spCas9), the amino acid sequence of which is shown below. The following information is provided. JPEG2025143260000003.jpg166165 (single underline: HNH domain; double underline: RuvC domain)

[0129] The nuclease-inactivated Cas9 protein is interchangeably referred to as the “dCas9” protein (nuclease-“ It may also be referred to as "dead" Cas9 or catalytically inactive Cas9. Methods for generating a Cas9 protein (or a fragment thereof) having the following structure are known (see, e.g., Jinek et al. al, Science. 337:816-821(2012); Qi et al, “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression”(2013) Cell. 28; 152 (5): 1173-83, the contents of each of which are incorporated herein by reference. For example, the DN of Cas9 The A cleavage domain is composed of two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA and binds to RuvC1. The subdomains cleave the non-complementary strand. Mutations within these subdomains allow Cas9 to For example, mutations D10A and H840A can inhibit the nuclease activity of S. pyogenes Cas9. completely inactivates the ATPase activity (Jinek et al., Science. 337:816-821(2012); Qi et al. l, Cell. 28;152(5): 1173-83 (2013)). In some embodiments, the Cas9 nuclease is inactivated. The Cas9 has an active (e.g., inactivated) DNA cleavage domain, i.e., the Cas9 is referred to as an "nCas9" protein. The nickase is called Cas9 (meaning "nickase"). In some embodiments, Cas9 For example, in some embodiments, proteins comprising fragments of the protein The gene contains one of two Cas9 domains: (1) the gRNA-binding domain of Cas9; (2) the C In some embodiments, a protein comprising Cas9 or a fragment thereof is Cas9 variants are those that share homology with Cas9 or its fragments. For example, a Cas9 variant may have at least about 70% identity to wild-type Cas9, at least about 80% identity to wild-type Cas9, or at least about 80% identity to wild-type Cas9. 0% identity, at least about 90% identity, at least about 95% identity, at least about 96% Identity, at least about 97% identity, at least about 98% identity, at least about 99% identity have at least about 99.5% identity, or at least about 99.9% identity. In some embodiments, the Cas9 mutant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 2 9, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 4 In some embodiments, the Cas9 vector may have 9, 50, or more amino acid changes. The variant contains a fragment of Cas9 (e.g., the gRNA binding domain or the DNA cleavage domain) and The fragments have at least about 70% identity to the corresponding fragment of wild-type Cas9, and at least about 80% identity to the corresponding fragment of wild-type Cas9. has at least about 90% identity, has at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity have at least about 99% identity, have at least about 99.5% identity, and In some embodiments, the fragment has at least about 99.9% identity to the corresponding wild-type Cas At least 30%, at least 35%, at least 40%, at least 45%, at least 9 amino acids in length at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least At least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, is 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% .

[0130] In some embodiments, the fragment is at least 100 amino acids in length. In the above, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 60 0, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or It is at least 1300 amino acids in length.

[0131] In one embodiment, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes. (NCBI reference sequence: NC_017053.1, nucleotide and amino acid sequences are as follows): ATGGATAAGAAATACTCAATGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGATTATAAGGT TCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTG GCAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGT TATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGT GGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAA CTATCTATCATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCAT ATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCA GTTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTG CACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAGAAATGGCTTGTTTGGGAAT CTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTC AAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTA AGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCA ATGATTAAGCGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTA TAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATA AATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGC AAGCAACGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGA AGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTC CATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAA GTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGT ACTACCAAAACATAGTTTGCTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAGGGAA TGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACC GTTAAGCAATTAAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATT TAATGCTTCATTAGGCGCCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAG ATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGGGATGATTGAGGAAAGACTTAAAACATATGCT CACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGAT TAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTA TGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGATATTCAAAAAGCACAGGTGTCTGGACAAGGCCATAGTTTA CATGAACAGATTGCTAACTTAGCTGGCAGTCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAATTGTTGATGAACT GGTCAAAGTAATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGA AAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTT GAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTACAAAATGGAAGAGACATGTATGTGGACCAAGAATT AGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGACGATTCAATAGACAATA AGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAAC TATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTT GAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTT TGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAA TTAGTTTCTGACTTCCGAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTA TCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAG TTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAAT ATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGA AACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTG TCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAGAAATTCGGACAAGCTTATTGCT CGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAA GGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTG AAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATAT AGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCT GCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAA AACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATT TTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAA TATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAAC GATATACGTCTACAAAAGAAGTTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGAT TTGAGTCAGCTAGGAGGTGACTGA JPEG2025143260000004.jpg168163 (single underline: HNH domain; double underline: RuvC domain)

[0132] In one embodiment, the wild-type Cas9 has the following nucleotide and / or amino acid sequence: Corresponding to or containing columns: ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGT ACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAAGAATCTTATCGGTGCCCTCCTATTCG ATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGTCCGGAGAAGGTATACACGTCGCAAGAACCGAATATGT TACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGT CGAAGAGGACAAAACATGAACGGCACCCCATCTTTGGAAACATAGGATGAGGTGGCATATCATGAAAAGTACCCAA CGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCAT ATGATAAAGTTCCGTGGGCACTTTCCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCA GTTAGTACAAACCTATAATCAGTTGTTTGAAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCG CCCGCCTCTCTAAATCCCGACGGCTAGAAAAACCTGATCGCACAATTACCCGGAGAGAAAAAAATGGTTGTTCGGTAAC CTTATAGCGCTCTCACTAGGCCTGACACCAAATTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAG TAAGGACACGTACGATGACGATCTCGCAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCA AAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCCGCTTCA ATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATA TAAGGAAATATTCTTTGATCAGTCGAAAACGGGTACGCAGGTTATATTGACGGGCGGAGCGAGTCAAGAGGAATTCTACA AGTTTATCAAACCCATATTAGAGAATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGA AAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGA GGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTACTATGTGGGAC CCCTGGCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAA GTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCACTTTGACAAGAATTTACCGAACGAAAAAGT ATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCA TGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACA GTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATT TAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAG ATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCT CACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTAT CAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTA TGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTG CACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCT AGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGC AAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCT GTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGA ACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACA ATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAG AACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGG CTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGA TACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCA AATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGC TTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACA AAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCT AACATTATGAATTTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGG GGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACA TAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATC GCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGC AAAAGTTGAGAAGGGAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTT TTGAAAAGACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAG TATAGTCTGTTTGAGTTAGAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCCAAAGGGGAACGAACTCGC ACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAAC AGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTC ATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGA AAATATTATCCATTTGTTTACTCTTCCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCA AACGATACACTTCTACCAAGGAGGTGCTAGACCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATA GATTTGTCACAGCTTGGGGGTGACGGATCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGA TTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCTGCAGGA JPEG2025143260000005.jpg173168 (single underline: HNH domain; double underline: RuvC domain)

[0133] In some embodiments, the wild-type Cas9 is Cas9 from Streptococcus pyogenes (NC BI reference sequence: NC_002737.2 (nucleotide sequence below) and Uniprot reference sequence: Q99ZW2 (nucleotide sequence below) The amino acid sequence corresponds to the following: ATGGATAAGAAATACTCAATGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGT TCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTG ACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGT TATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGT GGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAA CTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCAT ATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCA GTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTG CACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAAT CTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTC AAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTA AGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCA ATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTA TAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATA AATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGC AAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGA AGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTC CATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAA GTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGT ACTACCAAAACATAGTTTGCTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAA TGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACC GTTAAGCAATTAAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATT TAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAG ATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCT CACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGAT TAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTA TGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTA CATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATT GGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCC AGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCT GTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGA ATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACA ATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAA AACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGG TTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAA TTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCT AATTAGTTTCTGACTTCCGAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGC GTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATA AAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCT AATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGG GGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATA TTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTTACCAAAAGAAATTCGGACAAGCTTATT GCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGC TAAGGTGGAAAGGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCT TTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAGACTTAATCATTAAACTACCTAAA TATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAGGGAAATGAGCTGGC TCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAAC AAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTT ATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGA AAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTA AACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATT GATTTGAGTCAGCTAGGAGGTGACTGA JPEG2025143260000006.jpg170167(SEQ ID NO: 1) (Single underline: HNH domain; double underline: RuvC domain)

[0134] In certain embodiments, Cas9 is from Corynebacterium ulcerans (NCBI Refs: NC_015683.1 , NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1) ; Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcu s iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psyc hroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref : YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni ( NCBI Ref: YP_002344900.1) or Neisseria meningitidis (NCBI Ref: YP_002342100 .1), or Cas9 from any other organism.

[0135] In some embodiments, dCas9 contains one or more nucleotides that inactivate Cas9 nuclease activity. It corresponds to, or contains part or all of, a mutated Cas9 amino acid sequence. For example, in some embodiments, the dCas9 domain contains the D10A and H840A mutations or another Ca mutation. In some embodiments, the dCas9 comprises a corresponding mutation in dCas9 (D10A and and H840A) contains the amino acid sequence: JPEG2025143260000007.jpg171167 (single underline: HNH domain; double underline: RuvC domain)

[0136] In some embodiments, the Cas9 domain comprises a D10A mutation, while the the residue at position 840 in the amino acid sequence provided herein, or The residue at the corresponding position in either sequence remains a histidine.

[0137] In other embodiments, D10A, e.g., resulting in nuclease-inactivated Cas9 (dCas9), and dCas9 variants having mutations other than H840A. For example, other amino acid substitutions at D10 and H840, or the nuclease domain of Cas9 may be used. Other substitutions within the domain (e.g., HNH nuclease subdomain and / or RuvC1 subdomain) In some embodiments, a variant or homolog of dCas9 is at least about 70% identity, at least about 80% identity, at least about 90% identity , at least about 95% identity, at least about 98% identity, at least about 99% identity, Those having at least about 99.5% identity, or at least about 99.9% identity are provided. In some embodiments, the amino acid sequence is about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, or Amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids Variants of dCas9 having amino acid sequences shorter or longer than 1000 or 10000 are provided. do.

[0138] In some embodiments, the Cas9 fusion proteins provided herein comprise a Cas9 protein. The full-length amino acid sequence of the protein, for example, one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein contain a full-length Cas9 sequence. Examples of Suitable Cas9 Domains and Cas9 Fragments Suitable amino acid sequences are provided herein, and further suitable sequences for Cas9 domains and fragments are , as will be apparent to those skilled in the art.

[0139] Additional Cas9 proteins (e.g., nuclease-dead Cas9 (dCas9), Cas9 nCas9, or nuclease-active Cas9, its variants and homologs It should be understood that within the scope of this disclosure are any and all Cas9 proteins, including: In some embodiments, Cas9 The protein is a nuclease-inactive Cas9 (dCas9). In some embodiments, the Cas9 protein The protein is Cas9 nickase (nCas9). In some embodiments, the Cas9 protein The quality is nuclease-active Cas9.

[0140] Exemplary catalytically inactive Cas9 (dCas9): DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHM IKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRIEMIEERLKTYAH LFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEGIGELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKN YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSN IMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA RKKDWDPKKYGGFDSPTVAYSVLVVAKVEGKKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID LSQLGGD

[0141] Exemplary catalyst of Cas9ニッカーゼ (nCas9): DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHM IKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEEV VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRIEMIEERLKTYAH LFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEGIGELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKN YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSN IMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA RKKDWDPKKYGGFDSPTVAYSVLVVAKVEGKKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID LSQLGGD

[0142] Exemplary catalyst activityCas9: DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHM IKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRIEMIEERLKTYAH LFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEGIGELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKN YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSN IMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID LSQLGGD.

[0143] In one embodiment, Cas9 is directed against archaeal organisms that comprise the domain and kingdom of unicellular prokaryotic microorganisms. In one embodiment, the Cas9 protein is derived from, for example, a fungus (e.g., nanoarchaea). , Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res . 2017 Feb 21. doi: 10.1038 / cr.2017.21 refers to CasX or CasY, and The entire contents of which are incorporated herein by reference. Many CRISPR-Cas systems, including Cas9, which was first reported in the archaeal domain of life, have been identified. This branched Cas9 protein was identified in the little-studied nanoarchaea. , discovered as part of an active CRISPR-Cas system, previously unknown in bacteria. Two systems, CRISPR-CasX and CRISPR-CasY, have been discovered that are the most efficient known to date. In some embodiments, Cas9 is a CasX or CasX-like protein. In some embodiments, Cas9 represents a variant of CasY or a variant of CasY. It represents nucleic acid programmable DNA binding protein (napDNAbp). Other RNA-guided DNA binding proteins may also be used, such as RNA-guided DNA binding proteins (RNA-guided DNA binding proteins), as disclosed herein. It should be understood that the range is

[0144] In certain embodiments, napDNAbp useful in the methods of the invention contain circular substitutions. , which is known and described, for example, in Oakes et al., Cell 176, 254-267, 2019 Below are exemplary circular permutations, where bolded sequences indicate Cas9-derived sequences and italicized sequences: The sequence represents the linker sequence and the underlined sequence represents the bipartite nuclear localization sequence. CP5 (with MSP “NGC=Pam Variant with mutations Regular Cas9 likes NGG” PID=Prot ein Interacting Domain and “D10A” nickase): JPEG2025143260000008.jpg171166

[0145] Polynucleotide programmable nucleotides that can be incorporated into base editors Non-limiting examples of binding domains include domains derived from CRISPR proteins, restriction nucleases, and the like. Enzymes, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases Examples include ZFNs.

[0146] In some embodiments, the nucleic acid of any of the fusion proteins provided herein The programmable DNA binding protein (napDNAbp) can be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. is at least 85%, at least 90%, or At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% In some embodiments, the napDNAbp comprises a naturally occurring amino acid sequence having identity to the napDNAbp. In some embodiments, the napDNAbp is a CasX or CasY protein present. At least 85%, at least at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least Contains amino acid sequences with at least 99.5% identity to Cas12b / C2c1 and CasX from other bacterial species. It should be understood that CasY and CasC may also be used in accordance with the present disclosure.

[0147] Cas12b / C2c1 (uniprot.org / uniprot / T0D7A2#2) sp|T0D7A2|C2C1_ALIAG CRISPR-associated endo- nuclease C2c1 OS = Alicyclobacillus acido- terrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) GN=c2c1 PE=1 SV=1 MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECDKTAEECKAELLERLRARQ VENGHRGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWE EEKEKAETRKSADRTADVLRALADFGLKPLMRVYTDSEMSSVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQ EYAKLVEQKNRFEQKNFVGQEHLVHLVNQLQQDMKEASPGLESKEQTAHYVTGRALRGSDKVFEKWGKLAPDAPFDLYDA EIKNVQRRNTRRFGSHDLFAKLAEPEYQALWREDASFLTRYAVYNSILRKLNHAKMFATFTLPDATAHPIWTRFDKLGGN LHQYTFLFNEFGERRHAIRFHKLLKVENGVAREVDDVTVPISMSEQLDNLLPRDPNEPIALYFRDYGAEQHFTGEFGGAK IQCRRDQLAHMHRRRGARDVYLNVSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGL LSGLRVMSVDLGLRTSASISVFRVARKDELKPNSKGRVPFFFPIKGNDNLVAVHERSQLLKLPGETESKDLRAIREERQR TLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPVDAANHMTPDWREAFENELQKLKSLHGICSDKEWMDAVYESVR RVWRHMGKQVRDWRKDVRSGERPKIRGYAKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREH IDHAKEDRLKKLADRIIMEALGYVYALDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELI NQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCTQEHNPEPFPWWLNKFVVEHTLDACPLRADDLIPTGEGEIF VSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQIRLRCDWGEVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYE RERGKKRRKVFAQEKLSEEEAELLVEADEAREKSVVLMRDPSGIINRGNWTRQKEFWSMV NQRIEGYLVKQIRSRVPLQ DSACENTGDI

[0148] CasX (uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53) >tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx protein OS = Sulfolobus islandicu s (strain HVE10 / 4) GN = SiH_0402 PE=4 SV=1 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIIL PLSNVKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAA AGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAV GQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSSSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNL NSDDGKVRDLKLISAYVNGELIRGEG

[0149] >tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS = Sulfolobus islandic us (strain REY15A) GN=SiRe_0771 PE=4 SV=1 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIIL PLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAA AGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAV GQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLN SDDGKVRDLKLISAYVNGELIRGEG

[0150] Deltaproteobacteria CasX MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAIL QVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVA EHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFL SKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAK PLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPK KPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMD EKEFYACEIQLQKWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTD GTDIKKSGKWQGLLYGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIG RDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQA AKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKL AYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELS AELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYK SGKQPFVGAWQAFYKRRLKEVWKPNA

[0151] CasY (ncbi.nlm.nih.gov / protein / APG80656.1) >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacte rium] MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKYPLYSSPSGGRTVPREIVSAINDDYVGLYGLSNFDDLYNAEKRNEE KVYSVLDFWYDCVQYGAVFSYTAPGLLKNVAEVRGGSYELTKTLKGSHLYDELQIDKVIKFLNKKEISRANGSLDKLKKD IIDCFKAEYRERHKDQCNKLADDIKNAKKDAGASLGERQKKLFRDFFGISEQSENDKPSFTNPLNLTCCLLPFDTVNNNR NRGEVLFNKLKEYAQKLDKNEGSLEMWEYIGIGNSGTAFSNFLGEGFLGRLRENKITELKKAMMDITDAWRGQEQEEELE KRLRILAALTIKLREPKFDNHWGGYRSDINGKLSSWLQNYINQTVKIKEDLKGHKKDLKKAKEMINRFGESDTKEEAVVS SLLESIEKIVPDDSADDEKPDIPAIAIYRRFLSDGRLTLNRFVQREDVQEALIKERLEAEKKKKPKKRKKKSDAEDEKET IDFKELFPHLAKPLKLVPNFYGDSKRELYKKYKNAAIYTDALWKAVEKIYKSAFSSSLKNSFFDTDFDKDFFIKRLQKIF SVYRRFNTDKWKPIVKNSFAPYCDIVSLAENEVLYKPKQSRSRKSAAIDKNRVRLPSTENIAKAGIALARELSVAGFDWK DLLKKEEHEEYIDLIELHKTALALLLAVTETQLDISALDFVENGTVKDFMKTRDGNLVLEGRFLEMFSQSIVFSELRGLA GLMSRKEFITRSAIQTMNGKQAELLYIPHEFQSAKITTPKEMSRAFLDLAPAEFATSLEPESLSEKSLLKLKQMRYYPHY FGYELTRTGQGIDGGVAENALRLEKSPVKKREIKCKQYKTLGRGQNKIVLYVRSSYYQTQFLEWFLHRPKNVQTDVAVSG SFLIDEKKVKTRWNYDALTVALEPVSGSERVFVSQPFTIFPEKSAEEEGQRYLGIDIGEYGIAYTALEITGDSAKILDQN FISDPQLKTLREEVKGLKLDQRRGTFAMPSTKIARIRESLVHSLRNRIHHLALKHKAKIVYELEVSRFEEGKQKIKKVYA TLKKADVYSEIDADKNLQTTVWGKLAVASEISASYTSQFCGACKKLWRAEMQVDETITTQELIGTVRVIKGGTLIDAIKD FMRPPIFDENDTPFPKYRDFCDKHHISKKMRGNSCLFICPFCRANADADIQASQTIALLRYVKEEKKVEDYFERFRKLKN IKVLGQMKKI

[0152] The term "Cas12" or "Cas12 domain" refers to the Cas12 protein or a fragment thereof (e.g., For example, an active, inactive, or partially active DNA cleavage domain of Cas12 and / or Cas Cas12 refers to an RNA-guided nuclease containing a gene encoding ... Cas12 nuclease belongs to the type V CRISPR / Cas system. d) regularly interspaced short palindromic repeat (REG)-associated nucleases An exemplary Bacillus hisashii Cas 12b (BhCas12b) Cas 12 domain sequence is shown below: Provided to. MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAE IQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRW YNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQAL ERFLSWESWNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWL KMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPIN HPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKH AFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKE LTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGET LVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWV AFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHL NALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGE IYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDR KCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIK KGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSS KQSMKRPAATKKAGQAKKKK.

[0153] An amino acid sequence having at least 85% identity to the BhCas12b amino acid sequence is also included. Also useful in the methods of the present invention.

[0154] "Cytidine deaminase" is a enzyme that converts amino groups into carbonyl groups through a deamination reaction. In one embodiment, the term "catalyzed polypeptide" refers to a polypeptide or fragment thereof that is capable of catalyzing , cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine PmCDA1 (Petromyzon marinus cytosine deaminase) from Petromyzon marinus 1, "PmCDA1"), or A derived from a mammal (e.g., human, pig, cow, horse, monkey, etc.) ID (activation-induced cytidine deaminase; AICDA), and APOBEC are exemplary cytidine deaminase It is minase.

[0155] The term "conservative amino acid substitution" or "conservative variation" refers to a mutation in which an amino acid is replaced by another amino acid that shares a common characteristic. It refers to the substitution of an amino acid with another amino acid that has the same properties. It defines the common properties between individual amino acids. A functional method for determining the normality of amino acid changes between corresponding proteins of homologous organisms is The goal is to analyze the frequency of the results (Schulz, GE and Schirmer, RH, Principles of f Protein Structure, Springer-Verlag, New York (1979). According to such analysis, Amino acids within a group are preferentially exchanged with each other, thus affecting the overall protein structure. Define the group of amino acids that are most similar to each other in their effect on the (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include: For example, the amino acids arginine to lysine and the like that can maintain a positive charge are used. Reverse; aspartic acid to glutamic acid and vice versa, which can maintain a negative charge; free threonine to serine, which maintains the -OH; and asparagine to asparagine, which maintains the free NH Examples include amino acid substitutions such as glutamine.

[0156] The terms "coding sequence" or "protein-coding sequence" are used interchangeably herein. " refers to a segment of a polynucleotide that encodes a protein. The sequence is bounded by a start codon near the 5' end and a stop codon near the 3' end. The coding sequence is also called an open reading frame.

[0157] As used herein, the terms "deaminase" or "deaminase domain" and "deaminase domain" refer to refers to a protein or enzyme that catalyzes a deamination reaction. The enzyme is an adenosine dehydrogenase that catalyzes the hydrolytic deamination of adenine to hypoxanthine. In some embodiments, the deaminase is an adenosine or adenine (A) deaminase. adenosine deaminase, which catalyzes the hydrolytic deamination of α- and β-inosine(I) In some embodiments, the deaminase or deaminase domain is an adenosine deaminase. enzymes that convert adenosine or deoxyinosine to inosine or deoxyinosine, respectively. In some embodiments, adenosine deamination This enzyme catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., genetically engineered adenosine deaminases) aminase, evolved adenosine deaminase) can be derived from any organism, including bacteria. In some embodiments, the adenosine deaminase is obtained from Escherichia coli, Staphylococcus aureus, us aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae ae, or Caulobacter crescentus.

[0158] In one embodiment, the adenosine deaminase is TadA deaminase. In some embodiments, the TadA deaminase is a TadA variant. The dA variant is TadA*8. In one embodiment, the deaminase or deaminase The domains may be, for example, human, chimpanzee, gorilla, monkey, cow, dog, rat, or mammal. In some embodiments, the deaminase is a variant of a naturally occurring deaminase from an organism such as a mouse. The enzyme or deaminase domain is non-naturally occurring. For example, in some embodiments In this embodiment, the deaminase or deaminase domain is At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least At least 92%, at least 93%, at least, at least 94%, at least 95%, at least 96%, At least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, At least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99 0.7%, at least 99.8%, or at least 99.9% identity. For example, Zedaima is registered in international PCT application numbers PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 0583 44 (WO2017 / 070632), each of which is incorporated herein by reference in its entirety. Komor, AC, et al., “Programmable editing of a target base n genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage”Nature 551, 464-471 (2017); Komor, AC, et al., “Im proved base excision repair inhibition and bacteriophage Mu Gam protein yields C :G-to-T:A base editors with higher efficiency and product purity”Science Advanc es 3:eaao4774 (2017) and Rees, HA, et al., “Base editing: precision chemist ry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 Dec; 19(12):770-788. See also doi: 10.1038 / s41576-018-0059-1 (the entire contents of which are hereby incorporated by reference). (Incorporated into the specification).

[0159] "Detection" refers to identifying the presence, absence, or amount of an analyte to be detected. In an embodiment, sequence variations in a polynucleotide or polypeptide are detected. In another embodiment, the presence of indels is detected.

[0160] A "detectable label" means a label that, when attached to a molecule of interest, is detectable by spectroscopic, photochemical, or biochemical means. "detectable" refers to a composition that renders the latter detectable through biological, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent Photochromic dyes, electron-dense reagents, enzymes (e.g., commonly used in enzyme-linked immunosorbent assays (ELISAs)) These include hydroxybenzoates, biotin, digoxigenin, or haptens.

[0161] "Disease" means any condition that damages or interferes with the normal function of a cell, tissue, or organ. Or it means disability.

[0162] As used herein, the term "effective amount" refers to an amount sufficient to induce a desired biological response. The term "amount of biologically active agent" refers to the amount of biologically active agent used to practice the present invention for the therapeutic treatment of disease. The effective amount of active agent(s) administered will depend on the mode of administration, the age, weight, and general health of the subject. Ultimately, your doctor or veterinarian will determine the appropriate amount and dosage. Such an amount is referred to as an "effective" amount. In one embodiment, an effective amount is a dose that is administered to a cell (e.g., an iPSC). A gene of interest in a cell (in vitro or in vivo) is prepared by subjecting the gene to a gene encoding the gene of interest to a gene encoding the gene of interest. Base editors (e.g., programmable DNA binding proteins, nucleobase editors, and In some embodiments, the fusion proteins provided herein are An effective amount of a protein, e.g., an nCas9 domain and a deaminase domain (e.g., an adenosine triphosphate deaminase domain) is administered. An effective amount of a nucleobase editor comprising a nucleobase editor (e.g., cytidine deaminase or cytidine deaminase) is Induce editing of target sites that are specifically bound and edited by the nucleobase editor of In one embodiment, an effective amount refers to an amount of a fusion protein sufficient to have a therapeutic effect (e.g., (e.g., reducing or controlling a disease or its symptoms or conditions) Such a therapeutic effect may be felt in all cells of a subject, tissue, or organ. It need not be sufficient to alter the gene of interest in the subject, tissue, or organ, but rather to Alter the gene of interest in approximately 1%, 5%, 10%, 25%, 50%, 75% or more of the cells present. All you have to do is

[0163] In some embodiments, the fusion proteins provided herein (e.g., nCas9 domains) are Main and deaminase domains (e.g., adenosine deaminase, cytidine deaminase) An effective amount of a nucleobase editor, including a nucleobase editor (e.g., a nucleobase editor enzyme), is a fusion protein sufficient to induce editing of a target site that is specifically bound and edited by As will be appreciated by those skilled in the art, the term "quantity" refers to the amount of an agent (e.g., a fusion protein, a nucleic acid, ase, hybrid protein, protein dimer, protein (or protein An effective amount of a complex of a dimer and a polynucleotide, or a polynucleotide, can be, for example, , a desired biological response, e.g., a specific allele, genome, or target region to be edited. Various factors, such as the location, the cell or tissue being targeted, and / or the agent being used, may be involved. This may vary depending on the child.

[0164] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule, which portion is identical to that of a reference nucleic acid At least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% of the entire length of the molecule or polypeptide , or 90%. The fragments may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 3 nucleotides or amino acids. do.

[0165] "Guide RNA" or "gRNA" is a polynucleotide that can be specific for a target sequence. a programmable nucleotide-binding domain protein (e.g., Cas9 or Cpf1) In one embodiment, the term "polynucleotide" refers to a polynucleotide that can form a complex with a The guide polynucleotide is a guide RNA (gRNA). gRNA is a complex of two or more RNAs. It can exist as a single RNA molecule or as a single RNA molecule. The gRNAs present are sometimes called single guide RNAs (sgRNAs), but "gRNA" refers to a single Interchangeable to refer to guide RNAs that exist as a single molecule or as a complex of two or more molecules. Typically, gRNAs exist as a single RNA species, and are used in a variety of ways. (1) They are homologous to the target nucleic acid. (2) a domain that shares a common property (e.g., directs binding of the Cas9 complex to the target); and In one embodiment, the domains are the s9 protein-binding domains. In (2), the sequence corresponds to the sequence known as tracrRNA and contains a stem-loop structure. In some embodiments, domain (2) is selected from the group consisting of the amino acid sequence of Jinek et al., Science 337:816-821(201 2) tracrRNA as provided in (the entire contents of which are incorporated herein by reference) Other examples of gRNAs (e.g., those containing domain 2) are listed on September 6, 2013. In a filed U.S. provisional patent application entitled "Switchable Cas9 Nucleases and Uses Thereof," U.S. SSN 61 / 874,682 and "Delivery System For Functional Numerical System" filed September 6, 2013 The entire disclosure of each of these patent applications can be found in U.S. Provisional Patent Application No. USSN 61 / 874,746 entitled "Patent Application No. 61 / 874,746," which is hereby incorporated by reference. The contents of which are incorporated herein by reference. In some embodiments, the gRNA A gRNA containing two or more of the main (1) and (2) sequences may be referred to as an "extended gRNA." The gRNA binds to two or more Cas9 proteins and expresses two or more Cas9 proteins as described herein. The gRNA binds to the target nucleic acid at a different region on the target site. sequence, which mediates binding of the nuclease / RNA complex to the target site, :Provides sequence specificity of the RNA complex.

[0166] "Hybridization" means hydrogen bonding between complementary nucleobases, as defined by Watson- It can be a Crick, Hoogsteen or reversed Hoogsteen hydrogen bond. For example, Adenine and thymine are complementary nucleobases that form hydrogen bonds to form pairs.

[0167] The term "inhibitor of base repair" or "IBR" refers to a nucleic acid Inhibiting the activity of repair enzymes, such as base excision repair (BER) enzymes In one embodiment, IBR refers to a protein capable of inhibiting inosine base excision repair. Examples of inhibitors of base repair include APE1, Endo III, Endo IV, Endo V, and Endo o VIII, Fpg, hOGGl, hNEILl, T7 Endol, T4 PDG, UDG, hSMUGL, and hAAG inhibitors In one embodiment, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is catalytically inactive EndoV or catalytically inactive hAAG. In one embodiment, the base repair inhibitor is an inhibitor of Endo V or hAAG. , the base repair inhibitor is catalytically inactive EndoV or catalytically inactive hAAG.

[0168] In some embodiments, the base repair inhibitor is a uracil glycosylase inhibitor (UGI). UGI is a gene that can inhibit the base excision repair enzyme uracil-DNA glycosylase. In some embodiments, the UGI domain refers to a protein that is a wild-type UGI or In some embodiments, the UGI proteins provided herein comprise a fragment of U In one embodiment, the present invention includes a fragment of GI and a protein homologous to UGI or a UGI fragment. The base repair inhibitor is an inhibitor of inosine base excision repair. In the present study, base repair inhibitors were identified as "catalytically inactive inosine-specific nucleases" or "dead" nucleases. Without wishing to be bound by any particular theory, However, catalytically inactive inosine glycosylases (e.g., alkyladenine glycosylases) Abasic amino acid sequence (AAG) can bind to inosine but also creates an abasic site. It is also unable to remove the inosine moiety, thereby blocking the newly formed inosine moiety from DNA damage / repair. In some embodiments, catalytically inactive inositols are sterically blocked from the cleavage mechanism. Inosine-specific nucleases can bind to inosine in nucleic acids but do not cleave the nucleic acid. Representative, non-limiting examples of catalytically inactive inosine-specific nucleases include: Catalytically inactive alkyl adenosine glycosylase (AAG nuclease) (e.g., from humans) and catalytically inactive endonuclease V (EndoV nuclease) (e.g., from E. coli). In some embodiments, catalytically inactive AAG nucleases are used. The AAG nuclease contains an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0169] By "increase" is meant a positive change of at least 10%, 25%, 50%, 75%, or 100%.

[0170] An "intein" excises itself and releases the remaining fragment (the extein) (extein)) to form peptide bonds in a process known as protein splicing Inteins are fragments of proteins that can be linked together using a "protein intron." The intein excises itself and links the rest of the protein. The process is referred to herein as "protein splicing" or "intein-mediated In some embodiments, precursor proteins (integrins) are spliced. Inteins (intein-containing proteins before intein-mediated protein splicing) Such inteins are referred to herein as split inteins. These are called split inteins (e.g., split intein-N and split intein-C). In Bacteria, DnaE, ​​the catalytic subunit a of DNA polymerase III, is expressed in two separate genes. Encoded by the genes dnaE-n and dnaE-c. Encoded by the dnaE-n gene The intein may be referred to herein as "intein N." The loaded intein may be referred to herein as "intein C."

[0171] Other intein systems can also be used. For example, the dnaE intein, i.e., Cfa-N (e.g., Based on the intein pair Cfa-C (e.g., split intein-N) and Cfa-C (e.g., split intein-C) Synthetic inteins have been described (see, e.g., Stevens, J. Med. Chem. Soc. 1999, 144:111-112, which is incorporated herein by reference). s et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5). Use in accordance with this disclosure Non-limiting examples of intein pairs that can be used include Cfa DnaE intein, Ssp GyrB intein, and Intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein Cne intein, and Cne Prp8 intein (see, e.g., U.S. Pat. No. 6,223,629, incorporated herein by reference). Examples include those described in Patent No. 8,394,604.

[0172] Exemplary nucleotide and amino acid sequences of inteins are provided. DnaE Intein-N DNA: TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTCTGCCAATCGGG AAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCGATAACAATGGTAACATTTATACTCAGCCAGTTGCCCA GTGGCACGACCGGGGAGAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAGGGCCACTAAGGACCACA AATTTATGACAGTCGATGGCCAGATGCTGCCTATAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAAC CTTCCTAAT DnaE Intein-N Protein: CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDR GEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN DnaE Intein-C DNA: ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTATGA TATTGG AGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTCATAGCTTCTAAT Intein-C: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN Cfa-N DNA: TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCCTATTGGAAAGATTGTCGAA GAGAGAATTGAATGCACAGTATATACTGTAGACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGGCACAATCG CGGCGAACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACGAGCAACTAAAGATCATAAATTCATGACCA CTGACGGGCAGATGTTGCCAATAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTGCCA Cfa-N Protein: CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQ MLPIDEIFERGLDLKQVDGLP Cfa-C DNA: ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAGGAAAGTAAAGATAATATCT CGAAAAAGCTTGGTACCCAAAATGTCTATGATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGT AGCCAGCAAC Cfa-C protein: MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN

[0173] To join the N-terminal part of the split Cas9 with the C-terminal part of the split Cas9, intein N and intein B were used. The tein C can be fused to the N-terminal end of the split-Cas9 and the C-terminal end of the split-Cas9, respectively. For example, In some embodiments, intein-N is C-terminal to the N-terminal portion of the split Cas9. The split-Cas9 is fused to the intein-N domain, forming the structure N--[N-terminal portion of split-Cas9]-[intein-N]--C. In some embodiments, the intein-C is located at the N-terminus of the C-terminal portion of the split Cas9. The intein is fused to the C-terminal portion of the split Cas9, forming the structure N-[intein-C]-[C-terminal portion of the split Cas9]-C. Intein for linking the protein (e.g., split-Cas9) to which the intein is fused The mechanism of tein-mediated protein splicing is described, for example, in the publications As described in Shah et al., Chem Sci. 2014; 5(1):446-461, the present invention Methods for designing and using inteins are known in the art. and are disclosed, for example, in WO2014004336, WO2017132580, US20150344549 and US20180127780. and US Pat. No. 6,263,999, each of which is incorporated herein by reference in its entirety.

[0174] The terms "isolated," "purified," or "biologically pure" refer to a substance that is in its native state. Substances that have been removed to varying degrees from components normally associated with them when found in the natural state. "Isolated" refers to the degree of separation from the original source or surrounding environment. "Purified" refers to the degree of separation from the original source or surrounding environment. A "purified" or "biologically pure" protein is one that is free from impurities. that the substance does not materially affect the biological properties of the protein or cause other adverse consequences. In other words, the nucleic acid or peptide of the present invention is or, if produced by recombinant DNA technology, cellular material, viral material, or culture medium. If the substance is not naturally contained in the substance, or if it is chemically synthesized, it may contain chemical precursors or other chemical substances. Purity and homogeneity are typically determined by analytical methods. Chemical techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography The term "purified" refers to the degree to which a nucleic acid or protein is purified by electrophoresis. This can mean that the resulting protein essentially produces one band. For proteins that can undergo glycosylation, the different modifications are purified separately. This can result in different isolated proteins that can be isolated.

[0175] An "isolated polynucleotide" is a nucleic acid molecule that does not occur in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived. It means nucleic acid (e.g., DNA) that does not contain the genes adjacent to the gene. The term refers to, for example, those incorporated into vectors; autonomously replicating plasmids or viruses. integrated into the genomic DNA of prokaryotes or eukaryotes; or independent of other sequences another molecule (e.g., cDNA generated by PCR or restriction endonuclease digestion) or genomic or cDNA fragments). RNA molecules transcribed from DNA molecules, as well as hybrids encoding additional polypeptide sequences. It contains recombinant DNA that is part of the hybrid gene.

[0176] An "isolated polypeptide" is a polypeptide of the invention separated from components that naturally accompany it. Typically, a polypeptide is a polypeptide that is a protein with which it is naturally associated. A substance is isolated if it is at least 60% by weight free from substances and naturally occurring organic molecules. Preferably, the preparation comprises at least 75% by weight of soluble fiber, more preferably at least 90% by weight of soluble fiber, most preferably at least 10% by weight of soluble fiber. or at least 99% of the isolated polypeptide of the present invention. can be prepared by, for example, extraction from a natural source, expression of a recombinant nucleic acid encoding such a polypeptide, or the like. or by chemically synthesizing the protein. Purity can be achieved by any suitable method. Suitable methods, such as column chromatography, polyacrylamide gel electrophoresis, or can be measured by HPLC analysis.

[0177] As used herein, the term "linker" refers to a molecule that binds two molecules or moieties (e.g., a Two components of a protein or ribonucleocomplex, or two domains of a fusion protein In, for example, a polynucleotide programmable DNA binding domain (e.g., dCas9) Deaminase domains (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase), or the napDNAbp domain (e.g. Cas12b) and a deaminase domain (e.g., adenosine deaminase or cytidine deaminase) Covalent linkers (e.g., covalent bonds), non-covalent linkers, chemical bonds, etc., linking the In certain embodiments, the linker can refer to a chemical group, a molecule, or a molecule that binds to the Cas protein. The linker is adjacent to the deaminase domain inserted within the protein or a fragment thereof. Connecting different components or parts of a component in a editor system For example, in some embodiments, the linker can be a polynucleotide protease. a guide polynucleotide binding domain for a grammable nucleotide binding domain; and In some embodiments, the linker can link the catalytic domains of the deaminase. The ISPR polypeptide and the deaminase can be linked. The linker can link the Cas9 and the deaminase. In some embodiments, the linker is In some embodiments, the linker can link the dCas9 and the deaminase. The s9 and the deaminase can be linked. For example, in one embodiment, the linker can be Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h In some embodiments, the linker may link the guidepoietin and the deaminase. In some embodiments, the oligonucleotide and the deaminase can be linked. The linker is a base editor system having a deaminating component and a polynucleotide programming component. In some embodiments, the linkable nucleotide binding moieties can be linked. The anchor connects the RNA-binding moiety of the deaminating component of the base editor system with the napDNAbp component. In some embodiments, the linker can be used to decouple the base editor system. The RNA-binding portion of the amination component and the polynucleotide-programmable nucleotide bond In some embodiments, the linker can be a base element. The RNA-binding moiety of the deaminating component of the RNA-binding protein system and the polynucleotide programmable nuclease A linker is a molecule that can link two groups, molecules, or a combination of two or more ... or other moieties, and are disposed between or sandwiched by them and are bonded covalently or non-covalently They are connected to each other through binding interactions, thus allowing the two to be linked together. In various embodiments, the linker can be an organic molecule, group, polymer, or chemical moiety. In embodiments, the linker can be a polynucleotide. In some embodiments, the linker may be a DNA linker. In some embodiments, the linker may be an RNA linker. In such a case, the linker can include an aptamer capable of binding to the ligand. In some embodiments, the ligand is a carbohydrate, peptide, protein, or nucleic acid. In some embodiments, the linker can be an aptamer derived from a riboswitch. The riboswitch from which the aptamer is derived can be theophylline riboswitch. , thiamine pyrophosphate (TPP) riboswitch, adenosine cobalamin (AdoCbl) riboswitch switch, S-adenosylmethionine (SAM) riboswitch, SAH riboswitch, flavin mononucleotide Nucleotide (FMN) riboswitch, tetrahydrofolate riboswitch, lysine riboswitch, Glycine riboswitch, purine riboswitch, GlmS riboswitch, or prequeosine 1 (PreQ1) riboswitch. In some embodiments, the linker can be selected from a polypeptide The aptamer may comprise an aptamer bound to a protein domain, such as a polypeptide ligand. In some embodiments, the polypeptide ligand comprises a K homology (KH) domain, an MS2 coat protein, or a phosphodiesterase (MPS) domain. Protein domain, PP7 coat protein domain, SfMu Com coat protein domain, Sterile α motif, telomerase Ku binding motif and Ku protein, telomerase Sm7 The Sm7 protein binding motif may be an Sm7 protein binding motif or an RNA recognition motif. Thus, the polypeptide ligand can be part of a base editor system component. The base editing component may comprise a deaminase domain and an RNA recognition motif.

[0178] In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or In some embodiments, the linker may be about 5 to 100 amino acids in length. For example, lengths of approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-30 , 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acids. In some embodiments, the linker has a length of about 100-150, 150-200, 200-250, 250-300, It can be 300-350, 350-400, 400-450, or 450-500 amino acids. Shorter linkers are also contemplated.

[0179] In some embodiments, the linker comprises an RNA programmable The gRNA-binding domain of a nuclease and a nucleic acid editing protein (e.g., cytidine or adenine) In some embodiments, the linker connects the catalytic domain of dCas 9 and the nucleic acid editing protein. For example, the linker may be a linker that connects two groups, molecules, or other Located between or flanked by two groups, molecules, or other moieties, are linked to each other via a covalent bond, thus linking the two. The anchor is an amino acid or multiple amino acids (eg, a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5 to 200 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 95, 100, 150, 100, 250, 350, 400, 550, 600, 700, 800, 950, 1000, 1500, 1000, 2500, 3500, 4000, 5 ...6000, 7000, 8000, 9500, 1000, 1 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 7 0, 75, 80, 85, 90, 90, 95, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150 , 160, 175, 180, 190, or 200 amino acids. is also contemplated.

[0180] In some embodiments, the nucleobase editor domain is SGGSSGSETPGTSESATPESSGGS, SG GSSGGSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA The amino acid sequence of PGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS In some embodiments, the nucleobase editor domain is fused via a linker comprising The XTEN linker is fused via a linker containing the amino acid sequence SGSETPGTSESATPES, which may also be referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. , the linker is (SGGS) n , (GGGS) n , (GGGGS) n , (G) n、 (EAAAK) n , (GGS) n ,SGSETPGTSESAT PES or (XP) n motif, or any combination thereof, where n is a unique In some embodiments, n is an integer between 1 and 30, and X is any amino acid. is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0181] In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker has the amino acid sequence SGGSSGGSSSGSETP In some embodiments, the linker comprises GTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the linker has the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSG In some embodiments, the linker comprises GSSGGSSGGSSGSETPGTSESATPESSGGS SGGS. In some embodiments, the linker has the amino acid sequence PGSPAGSPTSTEEGTSESATP. Contains ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0182] A "marker" is any molecule that has an altered expression level or activity that is associated with a disease or disorder. The term "protein" refers to any protein or polynucleotide.

[0183] As used herein, the term "mutation" refers to a change in a sequence, e.g., a nucleic acid or amino acid sequence. The substitution of a residue in a sequence of amino acids by another residue, or the deletion of one or more residues in the sequence. Mutations, as used herein, are typically made by identifying the original residue and then and identifying the newly substituted residue, Various methods for making amino acid substitutions (mutations) are provided herein. are well known in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012). In some embodiments, the present disclosure The proposed base editor generates a significant number of unintended mutations, e.g., unintended point mutations. without introducing a "purposeful mutation," e.g., a point mutation, into a nucleic acid (e.g., a nucleic acid in a subject's genome) In some embodiments, the intended mutation can be efficiently generated. The mutation is a guide polynucleotide specifically designed to produce the intended mutation. (e.g., gRNA) bound to a specific base editor (e.g., a cytidine base editor or This mutation is caused by an adenosine base editor (ABE).

[0184] Generally, the amino acid sequence of the present invention is made or identified in a sequence (e.g., an amino acid sequence described herein). The mutations detected are numbered relative to the reference (or wild-type) sequence, i.e., the sequence that does not contain the mutation. Those skilled in the art will recognize mutations in amino acid and nucleic acid sequences relative to a reference sequence. It will be easy to understand how to determine the position.

[0185] The term "non-conservative mutation" refers to amino acid substitutions between different groups, e.g. For example, tryptophan to lysine, or serine to phenylalanine. The non-conservative amino acid substitutions do not disrupt or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions are preferred because they improve the biological activity of the functional variant compared to the wild-type protein. The biological activity of the functional variant can be enhanced so that it is increased compared to the protein. do.

[0186] The term "nuclear localization sequence," "nuclear localization signal," or "NLS" refers to the localization of a protein. The term "nuclear localization sequence" refers to an amino acid sequence that promotes import into the cell nucleus. For example, WO / 2001 / 038547 filed on November 23, 2000 and published on May 31, 2001 is incorporated herein by reference. and Plank et al. in published international PCT application PCT / EP 2000 / 011690, in which No. 6,239,493, which is incorporated herein by reference for its disclosure of exemplary nuclear localization sequences. In embodiments, the NLS may be any of the NLSs described, for example, in Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4 172. In some embodiments, the NLS is Sequence KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR , RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0187] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a nucleic acid molecule comprising a nucleobase and Compounds containing an acidic moiety, such as nucleosides, nucleotides, or polynucleotides Typically, a polymeric nucleic acid, e.g., a nucleic acid molecule containing three or more nucleotides is a linear sequence in which adjacent nucleotides are linked to each other via phosphodiester bonds. In some embodiments, a "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or or nucleosides). In some embodiments, a "nucleic acid" refers to three or more individual nucleosides. As used herein, the term "oligonucleotide" refers to an oligonucleotide chain containing nucleotide residues. "Nucleotide" and "polynucleotide" refer to a polymer of nucleotides (e.g., may be used interchangeably to refer to a chain of at least three nucleotides. In this context, "nucleic acid" encompasses RNA and single- and / or double-stranded DNA. For example, genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome It may naturally occur in the context of a chromatid, or other naturally occurring nucleic acid molecule. On the other hand, nucleic acid molecules can be, for example, non-naturally occurring molecules, recombinant DNA or RNA, artificial chromosomes, Engineered genomes, or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids, or or non-naturally occurring molecules containing non-naturally occurring nucleotides or nucleosides Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms may be used interchangeably. Nucleic acids include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. purified from the source, produced using a recombinant expression system and optionally purified, or chemically synthesized In the case of chemically synthesized molecules, the nucleic acid may, where appropriate, be Nucleotides such as analogs with chemically modified bases or sugars, and backbone modifications, are also suitable. Nucleic acid sequences are shown in the 5' to 3' direction unless otherwise indicated. In some embodiments, nucleic acids contain natural nucleosides (e.g., adenosine, thymidine, guanine, adenosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine cytidine, and deoxycytidine; nucleoside analogs (e.g., 2-aminoadenosine, 2-thiocytidine); Othymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2- Aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- Propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanine , O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose , 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages) or Includes these.

[0188] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" refers to Used interchangeably with "polynucleotide programmable nucleotide binding domain" A guide nucleic acid or guide polynucleotide that guides the napDNAbp to a specific nucleic acid sequence. It refers to a protein that associates with a nucleic acid (e.g., DNA or RNA) such as a nucleic acid (e.g., gRNA). In embodiments, a polynucleotide-programmable nucleotide binding domain is a polynucleotide-programmable DNA binding domain. In the present invention, the polynucleotide-programmable nucleotide binding domain is In one embodiment, the RNA-binding domain is programmable by oligonucleotides. The polynucleotide-programmable nucleotide binding domain is a Cas9 protein. The Cas9 protein binds to a specific DNA sequence complementary to the guide RNA. In some embodiments, the napDNAbp can bind to a guide RNA that guides the Cas9 domain. Main, e.g., nuclease-active Cas9, Cas9 nickase (nCas9), or nuclease Inactive Cas9 (dCas9). Non-limiting examples of nucleic acid programmable DNA binding proteins. Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, and Cas12c / C2c3 Cas enzymes include Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. Non-limiting examples include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12) ), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / Ca sX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5 e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3 , Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Cs x1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa 1, Csa2, Csa3, Csa4, Csa5, type II Cas effector protein, type V Cas effector protein Cas effector proteins, type VI Cas effector proteins, CARF, DinG, and their homologs Other nucleic acid programming may be used. Also included are DNA binding proteins that may not be specifically listed in this disclosure. It is within the scope of this disclosure, e.g., Makarova et al. CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 Oct;1:325-336. doi: 10.10 89 / crispr.2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas system s” Science. 2019 Jan 4;363(6422):88-91. See doi: 10.1126 / science.aav7271 (see the entire contents of each of which are incorporated herein by reference).

[0189] The terms "nucleobase," "nitrogenous base," or "base" are used interchangeably herein. refers to nitrogen-containing biological compounds that form nucleosides, which are nucleotides The ability of nucleobases to form base pairs and stack with each other is directly related to the synthesis of ribonucleic acid ( Adenine (A) is a nucleotide that gives rise to long helical structures such as RNA and deoxyribonucleic acid (DNA). The five nucleic acid bases are cytosine (C), guanine (G), thymine (T), and uracil (U). They are called primary or standard bases. Adenine and guanine are derived from purines, while cytosine, Uracil and thymine are derived from pyrimidines. DNA and RNA contain modified other (non-primary) bases. Non-limiting exemplary modified nucleobases include hypoxanthine, xanthine, , 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydroxybenzoates. Hypoxanthine and xanthine are produced in the presence of mutagens. Both can be produced by deamination (replacement of an amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can be generated by deamination of cytosine. A "nucleoside" is a nucleic acid base and a five-carbon atom. They consist of a sugar (ribose or deoxyribose). Examples of nucleosides include adenosine , guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, These include deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Nucleosides with modified nucleobases include inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), psoriasis Nucleotides consist of a nucleic acid base, a pentose sugar (ribose or is deoxyribose), and at least one phosphate group.

[0190] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" refers to napDNA Associate with a nucleic acid (e.g., DNA or RNA) such as a guide nucleic acid that directs the Abp to a specific nucleic acid sequence For example, the Cas12 protein is a protein that binds the Cas12 protein to a guide RNA complementary to the guide RNA. In some embodiments, the nucleic acid sequence can be linked to a guide RNA that directs the nucleic acid sequence to a specific DNA sequence. , napDNAbp is a Cas12 domain, e.g., a nuclease-active Cas12 domain. Examples of bps include Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, and Ca Other napDNAbps are also specifically listed in this disclosure. Although this may not be possible, it is within the scope of this disclosure. on and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 Oct ;1:325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., “Functionally diverse ty pe V CRISPR-Cas systems” Science. 2019 Jan 4;363(6422):88-91. doi: 10.1126 / scie See nce.aav7271, the entire contents of each of which are incorporated herein by reference.

[0191] The terms "nucleobase editing domain" or "nucleobase editing protein" are used herein. When used in the formula, cytosine (or cytidine) to uracil (or uridine) or hypoxia to thymine (or thymidine) and from adenine (or adenosine) Deamination to sansine (or inosine), and non-templated nucleotide addition and insertion Proteins or enzymes that can catalyze nucleobase modifications in RNA or DNA, such as refers to an enzyme. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., For example, adenine deaminase or adenosine deaminase; or cytidine deaminase In some embodiments, the nucleobase editing domain is a nucleobase editing domain (e.g., a nucleobase editing domain ... The enzymes may contain multiple deaminase domains (e.g., adenine deaminase or adenosine deaminase). aminase and cytidine or cytosine deaminase). The acid-base editing domain may be a naturally occurring nucleobase-editing domain. In embodiments, the nucleobase-editing domain is engineered from a naturally occurring nucleobase-editing domain. The nucleobase editing domain may be engineered or evolved in bacteria, Any living organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse For example, the nucleobase-editing protein can be derived from the protein described in International PCT Application No. PCT / 2017 / 0453 81 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), which each of which is incorporated herein by reference in its entirety. “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmabl e base editing of A·T to G·C in genomic DNA without DNA cleavage”Nature 551, 464-471 (2017); and Komor, AC, et al., “Improved base excision repair inhi bition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with high See also “Efficiency and product purity” Science Advances 3:eaao4774 (2017) the entire contents of which are incorporated herein by reference).

[0192] As used herein, "obtaining," as in "obtaining a drug," means This includes synthesizing, purchasing, or otherwise obtaining the drug.

[0193] As used herein, a "patient" or "subject" refers to a person who has been diagnosed with a disease or disorder. Mammals at risk of developing them or suspected of having or developing them In certain embodiments, the term "patient" refers to a subject or individual suffering from a disease or disorder. refers to a mammalian subject with a higher than average likelihood of developing a disease. Exemplary patients include humans, non-human primates, cats, dogs, pigs, cattle, horses, camels, llamas, goats, sheep, rodents (e.g. mice, rabbits, rats, guinea pigs) and will benefit from the treatments disclosed herein. Exemplary human patients include males and / or females. could be.

[0194] A "patient in need thereof" or a "subject in need thereof" is used herein to refer to a person with a disease or have been diagnosed with, are at risk of having, or may have a disability or disorder It is referred to as a patient who has been predetermined or is suspected of having it.

[0195] "Pathogenic mutation," "pathogenic variant," "disease-causing mutation," "disease-causing variant" The terms "deleterious mutation" or "predisposing mutation" refer to a mutation that is associated with a particular disease or disorder. Refers to a genetic change or mutation that increases an individual's susceptibility or predisposition. A pathogenic variant is a mutation that results in at least one wild-type mutation in the protein encoded by the gene. This includes those in which an amino acid has been replaced with at least one pathogenic amino acid.

[0196] The term "pharmaceutically acceptable carrier" refers to a liquid or solid filler, diluent, excipient, manufacturing agent, or Auxiliaries (e.g. lubricants, magnesium talc, calcium stearate or stearic acid zinc, or stearic acid), or solvent encapsulating materials, etc., in certain parts of the body (e.g. transport or transport of a compound from one site (e.g., a delivery site) to another site (e.g., an organ, tissue, or part of the body) Pharmaceutically acceptable materials, compositions, or vehicles involved in the delivery or transport of pharmaceuticals. A commercially acceptable carrier is one that is "compatible" with the other ingredients of the formulation and is not deleterious to the tissues of the subject. "Acceptable" (e.g., physiological compatibility, sterility, physiological pH, etc.). Terms such as "pharmaceutically acceptable carrier," "vehicle," and the like are used interchangeably herein. do.

[0197] The term "pharmaceutical composition" may refer to a composition formulated for pharmaceutical use.

[0198] The terms "protein," "peptide," "polypeptide," and their grammatical equivalents are used interchangeably herein and are linked to each other by a peptide (amide) bond. The term refers to a polymer of amino acid residues bound together in a single chain. It typically refers to a protein, peptide, or polypeptide. A protein, peptide, or polypeptide is at least three amino acids in length. A polypeptide can refer to an individual protein or a group of proteins. One or more amino acids in a protein, peptide, or polypeptide may have, for example, a carbohydrate group, a hydrophobic group, xyl group, phosphate group, farnesyl group, isofarnesyl group, fatty acid group, linkage They may be modified by the addition of chemical entities such as anchors, functionalization, or other modifications. Proteins, peptides, or polypeptides may also be single molecules or multi-molecular. The protein, peptide, or polypeptide may be a naturally occurring It may be merely a fragment of a protein or peptide. Peptides may be naturally occurring, recombinant, or synthetic, or any of the foregoing. As used herein, the term "fusion protein" refers to a combination of: Hybrid polypeptides containing protein domains from at least two different proteins One protein is the amino-terminal (N-terminal) portion of the fusion protein or the capsid. can be located at the carboxy-terminus (C-terminus) of a protein, thus The protein forms a terminal or carboxy-terminal fusion protein. a nucleic acid binding domain (e.g., a nucleic acid binding domain that induces binding of a protein to a target site) Cas9 gRNA binding domain and nucleic acid cleavage domain, or nucleic acid editing protein In some embodiments, the protein may comprise a proteinaceous moiety, e.g., a catalytic domain of For example, an amino acid sequence constituting a nucleic acid binding domain and an organic compound, such as a nucleic acid cleaving agent In some embodiments, proteins include compounds that can act as nucleic acids (e.g., RNA). or DNA) are complexed with or associated with nucleic acids. Any protein can be produced by any method known in the art. For example, the proteins provided herein can be prepared via recombinant protein expression and purification. This is particularly suitable for fusion proteins containing peptide linkers. Methods for recombinant protein expression and purification are well known and are described in Green and Samb rook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Labora and those described by the University of California Press, Cold Spring Harbor, NY (2012). the entire contents of which are incorporated herein by reference.

[0199] The polypeptides and proteins disclosed herein (including functional portions thereof and their functional variants) (including riboflavin) contains synthetic amino acids in place of one or more naturally occurring amino acids Such synthetic amino acids are known in the art and include, for example, aminocyclo Hexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylacetone aminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenyl phenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine phenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-Naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline 2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, Aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyl-lysine, 6- Hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, aminocyclohexyl cyclohexanecarboxylic acid, aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid carboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diamino These include monopropionic acid, homophenylalanine, and α-tert-butylglycine. Polypeptides and proteins are derived from post-translational modifications of one or more amino acids of the polypeptide construct. Non-limiting examples of post-translational modifications include phosphorylation, acetylation, and the like. and acylation, including formylation, glycosylation (including N-linked and O-linked), amino Derivatization, hydroxylation, alkylation including methylation and ethylation, ubiquitination, pyrolysis, Addition of lidocaine carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation ylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation and iodination.

[0200] "Polynucleotide programmable nucleotide binding domain" or "nucleic acid programmable nucleotide binding domain" The term "programmable DNA binding protein (napDNAbp)" refers to a polynucleotide programmable DNA binding protein. A guide polynucleotide (e.g., a nucleotide-binding domain) that directs the targetable nucleotide binding domain to a specific nucleic acid sequence. It refers to a protein that binds to nucleic acids (e.g., DNA or RNA) such as guide RNA. In some embodiments, the polynucleotide-programmable nucleotide binding domain The DNA is a polynucleotide programmable DNA binding domain. In one embodiment, the polynucleotide programmable nucleotide binding domain is In some embodiments, the polynucleotide is a programmable RNA binding domain. The nucleotide-programmable nucleotide-binding domain is the Cas12 protein.

[0201] The term "recombinant" as used herein with respect to a protein or nucleic acid means that the protein or nucleic acid is It refers to proteins or nucleic acids that do not occur in nature but are the product of human engineering. For example, In some embodiments, the recombinant protein or nucleic acid molecule is any naturally occurring At least one, at least two, at least three, at least four, or at least at least five, at least six, or at least seven amino acid or nucleotide mutations Contains an octide sequence.

[0202] "Decrease" means a negative change of at least 10%, 25%, 50%, 75%, or 100% .

[0203] "Reference" means a standard or control condition. , the reference is a wild-type or healthy cell. In other embodiments, including but not limited to, the reference are not exposed to the test conditions or are exposed to placebo or normal saline, medium, or buffer. and / or exposed to a control vector that does not carry the polynucleotide of interest, It is a physiological cell.

[0204] A "reference sequence" is a defined sequence used as a basis for sequence comparison. It can be a subset or the entirety of a particular sequence; for example, a full-length cDNA or gene sequence. A segment of a gene, or the complete cDNA or gene sequence. For polypeptides, see the reference polypeptide. The length of the peptide sequence is generally at least about 16 amino acids, at least about 20 amino acids, At least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is generally at least about 50 nucleotides, At least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides, or about 300 nucleotides thiazolinone, ... In one embodiment, the reference sequence is the wild-type sequence of the protein of interest. The sequence is a polynucleotide sequence that encodes the wild-type protein.

[0205] The terms "RNA programmable nuclease" and "RNA-guided nuclease" refer to Used in conjunction with (e.g., bound to or associated with) one or more non-target RNAs In some embodiments, the RNA-programmable nuclease, when complexed with RNA, Typically, the bound RNA is called a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. A gRNA that exists as a single RNA molecule is called a single guide RNA (sgRNA). Although sometimes referred to as "gRNA," "gRNA" can occur as a single molecule or as a complex of two or more molecules. are used interchangeably to refer to guide RNAs present as a single RNA species. The gRNA present in the target nucleic acid has (1) a domain that shares homology with the target nucleic acid (e.g., a domain that facilitates Cas9 duplication to the target). and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) is directed against a sequence known as tracrRNA. For example, in some embodiments, domain (2) comprises: Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. The gRNA (e.g., domain) is identical to or homologous to the tracrRNA provided in the Other examples of compounds containing 2 include "Switchable Cas9 Nucleases and Us" filed on September 6, 2013. No. 61 / 874,682, filed September 6, 2013, entitled "Methods Thereof," U.S. Provisional Patent Application USSN 080200004 entitled "Delivery System For Functional Nucleases" No. 61 / 874,746, the entire contents of each of which are incorporated herein by reference. In some embodiments, the gRNA comprises two or more of domains (1) and (2), For example, an extended gRNA may be a gene that is expressed as a sequence of a gene, such as a nucleotide sequence, a sequence of a gene, or a sequence of ... For example, two or more Cas9 proteins can be bound to target nuclei in two or more different regions. The gRNA contains a nucleotide sequence complementary to the target site, which binds to the target site. It mediates the binding of the nuclease / RNA complex and provides sequence specificity for the nuclease:RNA complex. Provide.

[0206] In some embodiments, the RNA programmable nuclease is a Cas9 enzyme (CRISPR-associated system). endonuclease, such as Cas9 (Casnl) from Streptococcus pyogenes (e.g., For example, "Complete genome sequence of an Ml strain of Streptococcus pyogenes." Ferret ti JJ et al., Proc. Natl. Acad. Sci. USA 98:4658-4663(2001); "CRISPR RNA ma turation by trans-encoded small RNA and host factor RNase III." Deltcheva E. et al., Nature 471:602-607(2011)).

[0207] RNA-programmable nucleases (e.g., Cas9) use RNA:D to target DNA cleavage sites. Since NA hybridization is used, these proteins can, in principle, be used as guide RNAs. Any sequence specified by A can be targeted. For site-specific cleavage, C Methods using RNA-programmable nucleases such as as9 (e.g., to modify genomes) For this purpose, methods for generating multiplexed genomic DNA (e.g., Cong, L. et al., Multiplex gene expression) are known in the art (see, e.g., Cong, L. et al., Multiplex gene expression). ome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013 ); Hwang, WY et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al., RNA-programme d genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, JE et al., G enome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al., RNA-guided editing of bacterial genomes using CRISPR-Cas systems. See Nature biotechnology 31, 233-239 (2013). the entire contents of each of which are incorporated herein by reference).

[0208] The term "single nucleotide polymorphism (SNP)" refers to a single nucleotide variation that occurs at a specific position in the genome. where each mutation is present in the population to a noticeable extent (e.g., >1%). For example, At certain base positions in the human genome, C nucleotides can occur in most individuals, In a small number of individuals, the position is occupied by A. This means that there is a SNP at this particular position, and C or A, meaning that two nucleotide variations are alleles at this position. SNPs underlie differences in susceptibility to disease, affecting the severity of the disease and the body's response to treatment. SNPs are found in the coding region of genes, non-coding regions of genes, and so on. It may be present in a gene region, or in an intergenic region (the region between genes). In this case, SNPs within the coding sequence may affect the identity of the protein produced due to the degeneracy of the genetic code. SNPs in the coding region are of two types: synonymous SNPs and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, whereas non-synonymous SNPs affect the amino acid sequence of a protein. There are two types of nonsynonymous SNPs: missense and nonsense. SNPs not in the coding region affect gene splicing, transcription factor binding, and messenger receptor (MR) activity. These types of SNPs can affect the degradation of RNA or the sequence of non-coding RNA. The gene expression that is affected is called an eSNP (expressed SNP) and can be upstream or downstream of the gene. Single nucleotide variants (SNVs) are single nucleotide variations with unlimited frequency and occur in somatic Somatic single base variations may also be referred to as single base modifications.

[0209] "Specifically bind" means to recognize and bind to the polypeptide and / or nucleic acid molecule of the present invention. and bind to other molecules in the sample (e.g., biological sample) but do not substantially recognize or bind to other molecules in the sample. Nucleic acid molecules, polypeptides, or complexes thereof (e.g., nucleic acid programmable DNA binding domain and guide nucleic acid), compound, or molecule.

[0210] Nucleic acid molecules useful in the methods of the present invention may encode a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules include any nucleic acid molecule that is 100% identical to the endogenous nucleic acid sequence. Typically, but not necessarily, substantial identity is shown. A polynucleotide having a nucleotide sequence typically hybridizes with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention can be modified to The present invention also includes any nucleic acid molecule encoding an endogenous nucleotide sequence or a fragment thereof. It need not be 100% identical to the nucleic acid sequence, but typically will show substantial identity. Polynucleotides having "substantial identity" to a double-stranded nucleic acid molecule typically have a small number of double-stranded nucleic acid molecules. "Hybridize" means to hybridize with at least one of the strands of a given molecule. Complementary polynucleotide sequences (e.g., those described herein) are synthesized under various stringency conditions. This means that a double-stranded molecule is formed between the two genes (or a part of them). For example, Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R (1987) Methods Enzymol. 152:507).

[0211] For example, a stringent salt concentration is typically less than about 750 mM NaCl and 75 mM citrate. trisodium, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, more preferably Preferably, the concentration is less than about 250 mM NaCl and 25 mM trisodium citrate. See hybridization can be obtained in the absence of organic solvents, e.g., formamide. whereas high stringency hybridization requires at least about 35% formaldehyde. It can be obtained in the presence of at least about 50% formamide. Stringent temperature conditions are typically at least about 30°C, more preferably at least about 37°C. 0 C, and most preferably at least about 42°C. The concentration of detergent (e.g., sodium dodecyl sulfate (SDS)) and carrier DNA content Various additional parameters, such as inclusion or exclusion, are well known to those skilled in the art. By combining these various conditions, various levels of stringency can be achieved. In one embodiment, hybridization is achieved at 30° C. in 750 mM NaCl, 75 In another embodiment, hybridization occurs in 10 mM trisodium citrate and 1% SDS. The solution was incubated at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide. In another embodiment, the hybridization occurs in 100 μg / ml denatured salmon sperm DNA (ssDNA). Hybridization was performed at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% S The reaction takes place in 500 μg / ml ssDNA, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions are The alternatives will be readily apparent to those skilled in the art.

[0212] For most applications, the washing steps that follow hybridization are also stringent. Wash stringency conditions are defined by salt concentration and temperature. As mentioned above, wash stringency can be increased by decreasing the salt concentration or increasing the temperature. This can be increased by increasing the thickness of the strip for the cleaning process. The appropriate salt concentration is preferably less than about 30 mM NaCl and 3 mM trisodium citrate. and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the process are typically at least about 25°C, more preferably In one embodiment, the temperature is at least about 42°C, and even more preferably at least about 68°C. Washing steps were performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash steps are carried out in 15 mM NaCl, 1.5 mM NaCl, at 42°C. 10 mM trisodium citrate, and 0.1% SDS. Washing steps were performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations of these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton et al. nd Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wil ey Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular C loning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York It has been done.

[0213] "Split" means divided into two or more pieces.

[0214] A "split Cas9 protein" or "split-Cas9" is a protein that is composed of two separate nucleotides. Cas9 protein provided as N-terminal and C-terminal fragments encoded by the sequences The polypeptides corresponding to the N-terminal and C-terminal parts of the Cas9 protein are spliced ​​together. In certain embodiments, the Cas9 protein can be reconstituted to form a "reconstituted" Cas9 protein. The Cas9 protein is described, for example, in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-9 49, 2014, or Jiang et al. (2016) Science 351: 867-871. PDB As described in file: 5F9R (each of which is incorporated herein by reference), In some embodiments, the protein is split into two fragments within a disordered region of the protein. The protein binds to the SpCas9 protein between approximately amino acids A292-G364, F445-K483, or E565-T637. At any C, T, A, or S within the region, or any other Cas9, Cas9 variant ( For example, nCas9, dCas9), or other nap DNA fragments at corresponding positions in the two fragments. In some embodiments, the protein is split into SpCas9 T310, T313, A456, S469, or is split into two fragments at C574. In some embodiments, the protein is split into two fragments. The process of dividing a protein into multiple fragments is called "splitting" the protein. do.

[0215] In other embodiments, the N-terminal portion of the Cas9 protein is S. pyogenes Cas9 wild type (SpC as9) (NCBI Reference Sequence: NC_002737.2, Uniprot Reference Sequence: Q99ZW2) amino acids 1 to 573 The C-terminal part of the Cas9 protein contains amino acids 1 to 637, and the C-terminal part of the Cas9 protein contains amino acids 574 to 1368 of the wild-type SpCas9. includes the part 638 to 1368.

[0216] The C-terminal part of the split Cas9 is ligated with the N-terminal part of the split Cas9 to form the complete Cas9 tag. In some embodiments, the C-terminus of the Cas9 protein can form a protein. The end portion begins where the N-terminal portion of the Cas9 protein ends. In some embodiments, the C-terminal portion of the split Cas9 is amino acids (551-651)-1368 of spCas9. "(551-651) -1368" refers to the amino acids between 551 and 651 (inclusive). This means that the C-terminal portion of the split Cas9 begins with amino acid 1368 and ends with amino acid 1368. The amino acids 551-1368, 552-1368, 553-1368, 554-1368, 555-1368, and 556-1368 of spCas9 are , 557-1368, 558-1368, 559-1368, 560-1368, 561-1368, 562-1368, 563-1368, 564-1368 , 565-1368, 566-1368, 567-1368, 568-1368, 569-1368, 570-1368, 571-1368, 572-1368 , 573-1368, 574-1368, 575-1368, 576-1368, 577-1368, 578-1368, 579-1368, 580-1368 , 581-1368, 582-1368, 583-1368, 584-1368, 585-1368, 586-1368, 587-1368, 588-1368 , 589-1368, 590-1368, 591-1368, 592-1368, 593-1368, 594-1368, 595-1368, 596-1368 , 597-1368, 598-1368, 599-1368, 600-1368, 601-1368, 602-1368, 603-1368, 604-1368 , 605-1368, 606-1368, 607-1368, 608-1368, 609-1368, 610-1368, 611-1368, 612-1368 , 613-1368, 614-1368, 615-1368, 616-1368, 617-1368, 618-1368, 619-1368, 620-1368 , 621-1368, 622-1368, 623-1368, 624-1368, 625-1368, 626-1368, 627-1368, 628-1368 , 629-1368, 630-1368, 631-1368, 632-1368, 633-1368, 634-1368, 635-1368, 636-1368 , 637-1368, 638-1368, 639-1368, 640-1368, 641-1368, 642-1368, 643-1368, 644-1368 , 645-1368, 646-1368, 647-1368, 648-1368, 649-1368, 650-1368, or 651-1368 In some embodiments, the split Cas9 protein may comprise either one of the following portions: The C-terminal portion of SpCas9 includes amino acids 574-1368 or 638-1368 of SpCas9.

[0217] "Subject" means a mammal, including a human, or a bovine, equine, canine, ovine, or Subjects include, but are not limited to, non-human mammals such as cats. Subjects include livestock, labor Domestic animals (cows, goats, chickens) that are raised to produce power and provide goods such as food , horses, pigs, rabbits, and sheep).

[0218] "Substantially identical" means that the amino acid sequence of a reference amino acid sequence (e.g., an amino acid sequence described herein) is substantially identical to the amino acid sequence of a reference amino acid sequence (e.g., an amino acid sequence described herein). any one of the sequences) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein) It means a polypeptide or nucleic acid molecule that exhibits at least 50% identity to In this form, such sequences are identified at the amino acid or nucleic acid level as sequences used for comparison. The sequence may have at least 60%, 80%, or 85%, 90%, 95% or even 99% identity in the sequence.

[0219] Sequence identity is typically determined using sequence analysis software (e.g., Genetics Computer Group , University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705 Sequence Analysis Software Package, BLAST, BESTFIT, GAP, or PILE UP / PRETTYBOX program). Such software is available with various permutations. By assigning degrees of homology to the sequences, deletions, and / or other modifications, it is possible to determine whether the sequences are identical or Similar sequences are matched. Conservative substitutions typically include substitutions within the following groups: leucine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, Paragine, glutamine; serine, threonine; lysine, arginine; phenylalanine In an exemplary approach to determining the degree of identity, the BLAST program You can use the RAM, -3 and e -100 Probability scores between indicate closely related sequences COBALT is used, for example, with the following parameters: a) Alignment parameters: Gap penalties -11, -1 and End-Gap penalties -5, -1 b) CDD parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conserved column s and Recompute on c) Query clustering parameters: Use query clusters on; Word Size 4; M ax cluster distance 0.8; Alphabet Regular. The EMBOSS Needle is used, for example, with the following parameters: a) Matrix: BLOSUM62; b) GAP OPEN: 10; c) GAP EXTEND: 0.5; d) OUTPUT FORMAT: pair; e) END GAP PENALTY: false; f) END GAP OPEN: 10; and g) END GAP EXTEND: 0.5.

[0220] The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a nucleobase editor. In one embodiment, the target site is a sequence that is targeted by a deaminase (e.g., a cytidine or or adenine deaminase) or a fusion protein containing it .

[0221] As used herein, the terms "treat," "treating," and "treatment" refer to "Treatment" and the like are intended to alleviate or improve a disorder and / or its associated symptoms. refers to the process of achieving a desired pharmacological and / or physiological effect. Treating a condition requires the complete elimination of the associated disorder, condition, or symptom. It will be understood that some aspects of the In some instances, the effect is therapeutic, i.e., the effect is, but is not limited to, the effect of treating a disease. Partially or completely reduce, diminish, eliminate or alleviate the disease and / or adverse symptoms resulting therefrom. In some embodiments, the effect is preventative, i.e., The effect is to protect or prevent the occurrence or recurrence of a disease or condition. The disclosed methods comprise administering a therapeutically effective amount of a composition as described herein. include.

[0222] "Uracil glycosylase inhibitor," or "UGI," is a protein that inhibits the uracil excision repair system. In one embodiment, the agent inhibits host uracil-DNA glycosylation. A protein or fragment thereof that binds to uracil and prevents the removal of uracil residues from DNA. In one embodiment, the UGI inhibits uracil-DNA glycosylase base excision repair enzyme. In some embodiments, the target protein is a protein, fragment, or domain thereof that can inhibit the target protein. In some embodiments, the UGI domain comprises wild-type UGI or a modified version thereof. In the present invention, the UGI domain comprises a fragment of the exemplary amino acid sequence provided below. In some embodiments, the UGI fragment comprises at least 60% of the exemplary UGI sequences provided below: At least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least In some embodiments, the UGI comprises an amino acid sequence that is at least 99%, or 100%, of the amino acid sequence of As described below, amino acids homologous to exemplary UGI amino acid sequences or fragments thereof may be used. In some embodiments, the UGI or a portion thereof comprises a sequence as described below. For example, at least 70%, at least 75% of the wild-type UGI or UGI sequence or a portion thereof , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or have 100% identity. Exemplary UGIs include the following amino acid sequences: >splP14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSD APEYKPWALVIQDSNGEN KIKML.

[0223] The term "vector" refers to a nucleic acid sequence that is introduced into a cell, resulting in a transformed cell. Vectors include plasmids, transposons, phages, viruses, and ribosomal An "expression vector" includes a vector that is expressed in a recipient cell. An expression vector is a nucleic acid sequence containing a nucleotide sequence that encodes a gene encoding a gene. Enhances expression of introduced sequences, such as promoter, secretory, and other sequences. and / or may contain additional nucleic acid sequences for facilitating

[0224] The compositions and methods provided herein may be used in combination with other compositions and methods provided herein. It can be combined with any one or more of the methods.

[0225] DNA editing is a method to correct disease states by correcting pathogenic mutations at the genetic level. Until recently, all DNA editing platforms induces DNA double-strand breaks (DSBs) at specific genomic sites and is partially repaired depending on endogenous DNA repair pathways. It functions by determining product outcomes in a probabilistic manner, resulting in a complex population of genetic products. Achieving accurate and user-defined repair outcomes via the homology-directed repair (HDR) pathway Although it is possible to achieve high resolution imaging using HDR in therapeutically relevant cell types, many challenges remain. Efficient repair is hindered. In reality, this pathway involves competing, error-prone, and non-correlated pathways. HDR is less efficient than the homo-end joining pathway. Furthermore, HDR is strictly confined to the G1 and S phases of the cell cycle. This limits the ability of DSBs to be repaired accurately in post-mitotic cells. Highly efficient genome sequencing in these populations in a user-defined and programmable manner It has proven difficult or impossible to alter

[0226] Superoxide dismutase 1 (SOD1) is an enzyme encoded by the SOD1 gene. SOD1 enzyme is a protein found in abundance in cells throughout the body. SOD1 enzyme is a protein found in the body. When bound to zinc (Zn), it forms what are called superoxide radicals or reactive oxygen species (ROS). Breaks down toxic charged oxygen molecules, which are by-products of normal cellular processes and can cause cellular damage It must be periodically degraded to avoid damage, transformation, or death. At least 200 mutations are associated with a condition characterized by progressive muscle weakness, loss of muscle mass, and loss of motor control. These have been found to cause amyotrophic lateral sclerosis (ALS), a condition that Most of the mutations in change a single amino acid in the superoxide dismutase enzyme. Mutations in the SOD1 gene cause 15 to 20 percent of familial ALS cases worldwide. Approximately half of all Americans with ALS, which is caused by a single gene mutation, have a mutation in the fifth gene responsible for the enzyme A specific mutation that replaces the amino acid residue alanine (A) with the amino acid residue valine (V), i.e., ALS caused by the A5V mutation is generally , resulting in a shorter life expectancy compared to ALS caused by other genetic mutations.

[0227] ALS is caused by the death of nerve cells (motor neurons) that control muscle movement. The reason why motor neurons are particularly susceptible to SOD1 gene mutations is currently unknown, but these The size of these types of neurons makes them more sensitive to disruption of normal SOD1 enzyme function. This may contribute to the death of motor neurons. Some possible embodiments include: (i) cells, particularly lymphocytes; Increased production of harmful superoxide radicals in endothelial cells and / or proteasomes (ii) increased production of other types of toxic radicals and increased cell death; and / or (iii) Aggregation of misfolded superoxide dismutase, which can be toxic to cells Accumulation of agglomerates (aggregates).

[0228] "SOD1 protein" refers to a protein having at least about 95% SOD1 activity relative to NCBI accession number NP_000445. % amino acid sequence identity to the polypeptide or fragment thereof. The SOD1 amino acid sequence is provided below. 1 MATKAVCVLK GDGPVQGIIN FEQKESNGPV KVWGSIKGLT EGLHGFHVHE FGDNTAGCTS 61 AGPHFNPLSR KHGGPKDEER HVGDLGNVTA DKDGVADVSI EDSVISLSGD HCIIGRTLVV 121 HEKADDLGKG GNEESTKTGN AGSRLACGVI GIAQ

[0229] "SOD1 polynucleotide" means a nucleic acid molecule that encodes the SOD1 protein or a fragment thereof. NCBI accession number NC_000021.9:31659622-31668931 (Homo sapiens Exemplary human SOD1 polynucleotides available on chromosome 21, GRCh38.p13 primary assembly) The genomic sequence of is provided below (SEQ ID NO: 3). JPEG2025143260000009.jpg245159JPEG2025143260000010.jpg249159JPEG2025143260000011.jpg249160JPEG2025143260000012.jpg208159

[0230] In the above SOD1 genomic nucleic acid sequence, the "ag" splice at the 5' end of exon 3 of the SOD1 gene The nucleic acid sequence of exon 3 of the SOD1 gene is shown in bold. It is shown in italics and double underlined in the SOD1 genomic sequence below.

[0231] Exemplary human SOD1 polynucleotide mRNA / cDNA available at NCBI Reference Sequence: NM_000454.4 The NA sequence is provided below: gcgtcgtag tctcctgcag cgtctggggt ttccgttgca gtcctcggaa ccaggacctc ggcgtggcct agcgagttat ggcgacgaag gccgtgtgcg tgctgaaggg cgacggccca gtgcagggca tcatcaattt cgagcagaag gaaagtaatg gaccagtgaa ggtgtgggga agcattaaag gactgactga aggcctgcat ggattccatg ttcatgagtt tggagataat acagcaggct gtaccagtgc aggtcctcac tttaatcctc tatccagaaa aacacggtggg ccaaaggatg aagagaggca tgttggagac ttgggcaatg tgactgctga caaagatggt gtggccgatg tgtctattga agattctgtg atctcactct caggagacca ttgcatcatt ggccgcacac tggtggtcca tgaaaaagca gatgacttgg gcaaaggtgg aaatgaagaa agtacaaaga caggaaacgc tggaagtcgt ttggcttgtg gtgtaattgg gatcgcccaa taaacattcc cttggatgta gtctgaggcc ccttaactca tctgttatcc tgctagctgt agaaatgtat cctgataaac attaaacact gtaatcttaa aagtgtaatt gtgtgacttt ttcagagttg ctttaaagta cctgtagtga gaaactgatt tatgatcact tggaagattt gtatagtttt ataaaactca gttaaaatgt ctgtttcaat gacctgtatt ttgccagact taaatcacag atgggtatta aacttgtcag aatttctttg tcattcaagc ctgtgaataa aaaccctgta tggcacttat tatgaggcta ttaaaagaat ccaaattcaa actaaaaaaa aaaaaaaaaa a

[0232] "Androgen receptor (AR) polynucleotide" means an androgen receptor polypeptide. An exemplary polynucleotide encoding an androgen receptor polynucleotide is any polynucleotide encoding an androgen receptor polynucleotide. The nucleotide is provided below (SEQ ID NO: 4): Homo sapiens androgen receptor (AR), transcript variant 1, mRNA (NM_000044.6) ATGGAAGTGCAGTTAGGGCTGGGAAGGGTCTACCCTCGGCCGCCGTCCAAGACCTACCGAGGAGCTTTCCAGAATCTGTT CCAGAGCGTGCGCGAAGTGATCCAGAACCCGGGCCCCAGGCACCCAGAGGCCGCGAGCGCAGCACCTCCCGGCGCCAGTT TGCTGCTGCTGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAA GAGACTAGCCCCAGGCAGCAGCAGCAGCAGCAGGGTGAGGATGGTTCTCCCCAAGCCCATCGTAGAGGCCCCACAGGCTA CCTGGTCCTGGATGAGGAACAGCAACCTTCACAGCCGCAGTCGGCCCTGGAGTGCCACCCCGAGAGAGGTTGCGTCCCAG AGCCTGGAGCCGCCGTGGCCGCCAGCAAGGGGCTGCCGCAGCAGCTGCCAGCACCTCCGGACGAGGATGACTCAGCTGCC CCATCCACGTTGTCCCTGCTGGGCCCCACTTTCCCCGGCTTAAGCAGCTGCTCCGCTGACCTTAAAGACATCCTGAGCGA GGCCAGCACCATGCAACTCCTTCAGCAACAGCAGCAGGAAGCAGTATCCGAAGGCAGCAGCAGCGGGAGAGCGAGGGAGG CCTCGGGGGCTCCCACTTCCTCCAAGGACAATTACTTAGGGGGCACTTCGACCATTTCTGACAACGCCAAGGAGTTGTGT AAGGCAGTGTCGGTGTCCATGGGCCTGGGTGTGGAGGCGTTGGAGCATCTGAGTCCAGGGGAACAGCTTCGGGGGGATTG CATGTACGCCCCACTTTTGGGAGTTCCACCCGCTGTGCGTCCCACTCCTTGTGCCCCATTGGCCGAATGCAAAGGTTCTC TGCTAGACGACAGCGCAGGCAAGAGCACTGAAGATACTGCTGAGTATTCCCCTTTCAAGGGAGGTTACACCAAAGGGCTA GAAGGCGAGAGCCTAGGCTGCTCTGGCAGCGCTGCAGCAGGGAGCTCCGGGACACTTGAACTGCCGTCTACCCTGTCTCT CTACAAGTCCGGAGCACTGGACGAGGCAGCTGCGTACCAGAGTCGCGACTACTACAACTTTCCACTGGCTCTGGCCGGAC CGCCGCCCCCTCCGCCGCCTCCCCATCCCCACGCTCGCATCAAGCTGGAGAACCCGCTGGACTACGGCAGCGCCTGGGCG GCTGCGGCGGCGCAGTGCCGCTATGGGGACCTGGCGAGCCTGCATGGCGCGGGTGCAGCGGGACCCGGTTCTGGGTCACC CTCAGCCGCCGCTTCCTCATCCTGGCACACTCTCTTCACAGCCGAAGAAGGCCAGTTGTATGGACCGTGTGGTGGTGGTG GGGGTGGTGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGAGGCGGGAGCTGTAGCCCCC TACGGCTACACTCGGCCCCCTCAGGGGCTGGCGGGCCAGGAAAGCGACTTCACCGCACCTGATGTGTGGTACCCTGGCGG CATGGTGAGCAGAGTGCCCTATCCCAGTCCCACTTGTGTCAAAAGCGAAATGGGCCCCTGGATGGATAGCTACTCCGGAC CTTACGGGGACATGCGTTTGGAGACTGCCAGGGACCATGTTTTGCCCATTGACTATTACTTTCCACCCCAGAAGACCTGC CTGATCTGTGGAGATGAAGCTTCTGGGTGTCACTATGGAGCTCTCACATGTGGAAGCTGCAAGGTCTTCTTCAAAAGAGC CGCTGAAGGGAAACAGAAGTACCTGTGCGCCAGCAGAAATGATTGCACTATTGATAAATTCCGAAGGAAAAATTGTCCAT CTTGTCGTCTTCGGAAATGTTATGAAGCAGGGATGACTCTGGGAGCCCGGAAGCTGAAGAAACTTGGTAATCTGAAACTA CAGGAGGAAGGAGAGGCTTCCAGCACCACCAGCCCCACTGAGGAGACAACCCAGAAGCTGACAGTGTCACACATTGAAGG CTATGAATGTCAGCCCATCTTTCTGAATGTCCTGGAAGCCATTGAGCCAGGTGTAGTGTGTGCTGGACACGACAACAACC AGCCCGACTCCTTTGCAGCCTTGCTCTCTAGCCTCAATGAACTGGGAGAGAGACAGCTTGTACACGTGGTCAAGTGGGCC AAGGCCTTGCCTGGCTTCCGCAACTTACACGTGGACGACCAGATGGCTGTCATTCAGTACTCCTGGATGGGGCTCATGGT GTTTGCCATGGGCTGGCGATCCTTCACCAATGTCAACTCCAGGATGCTCTACTTCGCCCCTGATCTGGTTTTCAATGAGT ACCGCATGCACAAGTCCCGGATGTACAGCCAGTGTGTCCGAATGAGGCACCTCTCTCAAGAGTTTGGATGGCTCCAAATC ACCCCCCAGGAATTCCTGTGCATGAAAGCACTGCTACTCTTCAGCATTATTCCAGTGGATGGGCTGAAAAATCAAAAATT CTTTGATGAACTTCGAATGAACTACATCAAGGAACTCGATCGTATCATTGCATGCAAAAGAAAAAATCCCACATCCTGCT CAAGACGCTTCTACCAGCTCACCAAGCTCCTGGACTCCGTGCAGCTATTGCGAGAGAGCTGCATCAGTTCACTTTTGAC CTGCTAATCAAGTCACACATGGTGAGCGTGGACTTTCCGGAAATGATGGCAGAGATCATCTCTGTGCAAGTGCCCAAGAT CCTTTCTGGGAAAGTCAAGCCCATCTATTTCCACACCCAG [Brief explanation of the drawings]

[0233] [Figure 1] Figures 1A-1C show plasmids. Figure 1A is an expression vector encoding the TadA7.10-dCas9 base editor. Figure 1B is a plasmid containing a nucleic acid molecule encoding a protein that confers chloramphenicol resistance (CamR) and spectinomycin resistance (SpectR). This plasmid also contains a kanamycin resistance gene disabled by two point mutations. Figure 1C is a plasmid containing a nucleic acid molecule encoding a protein that confers chloramphenicol resistance (CamR) and spectinomycin resistance (SpectR). This plasmid also contains a kanamycin resistance gene disabled by three point mutations. [Figure 2]Figure 2 shows an image of a bacterial colony transduced with the expression vector shown in Figure 1, which contains a nonfunctional kanamycin resistance gene. The vector contained an ABE7.10 variant generated using error-prone PCR. Bacterial cells expressing these "evolved" ABE7.10 variants were selected for kanamycin resistance using increasing concentrations of kanamycin. Bacteria expressing ABE7.10 variants with adenosine deaminase activity were able to correct the mutation introduced into the kanamycin resistance gene and restore kanamycin resistance. Kanamycin-resistant cells were selected for further analysis. [Figure 3] Figures 3A and 3B show editing of the regulatory region of the hemoglobin subunit gamma (HGB1) locus, a therapeutically relevant site for upregulation of fetal hemoglobin. Figure 3A is a diagram of a portion of the regulatory region of the HGB1 gene. Figure 3B quantifies the efficiency and specificity of adenosine deaminase variants. Editing was assayed at the hemoglobin subunit gamma 1 (HGB1) locus in HEK293T cells, which is a therapeutically relevant site for upregulation of fetal hemoglobin. The top panel shows nucleotide residues in the targeted region of the regulatory sequence of the HGB1 gene. A5, A8, A9, and A11 indicate the edited adenosine residues in HGB1. [Figure 4] Figure 4 shows the relative efficacy of adenosine base editors, including dCas9, that recognize non-canonical PAM sequences. The top panel shows the coding sequence for a hemoglobin subunit. The bottom panel shows the efficiency of adenosine deaminase variant base editors with guide RNAs of various lengths. [Figure 5] Figure 5 is a graph showing the efficiency and specificity of the ABE8 base editor, quantitating the percent editing at the intended target nucleotide and at unintended target nucleotides (bystanders). [Figure 6]Figure 6 is a graph showing the efficiency and specificity of the ABE8 base editor, quantitating the percent editing at intended target nucleotides and unintended target nucleotides (bystanders). [Figure 7] Figures 7A–7D show that eighth-generation adenine base editors mediate superior A·T to G·C conversion in human cells. Figure 7A shows an overview of adenine base editing: i) ABE8 creates an R-loop at the sgRNA targeting site in the genome; ii) TadA* deaminase chemically converts adenine to inosine through hydrolytic deamination of the ss-DNA portion of the R-loop; iii) Cas9 D10A nickase nicks the strand opposite the inosine-containing strand; iv) The inosine-containing strand can be used as a template during DNA replication; v) In the case of DNA polymerase, inosine preferentially base pairs with cytosine; and vi) After replication, inosine is replaced with guanosine. Figure 7B shows the architecture of ABE8.xm and ABE8.xd. Figure 7C shows three perspective views of E. coli TadA deaminase (PDB 1Z3A) aligned with S. aureus TadA (not shown) in complex with tRNA Arg2 (PDB 2B3J). Mutations identified over eight rounds of evolution are highlighted. Figure 7D is a graph showing the A·T to G·C base editing efficiency of the core ABE8 construct relative to the ABE7.10 construct across eight genomic sites in Hek293T cells. Values ​​and error bars reflect the mean and SD of three independent biological replicates performed on different days.

[0234] [Figure 8]Figures 8A–8C show that the Cas9 PAM variant ABE8 and the catalytically inactive Cas9 ABE8 variant mediate higher A·T to G·C conversion in human cells than the corresponding ABE7.10 variant. Values ​​and error bars reflect the mean and standard deviation (SD) of three independent biological replicates performed on different days. Figure 8A shows A·T to G·C conversion in Hek293T cells harboring NG-Cas9 ABE8s (-NG PAM). Figure 8B shows A·T to G·C conversion in Hek293T cells harboring Sa-Cas9 ABE8s (-NNGRRT PAM). Figure 8C shows A·T to G·C conversion in Hek293T cells harboring catalytically inactive dCas9-ABE8s (S. pyogenes Cas9 D10A, H840A). [Figure 9] Figures 9A-9E show a comparison of on-target and off-target editing frequencies between ABE7.10, ABEmax, and ABEmax with one BPNLS in Hek293T cells. Individual data points are shown for n=3 independent biological replicates performed on different days, and error bars represent standard deviation (SD). Figures 9A and 9B are graphs showing on-target DNA editing frequencies. Figures 9B and 9C are graphs showing the frequency of sgRNA-induced DNA off-target editing. Figure 9E is a graph showing RNA off-target editing frequencies. [Figure 10] Figures 10A-10B show the median A·T to G·C conversions and corresponding indel formation of TadA, C-terminal α-helical truncated ABE constructs in HEK293T cells. Figure 10A is a heatmap showing the median A·T to G·C editing conversions across eight genomic sites. Figure 10B is a heatmap showing indel formation. Delta residue values ​​correspond to the deletion position in TadA. Median values ​​generated from n=3 biological replicates. [Figure 11]Figure 11 is a heatmap showing the median A·T to G·C conversions of 40 ABE8 constructs across eight genomic sites in HEK293T cells. Medians were determined from two or more biological replicates. [Figure 12] Figure 12 is a heatmap showing the median % indels of 40 ABE8 constructs across eight genomic sites in HEK293T cells. Medians were determined from two or more biological replicates. [Figure 13] Figure 13 is a graph showing fold change in editing (ABE8:ABE7). Representation of average ABE8:ABE7 A·T→G·C editing across all A positions within targets at eight different genomic sites in Hek293T cells. Positions 2-12 indicate the location of the target adenine within the 20 nt protospacer, with position 20 immediately 5' to the -NGG PAM. [Figure 14] Figure 14 shows the ABE8 dendrogram, with the core ABE8 constructs selected for further study highlighted in black. [Figure 15] Figure 15 is a heatmap showing the median A⋅T to G⋅C conversions of the eight core ABE8 constructs across eight genomic sites in HEK293T cells. Medians were determined from three or more biological replicates.

[0235] [Figure 16] FIG. 16 is a heatmap showing the median indel frequencies of the eight core ABE8s tested at eight genomic sites in HEK293T cells. [Figure 17] Figure 17 is a heatmap showing the median A·T to G·C conversion of core NG-ABE8 construct 9 (-NG PAM) at six genomic sites in HEK293T cells. Median values ​​generated from n=3 biological replicates. [Figure 18]Figure 18 is a heatmap showing the median indel frequencies of core NG-ABE8 tested at six genomic sites in HEK293T cells. Median values ​​generated from n=3 biological replicates. [Figure 19] Figure 19 is a heatmap showing the median A·T to G·C conversion of the core Sa-ABE8 construct (-NNGRRT PAM) at six genomic sites in HEK293T cells. Site positions within the 22 nt protospacer are numbered from -2 to 20 (5' to 3'). Position 20 is 5' to the NNGRRT PAM. Median values ​​derived from n=3 biological replicates. [Figure 20] Figure 20 is a heatmap showing the median indel frequencies of core sa-ABE8 tested at 8 genomic sites in HEK293T cells. Median values ​​generated from n=3 biological replicates. [Figure 21] Figure 21 is a heatmap showing median A T to G C conversions of the core dC 9-ABE8-m construct at eight genomic sites in HEK293T cells. Dead Cas9 (dC 9) is defined as the D10A and H840A mutations in S. pyogenes Cas9. Median across three or more biological replicates. [Figure 22] Figure 22 is a heatmap showing median A T to G C conversions of the core dC9-ABE8-d construct at eight genomic sites in HEK293T cells. Dead Cas9 (dC9) is defined as the D10A and H840A mutations in S. pyogenes Cas9. Median values ​​generated from n≧3 biological replicates. [Figure 23]Figures 23A and 23B show the median indel frequencies of core dC9-ABE8 tested at eight genomic sites in HEK293T cells. Median values ​​generated from n≧3 biological replicates. Figure 23A is a heatmap showing indels indicated for the dC9-ABE8-m variant compared to ABE7.10. Figure 23B is a heatmap showing indels indicated for the dC9-ABE8-d variant compared to ABE7.10. [Figure 24] Figure 24 shows C·G to T·A editing by Hek293T cells treated with ABE8 and ABE7.10. Editing frequency for each site averaged across all C positions within the target. Cytosines within the protospacer are shaded. [Figure 25] Figures 25A-25H show DNA on-target editing and sgRNA-mediated DNA off-target editing by the ABE8 construct and an ABE8 construct with TadA mutations to improve DNA specificity. Individual data points are shown, and error bars represent the standard deviation (sd) for n=3 independent biological replicates performed on different days. Figures 25A and 25B are graphs showing the on-target DNA editing frequency for the core ABE8 construct compared to ABE7. Figures 25C and 25D are graphs showing the on-target DNA editing frequency for ABE8 with mutations that improve RNA off-target editing. Figures 25E and 25F are graphs showing the sgRNA-guided DNA off-target editing frequency for the core ABE8 construct compared to ABE7. Figures 25G and 25H are graphs showing the gRNA-guided DNA off-target editing frequency for ABE8 constructs with mutations that improve RNA off-target editing.

[0236] [Figure 26]Figure 26 is a graph showing indel frequencies at 12 previously identified sgRNA-dependent Cas9 off-target loci in human cells; individual data points are shown and error bars represent the s.d. for n=3 independent biological replicates performed on different days. [Figure 27] Figures 27A and 27B show A·T to G·C conversion in primary cells and the phenotypic results. Figure 27A is a graph showing A·T to G·C conversion at the -198 HBG1 / 2 site in ABE-treated CD34+ cells from two separate donors. NGS analysis performed 48 and 144 hours after treatment. The -198 HBG1 / 2 target sequence is shown with A7 highlighted. Percent A·T→G·C plotted for A7. Figure 27B is a graph showing the percentage of gamma globin formed as a percentage of alpha globin. Values ​​shown are from two different donors after ABE treatment and erythroid differentiation. [Figure 28] Figures 28A and 28B show A·T→G·C transversions at the −198 promoter site upstream of HBG1 / 2 in CD34+ cells treated with ABE8. Figure 28A is a heatmap showing the frequency of ABE8 A→G edits at 48 and 144 hours after editor treatment in CD34+ cells from two donors, where donor 2 is heterozygous for sickle cell disease. Figure 28B is a graphical representation of the distribution of total sequencing reads containing either the A7 edit alone or the (A7+A8) combined edit. [Figure 29] Figure 29 is a heat map showing indel frequencies at -198 in the gamma globin promoter in ABE8-treated CD34+ cells. Frequencies shown are from two donors at 48 and 144 hours. [Figure 30] FIG. 30 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of untreated differentiated CD34+ cells (donor 1). [Figure 31] FIG. 31 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE7.10-m. [Figure 32] FIG. 32 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE7.10-d. [Figure 33] Figure 33 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE8.8-m.

[0237] [Figure 34] FIG. 34 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE8.8-d. [Figure 35] FIG. 35 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE8.13-m. [Figure 36] FIG. 36 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE8.13-d. [Figure 37] FIG. 37 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE8.17-m. [Figure 38] FIG. 38 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE8.17-d. [Figure 39] FIG. 39 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE8.20-m. [Figure 40]FIG. 40 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE8.20-d. [Figure 41] Figure 41 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of untreated differentiated CD34+ cells (donor 2). Note: Donor 2 is heterozygous for sickle cell disease. [Figure 42] Figure 42 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE7.10-m. Note: Donor 2 is heterozygous for sickle cell disease. [Figure 43] Figure 43 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE7.10-d. Note: Donor 2 is heterozygous for sickle cell disease. [Figure 44] Figure 44 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE8.8-m. Note: Donor 2 is heterozygous for sickle cell disease. [Figure 45] Figure 45 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE8.8-d. Note: Donor 2 is heterozygous for sickle cell disease. [Figure 46] Figure 46 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE8.13-m. Note: Donor 2 is heterozygous for sickle cell disease.

[0238] [Figure 47]Figure 47 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE8.13-d. Note: Donor 2 is heterozygous for sickle cell disease. [Figure 48] Figure 48 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE8.17-m. Note: Donor 2 is heterozygous for sickle cell disease. [Figure 49] Figure 49 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE8.17-d. Note: Donor 2 is heterozygous for sickle cell disease. [Figure 50] Figure 50 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE8.20-m. Note: Donor 2 is heterozygous for sickle cell disease. [Figure 51] Figures 51A-51E show that editing by ABE8.8 at two independent sites reached over 90% editing before enucleation on day 11 after erythroid differentiation and approximately 60% gamma globin relative to alpha globin or total beta family globins on day 18 after erythroid differentiation. Figure 51A is a graph showing the average of ABE8.8 editing in two healthy donors in two independent experiments. Editing efficiency was measured with primers that distinguish between HBG1 and HBG2. Figure 51B is a graph showing the average of one healthy donor in two independent experiments. Editing efficiency was measured with primers that recognize both HBG1 and HBG2. Figure 51C is a graph showing ABE8.8 editing in a donor with a heterozygous E6V mutation. Figures 51D and 51E are graphs showing the increase in gamma globin in ABE8.8-edited cells. [Figure 52]Figures 52A and 52B show the percent editing (%Editing) using ABE variants to correct sickle cell mutations. Figure 52A is a graph showing screening of different editor variants with approximately 70% editing in SCD patient fibroblasts. Figure 52B is a graph showing CD34 cells from a healthy donor edited with the lead ABE variant, targeting a synonymous mutation A13 at the adjacent proline that lies within the editing window and serves as a proxy for editing of the SCD mutation. The ABE8 variant showed an average editing frequency of approximately 40% at proxy A13. [Figure 53] Figures 53A and 53B show RNA amplicon sequencing to detect intracellular A→I editing in RNA associated with ABE treatment. Individual data points are shown, and error bars represent the standard deviation (sd) for n=3 independent biological replicates performed on different days. Figure 53A is a graph showing the frequency of A→I editing in target RNA amplicons for the core ABE8 construct compared to ABE7 and Cas9 (D10A) nickase controls. Figure 53B is a graph showing the frequency of A→I editing in target RNA amplicons for ABE8 with mutations reported to improve RNA off-target editing.

[0239] [Figure 54] Figure 54 is a bar graph showing the total percent of A to G base editing at splice acceptor target sites (AG nucleic acid sites) achieved by the assayed adenosine base editors (ABE8 variants) compared to the control. The protospacer PAM sequence of the ABE evaluated was NGG ABE. [Figure 55]Figure 55 shows a schematic diagram of the exon 3 splice acceptor of the SOD1 genomic nucleic acid sequence as a target for an A-to-G nucleotide change that causes splice disruption of the SOD1 exon 3 transcript. Shown in the diagram are the genomic DNA location number (top) in the exon 3 region of the SOD1 nucleic acid sequence; the SOD1 genomic nucleic acid sequence in the exon 3 region; the intronic nucleic acid sequence 5' of exon 3; the target splice acceptor nucleic acid sequence 5' of exon 3; and the nucleic acid sequence of the corresponding guide RNA (gRNA), with the splice acceptor ("AG") nucleic acid immediately 5' of SOD1 exon 3 indicated by an upward arrow. Also shown are bystander adenosine (A) nucleobases (boxed) in the intronic sequence near the AG splice acceptor 5' of SOD1 exon 3. [Figure 56] Figure 56 is a table showing the efficiency of target site nucleobase modification in a target nucleic acid sequence observed after PCR and deep sequencing of DNA using the base editor system and methods described herein. Shown is the percentage efficiency of precise A-to-G nucleotide changes (modifications) in the target splice acceptor (AG) nucleic acid sequence located 5' of exon 3 of the SOD1 genomic nucleic acid sequence, as detected by PCR of genomic DNA in cells where base editing occurred, followed by deep sequencing (MySeq). Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid (in base pairs (bp)) is shown along the bottom of the table. The table shows an efficiency of approximately 81% (80.77%) of A-to-G conversion at the splice acceptor target nucleic acid site (position 6 along the target site) using the ABE8 base editor variant and the systems and methods described herein. Modification of bystander A nucleobases to G nucleobases (positions 2, 3, and 4 shown boxed to the left of the target A nucleobase in the splice acceptor) was minimal compared to editing of the target A in the splice acceptor nucleic acid sequence.

[0240] [Figure 57] Figures 57A-57L are tables showing the efficiency of target site nucleobase modification in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 58] Figure 58 is a bar graph showing the percentage of A to G base editing at Target Position 6 of the splice acceptor target site (AG nucleic acid site) achieved by an adenosine base editor (ABE8 variant) with assayed guide 20 compared to a control. The protospacer PAM sequence of the ABE evaluated was NGG ABE. [Figure 59] Figure 59 is a bar graph showing the percentage of A to G base editing at target position 5 (left) and the total percentage of A to G base editing at positions 2, 5, and 9 (right) in the splice acceptor target site (AG nucleic acid site) achieved by an adenosine base editor (ABE8 variant) using assayed guide 42 compared to a control. The protospacer PAM sequence of the ABE evaluated was NGG ABE. [Figure 60]Figure 60A is a bar graph showing the percentage of A to G base editing at target position 5 in a splice acceptor target site (AG nucleic acid site) achieved by an adenosine base editor (PV variant) with assayed guide 41 compared to a control. The protospacer PAM sequence for the ABE evaluated was NGT ABE. Figure 60B is a table showing the efficiency of target site nucleobase modification in a target nucleic acid sequence observed after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56 above. [Figure 61] Figure 61 is a bar graph showing the total percent of A to G base editing at splice acceptor target sites (AG nucleic acid sites) achieved by adenosine base editors (PV variants) with assayed guide 24 compared to controls. The protospacer PAM sequence of the ABE evaluated was NGA ABE. [Figure 62] Figure 62 shows SOD1 protein levels (left: Western blot, right: quantification) in HEK297T cells edited with ABE8.8 or ABE7.10 using guide 20. Beta actin was used as a control. [Figure 63] Figure 63 is a bar graph showing the percentage of C→T base editing at target position 8 in a splice acceptor target site (AG nucleic acid site) achieved by a cytidine base editor (PV variant) with assayed guide 19 compared to a control. The protospacer PAM sequence of the ABE evaluated was NGC CBE.

[0241] [Figure 64]Figure 64A is a bar graph (left) showing the percentage of A to G base editing at target position 5 in a splice acceptor target site (AG nucleic acid site) achieved by an adenosine base editor (PV variant) with assayed guide 41, compared to a control. The protospacer PAM sequence of the ABE evaluated was NGT ABE. On the right is a diagram showing the numerical genomic DNA positions in the region of exon 3 of the SOD1 nucleic acid sequence to which guides 41, 20, 40, and 21 are designed to bind. See also Figure 55 above. Figure 64B is a table showing the efficiency of target site nucleobase modification in a target nucleic acid sequence after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in DNA (in base pairs (bp)) along the target site nucleic acid is shown along the bottom of the table. See also Figure 56 above. [Figure 65] Figure 65 is a bar graph (left) showing the total percent A to G base editing at splice acceptor target sites (AG nucleic acid sites) achieved by adenosine base editors (ABE variants) with assayed guide 42 compared to a control. The protospacer PAM sequence of the ABE evaluated was NGG ABE. On the right is a diagram showing the numerical genomic DNA positions in the region of exon 3 of the SOD1 nucleic acid sequence to which guides 42, 24, and 25 are designed to bind. See also Figure 55 above. [Figure 66]Figure 66A is a bar graph showing the total percent of C to T base editing achieved by the cytidine base editor (BE4 VRQR) with assayed guide 40 compared to a control. The protospacer PAM sequence for the CBE evaluated was NGA CBE. Figure 66B is a table showing the efficiency of target site nucleobase modification in a target nucleic acid sequence observed after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 67] Figure 67 A is a bar graph showing the percentage of A to G base editing at target position 5 in a splice acceptor target site (AG nucleic acid site) achieved by an adenosine base editor (PV variant) with assayed guide 18 compared to a control. The protospacer PAM sequence of the ABE evaluated was NGT ABE. [Figure 68] Figure 68 is a table showing the efficiency of target site nucleobase modification in a target nucleic acid sequence after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above.

[0242] [Figure 69] Figure 69 is a bar graph showing the total percent of C to T base editing achieved by the cytidine base editor (BE4 VRQR) with guide 16 assayed compared to the control. The protospacer PAM sequence of the CBE evaluated was NGA CBE. [Figure 70]Figure 70 is a bar graph showing the percentage of C to T base editing at target position 4 achieved by a cytidine base editor (BE4 VRQR) with assayed guide 17 compared to a control. The protospacer PAM sequence of the CBE evaluated was an NGA CBE. [Figure 71] Figure 71 is a bar graph showing the percentage of C to T base editing at target position 5 achieved by a cytidine base editor (BE4 VRQR) with assayed guide 21 compared to a control. The protospacer PAM sequence of the CBE evaluated was an NGA CBE. [Figure 72] Figures 72A-72L are tables showing the efficiency of target site nucleobase modification in target nucleic acid sequences observed after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 73] Figures 73A-73C are tables showing the efficiency of target site nucleobase modification in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 74] Figures 74A-74C are tables showing the efficiency of target site nucleobase modification in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 75]Figures 75A-75D are tables showing the efficiency of target site nucleobase modification in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above.

[0243] [Figure 76] Figures 76A-76L are tables showing the efficiency of target site nucleobase modification in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 77] Figures 77A-77L are tables showing the efficiency of target site nucleobase modification in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 78] Figures 78A-78D are tables showing the efficiency of target site nucleobase modifications in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 79]Figures 79A-79L are tables showing the efficiency of target site nucleobase modification in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 80] Figure 80 shows that a stop codon was introduced into exon 1 of the androgen receptor when "CAG" was targeted using a cytidine base editor (CBE). [Figure 81] Figure 81 provides two graphs. The graph on the left shows the percent C to T editing by the specified cytidine base editor. The graph on the right shows the percent indel rate for each cytidine base editor. [Figure 82] Figures 82A-82I are tables showing the percentage efficiency of C→T editing in AR nucleic acid sequences using CBE base editor variants, as detected by PCR of genomic DNA in cells in which base editing occurred, followed by deep sequencing (MySeq). Figures 82A-82I show the percent C→T base editing at position 6 of the AR nucleic acid target site. Control reactions using water instead of CBE are shown.

[0244] [Figure 83]Figure 83A provides two graphs. The graph on the left shows that the introduction of a premature stop codon in exon 1 resulted in functional knockout of the androgen receptor in the majority of cells. The middle graph shows the percent indel rate for each cytidine base editor. The right is a diagram showing the numerical genomic DNA position in the region of exon 1 of the AR nucleic acid sequence where guide 8 is designed to bind. See also Figure 80 above. Figure 83B is a table showing the percentage efficiency of C→T editing in the AR nucleic acid sequence using CBE base editor variants, as detected by PCR followed by deep sequencing (MySeq) of genomic DNA in cells where base editing occurred. Figure 83B shows the percent C→T base editing at the target position in the AR nucleic acid target site. Control reactions using water instead of CBE are shown. [Figure 84] Figure 84A is a bar graph showing the percentage of C to T base editing at target position 6 achieved by cytidine base editors (BGX5, BGX27, BGX29, and BTX448) using guide 10 compared to controls (left) and the percentage indel rate (center). The protospacer PAM sequence of the CBE evaluated was NGG CBE. On the right is a diagram showing the numerical genomic DNA position in the region of exon 1 of the AR nucleic acid sequence to which guides 9 and 10 are designed to bind. See also Figure 80 above. Figure 84B is a table showing the efficiency of the percentage of C to T editing in AR nucleic acid sequences using CBE base editor variants, as detected by PCR followed by deep sequencing (MySeq) of genomic DNA in cells in which base editing occurred. Figure 83B shows the percent of C to T base editing at position 6 of the AR nucleic acid target site. Control reactions are shown in which water was used instead of CBE. [Figure 85]Figure 85 is a bar graph showing the total percent A to G base editing at splice acceptor target sites (AG nucleic acid sites) achieved by adenosine base editors (ABE8 variants) with assayed guide 8 (left) or guide 14 (right) compared to controls. The protospacer PAM sequence of the ABE evaluated was NGG ABE. [Figure 86] Figure 86A is a bar graph (top) and summary table (bottom) showing the percentage of C to T base editing at target position 8 achieved by a cytidine base editor (BE4 VRQR) with assayed guide 11 compared to a control. The protospacer PAM sequence for the CBE evaluated was NGA CBE. Figure 86B is a table showing the percentage efficiency of C to T editing in AR nucleic acid sequences using CBE base editor variants, as detected by PCR of genomic DNA in cells in which base editing occurred followed by deep sequencing (MySeq). Figure 86B shows the percent C to T base editing at position 8 in the AR nucleic acid target site. A control reaction is shown in which water was used instead of CBE.

[0245] [Figure 87] Figure 87A is a bar graph (top) and summary table (bottom) showing the percentage of C to T base editing at target position 5 achieved by a cytidine base editor (BE4 VRQR) with assayed guide 12 compared to a control. The protospacer PAM sequence for the CBE evaluated was NGA CBE. Figure 87B is a table showing the percentage efficiency of C to T editing in AR nucleic acid sequences using CBE base editor variants, as detected by PCR of genomic DNA in cells in which base editing occurred followed by deep sequencing (MySeq). Figure 87B shows the percent C to T base editing at position 5 of the AR nucleic acid target site. A control reaction is shown in which water was used instead of CBE. [Figure 88]Figure 88 is a bar graph showing the total percent of C to T base editing achieved by the cytidine base editor (BE4 VRQR) with assayed guide 15 compared to the control. The protospacer PAM sequence of the CBE evaluated was NGA CBE. Each nucleobase A, C, G, and T is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. [Figure 89] Figures 89A-89D are tables showing the efficiency of target site nucleobase modification in a target nucleic acid sequence after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Shown are the percentage efficiencies of precise C→T or A→G nucleotide changes (modifications) in the target nucleic acid sequence in exon 1 of the AR genomic nucleic acid sequence, as detected by PCR of genomic DNA in cells in which base editing occurred, followed by deep sequencing (MySeq). Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. [Figure 90] Figures 90A-90C are tables showing the efficiency of target site nucleobase modification in a target nucleic acid sequence after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Shown are the percentage efficiencies of precise C→T or A→G nucleotide changes (modifications) in the target nucleic acid sequence in exon 1 of the AR genomic nucleic acid sequence, as detected by PCR of genomic DNA in cells in which base editing occurred, followed by deep sequencing (MySeq). Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. [Figure 91]Figures 91A-91C are tables showing the efficiency of target site nucleobase modifications in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Shown are the percentage efficiencies of precise C→T or A→G nucleotide changes (modifications) in target nucleic acid sequences in exon 1 of the AR genomic nucleic acid sequence, as detected by PCR of genomic DNA in cells in which base editing occurred, followed by deep sequencing (MySeq). Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid (in base pairs (bp)) is shown along the bottom of the table. [Figure 92] Figures 92A and 92B are tables showing the efficiency of target site nucleobase modifications in target nucleic acid sequences observed after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Shown are the percentage efficiencies of precise C→T or A→G nucleotide changes (modifications) in target nucleic acid sequences in exon 1 of the AR genomic nucleic acid sequence, as detected by PCR of genomic DNA in cells in which base editing occurred, followed by deep sequencing (MySeq). Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. [Figure 93] Figures 93A-93X are tables showing the efficiency of target site nucleobase modification in a target nucleic acid sequence after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Shown are the percentage efficiencies of precise C→T or A→G nucleotide changes (modifications) in the target nucleic acid sequence in exon 1 of the AR genomic nucleic acid sequence, as detected by PCR of genomic DNA in cells in which base editing occurred, followed by deep sequencing (MySeq). Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. DETAILED DESCRIPTION OF THE INVENTION

[0246] The present invention provides novel adenine nucleotide sequences with increased efficiency for generating targeted nucleic acid base sequence modifications. Compositions comprising base editors (e.g., ABE8) and methods of using them are provided.

[0247] [Nucleobase Editor] A base editor for editing, modifying or altering a target nucleotide sequence of a polynucleotide. Disclosed herein are nucleic acid base editors or nucleobase editors. In certain embodiments, the present invention In certain embodiments, the base editors of the present invention modify the SOD1 polynucleotide. The base editor may introduce a stop codon or splice in the AR polynucleotide. Described herein are polynucleotide programmable nucleic acid sequences that disrupt the nucleic acid site. Nucleotide-binding domains (e.g., Cas9) and nucleobase-editing domains (e.g., adenosine deaminase). A programmable nucleotide-binding domain (e.g., Cas9) binds to the bound guide polypeptide. When combined with a nucleotide (e.g., gRNA), the bases of the bound guide nucleic acid and the target a target polynucleotide (through complementary base pairing between the bases of the polynucleotide sequence) capable of specifically binding to a sequence, thereby targeting the target nucleic acid desired to be edited. In some embodiments, the base editor can be localized to the target polynucleotide sequence. The nucleic acid sequence comprises single-stranded or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid. Includes hood.

[0248] Polynucleotide-programmable nucleotide-binding domains Polynucleotide programmable nucleotide binding domains also bind to nuclear RNA. It is understood that the present invention may include an acid programmable protein. The polynucleotide-programmable nucleotide binding domain is The nucleotide-binding domain can be linked to a nucleic acid that guides the RNA. Other DNA-binding proteins are also within the scope of this disclosure, although they are not specifically listed in this disclosure. It has not been done.

[0249] The polynucleotide-programmable nucleotide-binding domain of the base editor is The polynucleotide programmable vector itself can contain one or more domains. The nucleotide-binding domain capable of binding to the nuclease may include one or more nuclease domains. In some embodiments, the nucleic acid of a polynucleotide-programmable nucleotide binding domain The nuclease domain can comprise an endonuclease or an exonuclease. As used herein, the term "exonuclease" refers to an enzyme that liberates nucleic acids (e.g., RNA or DNA). The term "endonuclease" refers to a protein or polypeptide that can be digested from its termini. A "clease" is a nucleic acid that can catalyze (e.g., cleave) an internal region of a nucleic acid (e.g., DNA or RNA). In some embodiments, an endonuclease refers to a protein or polypeptide that It is capable of cleaving a single strand of double-stranded nucleic acid. Both strands of a double-stranded nucleic acid molecule can be cleaved. The programmable nucleotide binding domain can be a deoxyribonuclease. In some embodiments, the polynucleotide programmable nucleotide binding domain is a ribonucleotide. It may be a nuclease.

[0250] In one embodiment, the nucleotide of the polynucleotide-programmable nucleotide binding domain The cleavage domain can cleave zero, one, or two strands of a target polynucleotide. In one embodiment, the polynucleotide-programmable nucleotide binding domain The amino acid may comprise a nickase domain. " refers to a nucleic acid that can cleave only one of the two strands in a double-stranded nucleic acid molecule (e.g., DNA). a polynucleotide-programmable nucleotide-binding domain containing a cleavage domain; In some embodiments, the nickase is an active polynucleotide programmable nuclease. By introducing one or more mutations into the nucleotide binding domain, polynucleotide protease activity can be increased. Derived from a fully catalytically active (e.g., native) form of a programmable nucleotide-binding domain For example, polynucleotide programmable nucleotide binding domains can be used. If the gene contains a nickase domain derived from Cas9, the nickase domain derived from Cas9 The protein may contain a D10A mutation and a histidine at position 840. In embodiments, residue H840 retains catalytic activity, thereby cleaving a single strand of a nucleic acid duplex. In another example, the nickase domain from Cas9 contains an H840A mutation. while the amino acid residue at position 10 remains D. In some embodiments, the nickase may comprise all of the nuclease domain that is not required for nickase activity. or by removing a portion of the polynucleotide, programmable nucleotide binding It can be derived from a fully catalytically active (e.g., native) form of the domain. A nucleotide-programmable nucleotide-binding domain derived from Cas9 is used to identify nickases If the domain is included, the nickase domain from Cas9 is a RuvC domain or an HNH domain. It may contain a deletion of all or part of the domain.

[0251] The amino acid sequence of an exemplary catalytically active Cas9 is as follows: MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEGKKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD.

[0252] Thus, polynucleotides containing nickase domains can be used as programmable nucleotides. Base editors containing a base-binding domain can be used to bind to specific polynucleotide target sequences (e.g., binding domains). The DNA fragments are then ligated to the target nucleic acid, which then generates a single-stranded DNA break (a nick) at the target site (determined by the complementary sequence of the guide nucleic acid). In some embodiments, a nickase domain (e.g., a nickase domain derived from Cas9) can be used. Nucleic acid double-stranded target polynucleotides cleaved by base editors containing a base editor domain (a cleavage enzyme domain) The strand of the base sequence is the strand that is not edited by the base editor (i.e., (The strand cleaved by the cleavage is the opposite strand to the strand containing the base to be edited.) and base editors containing a nickase domain (e.g., a nickase domain derived from Cas9). can cleave the strand of a DNA molecule targeted for editing. In this state, the non-target strand is not cleaved.

[0253] Catalytically dead (i.e., unable to cleave target polynucleotide sequence) polynucleotides Base editors comprising nucleotide-programmable nucleotide-binding domains are also described herein. As used herein, the terms "catalytically dead" and "nuclease inactive" has one or more mutations and / or deletions that result in the inability to cleave a strand of nucleic acid. Polynucleotides with deletions are replaced to refer to programmable nucleotide binding domains. In some embodiments, catalytically dead polynucleotide proteases are used. A gram-capable nucleotide-binding domain base editor binds one or more nuclease domains Nuclease activity can be lost as a result of specific point mutations in the In the case of base editors containing the Cas9 domain, Cas9 is able to reverse the D10A and H840A mutations. Such a mutation would inactivate both nuclease domains. In another embodiment, catalytically dead polynucleotides are The nucleotide-programmable nucleotide-binding domain is a catalytic domain (e.g., RuvC1 and and / or the HNH domain). In embodiments, catalytically dead polynucleotide programmable nucleotide binding domains The mutants contain point mutations (e.g., D10A or H840A) as well as all or part of the nuclease domain. Contains some deletions.

[0254] Also provided herein are the following polynucleotide-programmable nucleotide binding domains: Catalytically dead polynucleotide programmable nucleic acid from a previously functioning version Mutations that can generate peptide-binding domains are also contemplated. In the case of dead Cas9 ("dCas9"), mutations other than D10A and H840A are present, leading to the nuclease Variants that result in inactive Cas9 are provided. Such mutations include, for example, D10 and other amino acid substitutions at H840 or other substitutions within the nuclease domain of Cas9. (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain) Additional suitable nuclease-inactive dCas9 domains are described in this disclosure and in the art. Such modifications would be apparent to those skilled in the art based on their knowledge and are within the scope of the present disclosure. Exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to: Contains D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (e.g., Prashant et al., CAS9 transcriptional activators for target specific ficity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference. incorporated into the book).

[0255] Polynucleotide programmable nucleotides that can be incorporated into base editors Non-limiting examples of binding domains include domains derived from CRISPR proteins, restriction nucleases, and the like. Enzymes, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases In some embodiments, the base editor is a CRISP gene for nucleic acids. R (i.e., Clustered Regularly Interspaced Short Palindromic Repeats)-mediated modifications In this case, natural or modified nucleic acids that can bind to a nucleic acid sequence via a binding guide nucleic acid are used. Polynucleotide-programmable nucleotide-binding domains containing proteins or portions thereof Such proteins are referred to herein as "CRISPR proteins." Thus, disclosed herein are methods for detecting CRISPR proteins, including all or part of the proteins. Polynucleotide base editors containing programmable nucleotide binding domains (i.e. That is, a base editor that contains all or part of the CRISPR protein as a domain (which is It is also called the "CRISPR protein-derived domain" of the base editor. The CRISPR protein-derived domain integrated into the CRISPR receptor is expressed as a wild-type or naturally occurring CRISPR protein. For example, as described below, CRISPR proteins can be modified relative to the CRISPR protein. The CRISPR domain may have one or more mutations, as compared to a wild-type or naturally occurring CRISPR protein, It may involve insertions, deletions, rearrangements and / or recombinations.

[0256] CRISPR provides defense against mobile genetic elements (viruses, transposable elements, conjugative plasmids) CRISPR clusters are composed of spacers, sequences that are complementary to the preceding mobile element. The CRISPR cluster contains a sequence of target nucleic acids, and the target invader nucleic acid. The CRISPR cluster is transcribed into CRISPR RNA (crRNA). In type II CRISPR systems, correct processing of pre-crRNA is essential for transcription. transcoding small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein tracrRNA guides the processing of pre-crRNA by ribonuclease 3. Cas9 / crRNA / tracrRNA then binds to a linear or circular dsDNA target complementary to the spacer. The target strand that is not complementary to the crRNA is first cleaved by the endonuclease. It is cleaved protease-wise and then exonucleolytically trimmed to 3'-5'. In the case of mitochondrial DNA, both the protein and the RNA are required for DNA binding and cleavage. A single guide RNA ("sgRNA", For example, Jinek M. et al., Science 337: 816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 targets a short motif (PAM or protospacer adjacent motif) in the CRISPR repeats. It helps us to recognize and distinguish between "self" and "non-self."

[0257] In some embodiments, the methods described herein involve recombinantly engineered The guide RNA (gRNA) is required for Cas binding. The required scaffold sequence and a user-defined approximately 20-base spacer that defines the genomic target to be modified. Therefore, those skilled in the art can identify the genomic target of Cas protein specificity. The gRNA targeting sequence can be varied to target the genome relative to the rest of the genome. This is determined in part by how specific it is to the target.

[0258] In some embodiments, the gRNA scaffold sequence is: GUUUUAGAGC UAGAAAU AGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAAGU GGCACCGAGU CGGUGCUUUU.

[0259] In some embodiments, the base editor comprises a CRISPR protein-derived domain. The main component is capable of binding to a target polynucleotide when combined with a binding guide nucleic acid. Endonucleases (e.g., deoxyribonucleases or ribonucleases) that can In some embodiments, the CRISPR protein incorporated into the base editor The protein-derived domain binds to the target polynucleotide when combined with the binding guide nucleic acid. In some embodiments, the base editor is a nickase that can bind to The CRISPR protein-derived domains integrated into the CRISPR protein bind to the CRISPR protein when combined with the guide nucleic acid. A catalytically dead domain is capable of binding to a target polynucleotide when In embodiments, a target polynucleotide that binds to a CRISPR protein-derived domain of a base editor is The nucleic acid is DNA, and in some embodiments, the nucleic acid comprises a CRISPR protein-derived domain of a base editor. The target polynucleotide that binds to the in is RNA.

[0260] The CAs proteins that can be used herein include class 1 and class 2. Non-limiting examples of proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5d, Cas5t, as5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also called Csn1 or Csx12), Cas10, Csy1, C sy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm 5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Cssx16, Cx16, Cx, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1 , Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i, CARF, DinG, and their homologs Unmodified CRISPR enzymes, like Cas9, are composed of two It has functional endonuclease domains, RuvC and HNH, and can have DNA cleavage activity. CRISPR enzymes target sequences, such as within the target sequence and / or the complementary strand of the target sequence. For example, CRISPR enzymes can induce cleavage of one or both strands of a target sequence. Approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 nucleotides from the first or last nucleotide of the string Induce breaks in one or both strands at 50, 100, 200, 500 base pairs or more. It is possible.

[0261] Loss of ability to cleave one or both strands of a target polynucleotide containing the target sequence To achieve this, vectors were used to encode CRISPR enzymes that were mutated relative to the corresponding wild-type enzyme. Cas9 can be a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from S. pyogenes). Cas9) and at least approximately 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93% %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology Cas9 can refer to a polypeptide having a wild-type exemplary Cas9 polypeptide ( For example, from S. pyogenes), at most, or at most, approximately, about 50%, 60%, 70% , 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or polypeptides having sequence homology. or deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any of these It can refer to modified forms of the Cas9 protein that may contain amino acid changes, such as combinations.

[0262] In some embodiments, the CRISPR protein-derived domain of the base editor is Corynebacterium diphtheria (NCBI Refs: NC_015683.1, NC_017317.1); NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021 284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (N CBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella ba ltica (NCBI Ref: NC_018010.1); Psychroflexus torquis (NCBI Ref: NC_018721.1); St reptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP _472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria meningiti dis (NCBI Ref: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus au The Cas9 fragment may comprise all or part of the Cas9 fragment derived from Cas9.

[0263] [Cas9 domain, a nucleobase editor] The sequence and structure of Cas9 nuclease are well known to those skilled in the art (see, e.g., "Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti et al., Pr oc. Natl. Acad. Sci. USA 98:4658-4663(2001); “CRISPR RNA maturation by trans -encoded small RNA and host factor RNase III.” Deltcheva E. et al., Nature 471: 602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptation See Jinek M. et al., Science 337:816-821 (2012). The entire contents of which are incorporated herein by reference.) Cas9 orthologs include, but are not limited to: It has been described in various species, including S. pyogenes and S. thermophilus, although not exclusively in the Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on the present disclosure. Such Cas9 nucleases and sequences are described in Chylinski, Rhun, and Charpentier, “ The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) R NA Biology 10:5, 726-737. The entire contents of which are incorporated herein by reference.

[0264] In some embodiments, nucleic acid programmable DNA binding proteins (napDNAbp) is a Cas9 domain. Non-limiting exemplary Cas9 domains are provided herein. The 9 domains are divided into nuclease-active Cas9 domains, nuclease-inactive Cas9 domains (dCas9 ), or Cas9 nickase (nCas9). In some embodiments, the Cas9 domain can be Nuclease activity domains. For example, the Cas9 domain binds both strands of a double-stranded nucleic acid (e.g., For example, it may be a Cas9 domain that cleaves both strands of a double-stranded DNA molecule. In some embodiments, the Cas9 domain comprises any one of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises any of the amino acid sequences described herein. At least 60%, at least 65%, at least 70%, at least 75%, or at least at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least or at least 97%, at least 98%, at least 99%, or at least 99.5% identical amino acid sequence. In some embodiments, the Cas9 domain comprises an amino acid sequence described herein. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 5, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 3 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more In some embodiments, the Cas9 domain comprises an amino acid sequence having a mutation. At least 10, at ... At least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 20 0, at least 250, at least 300, at least 350, at least 400, at least 500, a few At least 600, at least 700, at least 800, at least 900, at least 1000, at least amino acid sequences that have at least 1100 or at least 1200 identical stretches of amino acid residues Includes:

[0265] In some embodiments, proteins comprising fragments of Cas9 are provided. In embodiments, the protein includes one of the following two Cas9 domains: (1) Cas9 (2) a gRNA binding domain of Cas9; and (3) a DNA cleavage domain of Cas9. In some embodiments, Cas9 or a fragment thereof Proteins containing the fragments are referred to as "Cas9 variants." Cas9 variants are Cas9 or For example, a Cas9 variant may share at least about 70% homology with a wild-type Cas9. % 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, In some embodiments, the sequences are about 99.5% identical, or at least about 99.9% identical to each other. Cas9 mutants have the following advantages compared to wild-type Cas9: 4, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 3 4, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more In some embodiments, the Cas9 variant may have an amino acid change in the fragment of Cas9. The fragment contains a fragment of wild-type Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain), at least about 70% identical to the corresponding fragment, 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 In some embodiments, the fragment is about 99.5% identical, or at least about 99.9% identical. , at least 30%, at least 35%, at least 40% of the amino acid length of the corresponding wild-type Cas9; At least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least or at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 50 0, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 12 50, or at least 1300 amino acids in length.

[0266] In some embodiments, the Cas9 fusion proteins provided herein comprise a Cas9 protein. The full-length amino acid sequence of the protein, for example, one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein contain a full-length Cas9 sequence. Examples of Suitable Cas9 Domains and Cas9 Fragments Suitable amino acid sequences are provided herein, and further suitable sequences for Cas9 domains and fragments are , as will be apparent to those skilled in the art.

[0267] The Cas9 protein guides the protein to a specific DNA sequence complementary to its guide RNA. In one embodiment, the polynucleotide protease is capable of binding to a guide RNA. The mutatable nucleotide binding domain can be a Cas9 domain, e.g., a nuclease-active Cas9, Cas9 nickase (nCas9), or nuclease-inactive Cas9 (dCas9). Examples of tunable DNA-binding proteins include Cas9 (e.g., dCas9 and nCas9), CasX, C These include, but are not limited to, asY, Cpf1, Cas12b / C2C1, and Cas12c / C2C3. In one embodiment, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes ( NCBI reference sequence: NC_017053.1, nucleotide and amino acid sequences are as follows: ATGGATAAGAAATACTCAATGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGATTATAAGGT TCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTG GCAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGT TATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGT GGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAA CTATCTATCATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCAT ATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCA GTTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTG CACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAGAAATGGCTTGTTTGGGAAT CTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTC AAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTA AGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCA ATGATTAAGCGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTA TAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATA AATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGC AAGCAACGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGA AGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTC CATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAA GTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGT ACTACCAAAACATAGTTTGCTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAGGGAA TGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACC GTTAAGCAATTAAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATT TAATGCTTCATTAGGCGCCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAG ATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGGGATGATTGAGGAAAGACTTAAAACATATGCT CACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGAT TAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTA TGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGATATTCAAAAAGCACAGGTGTCTGGACAAGGCCATAGTTTA CATGAACAGATTGCTAACTTAGCTGGCAGTCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAATTGTTGATGAACT GGTCAAAGTAATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGA AAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTT GAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTACAAAATGGAAGAGACATGTATGTGGACCAAGAATT AGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGACGATTCAATAGACAATA AGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAAC TATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTT GAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTT TGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAA TTAGTTTCTGACTTCCGAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTA TCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAG TTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAAT ATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGA AACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTG TCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAGAAATTCGGACAAGCTTATTGCT CGTAAAAAGACTGGGATCCAAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAA GGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTG AAAAAATCCGATTGACTTTTAGAAGCTAAAGGATAAGGAAGTTAAAAGACTTAATCATTAAACTACCTAATAT AGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAATTACAAAAAGGAAATGAGCTGGCTCT GCCAAGCAAATGTGAATTTTTTATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAA AACAATTGTTTGTGGAGCAGCATAAGCATTATTTGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATT TTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAA TATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAAC GATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCCATCCATGGTCTTTGAAACACGCATTGAT TTGAGTCAGCTAGGAGGTGACTGA JPEG2025143260000013.jpg171169(Picture:HNHドメイン;Picture:RuvCドメイン)

[0268] In some embodiments, wild-type Cas9 contains the following nucleotides and / or amino acids: corresponding to or including the amino acid sequence: ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGT ACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAAGAATCTTATCGGTGCCCTCCTATTCG ATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGT TACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGT CGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAA CGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCAT ATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCA GTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCG CCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAAATGGGTTGTTCGGTAAC CTTATAGCGCTCTCACTAGGCCTGACACCAAATTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAG TAAGGACACGTACGATGACGATCTCGCAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCA AAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCCGCTTCA ATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATA TAAGGAAATATTCTTTGATCAGTCGAAAACGGGTACGCAGGTTATATTGACGGGCGGAGCGAGTCAAGAGGAATTCTACA AGTTTATCAAACCCATATTAGAGAATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGA AAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGA GGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTACTATGTGGGAC CCCTGGCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAA GTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCACTTTGACAAGAATTTACCGAACGAAAAAGT ATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCA TGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACA GTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATT TAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAG ATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCT CACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTAT CAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCATAGGAACTTTA TGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTG CACGAACATATTGCGAATCTTGCTGGGTTCGCCAGCCATCAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCT AGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGC AAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCT GTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGA ACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACA ATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAG AACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGG CTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGA TACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCA AAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGC TTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACA AAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCT AACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGG GGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACA TAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATC GCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGC AAAAGTTGAGAAGGGAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTT TTGAAAAGACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAG TATAGTCTGTTTGAGTTAGAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCCAAAGGGGAACGAACTCGC ACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAAC AGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTC ATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGA AAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCA AACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATA GATTTGTCACAGCTTGGGGGTGACGGATCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGA TTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCTGCAGGA JPEG2025143260000014.jpg167167 (single underline: HNH domain; double underline: RuvC domain)

[0269] In some embodiments, the wild-type Cas9 is Cas9 from Streptococcus pyogenes (NCBI Reference ce Sequence: NC_002737.2 (nucleotide sequence is as follows) and Uniprot Reference Sequence: Corresponding to Q99ZW2 (amino acid sequence is as follows): ATGGATAAGAAATACTCAATGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGT TCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTG ACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGT TATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGT GGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAA CTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCAT ATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCA GTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTG CACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAAT CTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTC AAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTA AGAATTTATCAGATGCTATTTTA...

Claims

1. 1. A method of treating a neurological disorder in a subject in need thereof, comprising: (i) administering to a subject an adenosine base (ii) a target polynucleotide or a nucleic acid sequence encoding the target polynucleotide; and administering to the subject a nucleic acid sequence encoding the adenosine base editor, comprising a programmable DNA binding domain and an adenosine deaminase domain, the guide polynucleotide directs the adenosine base editor to Produces single nucleobase alterations at splice sites of target genes associated with neurological disorders thereby treating said neurological disorder in a subject.

2. The adenosine deaminase is at amino acid position 82 or 83 in the numbering of SEQ ID NO:

2. The method of claim 1, wherein said amino acid substitution at 166 or a corresponding substitution thereto is included.

3. The single nucleobase modification results in an alternative sequence for the transcript encoded by the target gene.

3. The method of claim 1 or 2, which results in lysing.

4. The alternative splicing is carried out to remove the truncated transcript encoded by the target gene. The method of any one of claims 1 to 3, wherein the method produces a protein or a non-functional protein. 。

5. wherein the modification of the single nucleobase results in decreased expression of the target gene in the subject.

5. The method of any one of claims 1 to 4.

6. The target gene is the superoxide dismutase 1 (SOD1) gene, and the neurological disease is The method of any one of claims 1 to 5, wherein the disease is amyotrophic lateral sclerosis (ALS).

7. A method of treating amyotrophic lateral sclerosis (ALS) in a subject in need thereof, comprising administering to said subject The image comprises (i) a base editor or a nucleic acid sequence encoding the same, and (ii) a guide polynucleotide. administering a nucleotide or a nucleic acid sequence encoding the same; The base editor comprises a programmable DNA binding domain and a deaminase domain. Including The guide polynucleotide directs the adenosine base editor to A single nucleobase at the splice site of the superoxide dismutase 1 (SOD1) gene thereby treating ALS in said subject.

8. A method of treating amyotrophic lateral sclerosis (ALS) in a subject in need thereof, comprising administering to said subject The animal is provided with (i) a base editor or a nucleic acid sequence encoding the same, and (ii) a guide polynucleotide. administering a medicament or a nucleic acid sequence encoding the same to a subject; The base editor comprises a programmable DNA binding domain and a deaminase domain. Including The guide polynucleotide directs the adenosine base editor to resulting in a single nucleobase alteration in the superoxide dismutase 1 (SOD1) gene The single nucleic acid base modification results in a premature stop codon in the SOD1 gene, thereby to treat ALS in said subject.

9. 2. The deaminase is a nucleotide sequence encoding a nucleotide sequence at amino acid position 8 or 166 in the numbering of SEQ ID NO:

2. adenosine deaminase containing the amino acid substitutions listed above or corresponding substitutions. 7 or 8. The method according to claim 7 or 8.

10. The method according to any one of claims 7 to 9, wherein the single nucleobase modification is an A to G modification. Law.

11. The single nucleic acid base modification is in the splice acceptor site of the SOD1 gene. The method of any one of claims 7 to 10, wherein

12. The splice site is a splice acceptor at the 5' end of an exon of the SOD1 gene. The method of any one of claims 7 to 11, wherein the site is a site.

13. The exon of the SOD1 gene is exon 3 corresponding to SEQ ID NO: 3 or a variant thereof. The method of any one of claims 7 to 12, wherein

14. The exon 3 of the SOD1 gene is identified by the numbering in SEQ ID NO:3 in the SOD1 polynucleotide sequence. adjacent to the splice acceptor AG at nucleotide position 6828 of The method of any one of claims 7 to 13, wherein the

15. The exon of the SOD1 gene is exon 4 corresponding to SEQ ID NO: 3 or a variant thereof. The method of any one of claims 7 to 12, wherein

16. The single nucleobase modification results in a deletion of exons 3 to 5 of the human SOD1 gene corresponding to SEQ ID NO:

3. The method according to any one of claims 7 to 15, wherein the transcription product or a variant thereof is produced. method.

17. 8. The method of claim 7, wherein the expression of the SOD1 gene is reduced by at least 40% in the subject after said administration. The method of any one of claims 1 to 16.

18. The guide polynucleotide is a splice acceptor nucleic acid sequence of the SOD1 gene or 18. The method of claim 7, comprising a nucleic acid sequence complementary to a splice donor nucleic acid sequence. How to do it.

19. The guide polynucleotide comprises any nucleic acid sequence selected from Table 19 or Table 23. The method of any one of claims 7 to 18, comprising:

20. The guide polynucleotide is 5′-UUAAAGGAAAGUAAUGGACCAGU-3′, 5′-UAAAUAGGCUG UACCAGUGCAGG-3′, 5′-UUCAUUAUUAGGCAUGUUGGAGA-3′, 5′-AAAUAGGCUGUACCAGUGCAGGU-3 5'-UAUUAGGCAUGUUGGAGACUUGG-3', 5'-UAUUAGGCAUGUUGGAGACUUGG-3', 20. The method according to any one of paragraphs 7 to 19.

21. The target gene is the androgen receptor (AR) gene, and the neurological disease is spinal-bulbar muscular atrophy. The method of any one of claims 1 to 5, wherein the disease is SBMA.

22. 1. A method of treating spinal and bulbar muscular atrophy (SBMA) in a subject, comprising: (i) administering to a subject a base editor or a nucleic acid sequence encoding the same, and (ii) a guide polynucleotide or a nucleic acid sequence encoding the same. and administering to said subject a nucleic acid sequence encoding the The base editor comprises a programmable DNA binding domain and a deaminase domain. Including The guide polynucleotide directs the adenosine base editor to A single nucleobase alteration occurs at a splice site in the androgen receptor (AR) gene. thereby treating SBMA in the subject.

23. 1. A method of treating spinal and bulbar muscular atrophy (SBMA) in a subject, comprising: (i) administering to a subject a base editor or a nucleic acid sequence encoding the same, and (ii) a guide polynucleotide or a nucleic acid sequence encoding the same. and administering to said subject a nucleic acid sequence encoding the The adenosine base editor comprises a programmable DNA binding domain and a deamina containing a lyase domain, the guide polynucleotide directs the adenosine base editor to A method for producing a single nucleic acid base modification in the androgen receptor (AR) gene, comprising: The base modification results in a premature stop codon in the AR gene, thereby Methods for treating SBMA in elephants.

24. 22. The method of claim 21, wherein the nucleobase modification results in a CAG-TAG codon change in the AR gene. is the method described in 23.

25. Claim 22: The codon change is in exon 1 or exon 2 of the AR gene. The method of any one of claims 1 to 24.

26. The deaminase is at amino acid position 82 or 166 according to the numbering in SEQ ID NO:

2. The adenosine deaminase according to any one of claims 22 to 25, which comprises an amino acid substitution. method.

27. 27. The method of claim 22, wherein the single nucleobase modification is an A to G modification. How to do it.

28. The A to G nucleobase modification is in the splice acceptor site of the AR gene. The method of any one of claims 22 to 27, wherein

29. The splice site is a splice acceptor site located 5' of an exon of the AR gene. The method of any one of claims 22 to 28, wherein

30. The exon of the AR gene is exon 2 corresponding to SEQ ID NO: 4 or a variant thereof. The method of any one of claims 22 to 29.

31. The splice site is a splice donor site located 3' of an exon of the AR gene; 29. The method of any one of claims 22 to 28.

32. The exon of the AR gene is exon 1 corresponding to SEQ ID NO: 4 or a variant thereof. The method of any one of claims 22 to 28 and 31,

33. Claims 22-32, wherein the expression of the AR gene is reduced by at least 40% in the subject after said administration. The method according to any one of the preceding claims.

34. The guide polynucleotide is a splice acceptor nucleic acid sequence of the AR gene.

34. The method of claim 22, comprising a nucleic acid sequence complementary to a price donor nucleic acid sequence. How to do it.

35. wherein the guide polynucleotide comprises a nucleic acid sequence selected from Table 41A or 41B. The method of any one of claims 22 to 34.

36. The guide polynucleotide is 5'-ACUUACCGCAUGUCCCCGUAAGG-3', 5'- AGUGCAGUUA a nucleic acid selected from the group consisting of: 5'- GGGCUGGGAAGGG-3', 5'- AAGUGCAGUUAGGGCUGGGAAGG-3' The method of any one of claims 22 to 35, comprising the sequence

37. The method of any one of claims 1 to 36, wherein the subject is a mammal or a human.

38. 10. The method of claim 1, wherein the administration is via delivery to cells of the subject's central nervous system (CNS). The method of any one of claims 1 to 37.

39. The method of any one of claims 1 to 38, wherein the cell is a motor neuron.

40. A method for modifying a target gene or its regulatory element associated with a neurological disorder, comprising: The target gene or its regulatory element is treated by: (i) an adenosine base editor or and (ii) a guide polynucleotide or nucleic acid sequence encoding the same. and contacting the adenosine base editor with a programmable DNA sequence. A binding domain and an adenosine deaminase domain, and a target gene splice site, wherein the target gene splice site is a splice site of the target gene. A method for producing a single nucleobase modification in a nucleic acid molecule.

41. The adenosine deaminase is selected from the group consisting of adenosine deaminase and adenosine deaminase.

41. The method of claim 40, comprising an amino acid substitution at 66.

42. The single nucleobase modification inhibits alternative splicing of the transcript encoded by the target gene. target gene, truncated and / or non-functional protein encoded by the target gene and / or a protein, and / or a cell, resulting in a decrease in expression of a target gene when expressed therein. Item 40. The method according to item 41.

43. The target gene is the superoxide dismutase 1 (SOD1) gene, and the neurological disease is The method of any one of claims 40 to 42, wherein the disease is amyotrophic lateral sclerosis (ALS).

44. A method for regulating the expression of the superoxide dismutase (SOD1) gene, comprising: a gene or its regulatory element, (i) a base editor or a nucleic acid sequence encoding the same; and (ii) contacting the guide polynucleotide or a nucleic acid sequence encoding the same. This includes: The base editor comprises a programmable DNA binding domain and a deaminase domain. wherein the guide polynucleotide guides the adenosine base editor to A single nucleobase modification at a splice site in the peroxide dismutase 1 (SOD1) gene A way to bring about change.

45. A method for modifying the superoxide dismutase (SOD1) gene, comprising: or a regulatory element thereof, by combining (i) a base editor or a nucleic acid sequence encoding same, and and (ii) contacting the guide polynucleotide or a nucleic acid sequence encoding the same. Including, The guide polynucleotide directs the adenosine base editor to resulting in a single nucleobase alteration in the superoxide dismutase 1 (SOD1) gene wherein said single nucleobase modification results in a premature stop codon in the SOD1 gene.

46. 46. ​​The method of claim 44 or 45, wherein the single nucleobase modification is an A to G modification.

47. The nucleic acid base modification is in the splice acceptor site of the SOD1 gene.

47. The method according to any one of claims 44 to 46.

48. The splice site is a splice acceptor site located 5' of an exon of the SOD1 gene. The method of any one of claims 44 to 47, wherein

49. The exon of the SOD1 gene is exon 3 corresponding to SEQ ID NO: 3 or a variant thereof. The method of any one of claims 44 to 48, wherein

50. The exon 3 of the SOD1 gene is identified by the numbering in SEQ ID NO:3 in the SOD1 polynucleotide sequence. adjacent to the splice acceptor at nucleotide position 6828 of the sequence.

49. The method of any one of claims 49, or a variant thereof.

51. The exon of the SOD1 gene is exon 4 corresponding to SEQ ID NO: 3 or a variant thereof. The method of any one of claims 44 to 48, wherein

52. The single nucleobase modification results in a translocation that deletes exons 3 to 5 of the SOD1 gene corresponding to SEQ ID NO:

3.

52. The method of any one of claims 44 to 51, wherein the method produces a transcript or a variant thereof.

53. The guide polynucleotide is a splice acceptor nucleic acid sequence of the SOD1 gene or 53. The method of any one of claims 44 to 52, comprising a nucleic acid sequence complementary to a splice donor nucleic acid sequence. How to do it.

54. wherein the guide polynucleotide comprises a nucleic acid sequence selected from Table 19 or Table 23. Item 44. The method according to any one of items 44 to 53.

55. The guide polynucleotide is 5′-UUAAAGGAAAGUAAUGGACCAGU-3′, 5′-UAAAUAGGCUG UACCAGUGCAGG-3′, 5′-UUCAUUAUUAGGCAUGUUGGAGA-3′, 5′-AAAUAGGCUGUACCAGUGCAGGU-3 5'-UAUUAGGCAUGUUGGAGACUUGG-3', 5'-UAUUAGGCAUGUUGGAGACUUGG-3', 55. The method of any one of paragraphs 44 to 54.

56. The target gene is the androgen receptor (AR) gene, and the neurological disease is spinal-bulbar muscular atrophy. The method of any one of claims 40 to 42, wherein the disease is SBMA.

57. A method for regulating expression of the androgen receptor (AR) gene, comprising: The regulatory element comprises: (i) a base editor or a nucleic acid sequence encoding the same; and (ii) contacting the nucleic acid sequence with a guide polynucleotide or a nucleic acid sequence encoding the same; The base editor comprises a programmable DNA binding domain and a deaminase domain. wherein the guide polynucleotide guides the adenosine base editor to amplify resulting in a single nucleobase modification at a splice site in the steroid receptor (AR) gene, method.

58. A method for modifying the androgen receptor (AR) gene, comprising: (i) a base editor or a nucleic acid sequence encoding the same, and (ii) a guide contacting the polypeptide or nucleic acid sequence encoding the polypeptide with The base editor comprises a programmable DNA binding domain and a deaminase domain. wherein the guide polynucleotide guides the adenosine base editor to resulting in a single nucleobase alteration in the androgen receptor (AR) gene in a subject; wherein said single nucleobase modification results in a premature stop codon in said AR gene. 。

59. The deaminase is at amino acid position 82 or 166 according to the numbering in SEQ ID NO:

2.

59. The method of claim 57 or 58, wherein the adenosine deaminase comprises an amino acid substitution.

60. 60. The method of claim 57, wherein the deaminase is a cytidine deaminase. method.

61. 61. The method of any one of claims 57 to 60, wherein the single nucleobase modification is a C to T modification. How to do it.

62. Claims 57-6, wherein the nucleic acid base modification results in a CAG-TAG codon change in the AR gene.

2. The method of any one of claims 1 to 11.

63. Any of claims 57 to 62, wherein the codon change is in exon 1 or exon 2 of the AR gene. or the method described in any one of claims 1 to 5.

64. 64. The method according to any one of claims 57 to 63, wherein the modification of the single nucleobase is an A to G modification. How to post.

65. An A to G nucleobase alteration is present in the splice acceptor site of the AR gene 65. The method of any one of claims 57 to 64.

66. The splice site is a splice acceptor site located 5' of an exon of the AR gene. The method according to any one of claims 57 to 65, wherein

67. The exon of the AR gene is exon 2 corresponding to SEQ ID NO: 4 or a variant thereof. The method of any one of claims 57 to 66,

68. The splice site is a splice donor site located 3' of an exon of the AR gene; 66. The method of any one of claims 57 to 65.

69. The exon of the AR gene is exon 1 corresponding to SEQ ID NO: 4 or a variant thereof.

69. The method of any one of claims 57 to 65 and 68,

70. wherein the guide polynucleotide comprises a nucleic acid sequence selected from Table 41A or 41B.

70. The method of any one of claims 57 to 69.

71. The guide polynucleotide is a splice acceptor nucleic acid sequence of the AR gene.

71. The method of any one of claims 57 to 70, comprising a nucleic acid sequence complementary to a price donor nucleic acid sequence. How to do it.

72. wherein the guide polynucleotide comprises a nucleic acid sequence selected from Table 41A or 41B. The method of any one of claims 57 to 71.

73. The guide polynucleotide is 5'-ACUUACCGCAUGUCCCCGUAAGG-3', 5'- AGUGCAGUUA GGGCUGGGAAGGG-3′, 5′- AAGUGCAGUUAGGGCUGGGAAGG-3′, and their complementary strands 73. The method of any one of claims 57 to 72, comprising a nucleic acid sequence selected from:

74. The method of any one of claims 57-73, wherein said contacting is intracellular.

75. wherein the single nucleobase modification results in fewer than 15% indels in the genome of the cell. The method of any one of claims 57 to 74.

76. the single nucleobase modification results in less than 5% indels in the genome of the cell.

76. The method of any one of paragraphs 57 to 75.

77. the single nucleobase modification results in less than 2% indels in the genome of the cell.

77. The method of any one of paragraphs 57 to 76.

78. The method of any one of claims 57 to 77, wherein the cell is a mammalian cell or a human cell. Law.

79. 79. The method of any one of claims 57 to 78, wherein the cell is a central nervous system cell.

80. 80. The method of any one of claims 57 to 79, wherein the cell is a motor neuron.

81. The method of any one of claims 57 to 80, wherein said contacting is in a population of cells.

82. Claims 57-81, wherein after said contacting, at least 40% of the cell population comprises a single nucleobase modification. The method according to any one of the preceding claims.

83. Claims 57-82, wherein after said contacting, at least 50% of the cell population comprises a single nucleobase modification. The method according to any one of the preceding claims.

84. Claims 57-83, wherein after said contacting, at least 60% of the cell population comprises a single nucleobase modification. The method according to any one of the preceding claims.

85. Any of claims 57 to 84, wherein at least 85% of the cell population is viable after said contacting. The method described in claim 1.

86. 86. The method of any one of claims 57 to 85, wherein the population of cells is mammalian or human cells. How to post.

87. 87. The method of any one of claims 57 to 86, wherein said population of cells is central nervous system cells.

88. 88. The method of any one of claims 57 to 87, wherein the population of cells is motor neurons.

89. 89. Any one of claims 1 to 88, wherein the adenosine deaminase comprises TadA deaminase. The method described.

90. 90. The method of claim 89, wherein the adenosine deaminase is TadA7.

10.

91. The adenosine deaminase has a V28S mutation or a T166R mutation according to the numbering in SEQ ID NO:

2.

91. The method of claim 89 or 90, wherein the TadA comprises the mutation or a corresponding mutation.

92. The adenosine deaminase has the sequence Y147T, Y147R, Q154S, as numbered in SEQ ID NO: 2, Claims containing one or more mutations of Y123H and Q154R, or corresponding mutations thereof.

92. The method of any one of paragraphs 89 to 91.

93. The adenosine deaminase is selected from the group consisting of Y147T + Q154R; Y147T + Q154S; Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; Y 147R + Q154R +Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y123H + Y147R + Q154R + I76Y; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R or their corresponding mutations. The method of any one of claims 89 to 92.

94. The adenosine deaminase is selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156, and 15 94. Any one of claims 89 to 93, comprising a C-terminal deletion beginning at a residue selected from the group consisting of: The method described in section.

95. 95. The method of any one of claims 89 to 94, wherein the adenosine deaminase comprises a TadA dimer. How to do it.

96. Claims 89-95, wherein the adenosine deaminase comprises an adenosine deaminase monomer. The method according to any one of the preceding claims.

97. the polynucleotide-programmable DNA-binding domain is a Cas9 domain 97. The method of any one of claims 1 to 96.

98. 98. The method of Claim 97, wherein said Cas9 domain is a Cas9 nickase domain.

99. 99. The method of Claim 97 or 98, wherein the Cas9 domain comprises an SpCas9 domain.

100. the SpCas9 domain comprises D10A and / or H840A amino acids according to the numbering in SEQ ID NO: 1; The method of any one of claims 97 to 99, comprising an amino acid substitution or a corresponding amino acid substitution. Law.

101. The method of any one of claims 97-100, wherein said Cas9 domain comprises a SaCas9 domain. 。

102. 102. The method of claim 97, wherein the Cas9 domain has specificity for an engineered PAM. or the method described in any one of claims 1 to 5.

103. The Cas9 domain is selected from the group consisting of NGG, NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, and and NGC, where N is A, G, C, or or T, and R is A or G.

104. 104. A population of cells produced by any one of claims 1 to 103.

105. (i) a base editor or a nucleic acid sequence encoding the same; and (ii) a guide polynucleotide. and a nucleic acid sequence encoding the same, The base editor comprises a programmable DNA binding domain and a deaminase domain. Including The guide polynucleotide guides the adenosine base editor to This results in a single nucleobase modification at the splice site of sulfoxide dismutase 1 (SOD1). 、 Base editor system.

106. (i) a base editor or a nucleic acid sequence encoding the same; and (ii) a guide polynucleotide. and a nucleic acid sequence encoding the same, The base editor comprises a programmable DNA binding domain and a deaminase domain. Including The guide polynucleotide induces the adenosine base editor to A single nucleic acid base modification in the steroid dismutase 1 (SOD1) gene is introduced into the single nucleic acid base modification. Acid-base modifications leading to premature stop codons in the SOD1 gene Base editor system.

107. The deaminase is at amino acid position 82 or 166 according to the numbering in SEQ ID NO:

2.

107. The adenosine deaminase of claim 105 or 106, which is an adenosine deaminase containing an amino acid substitution. Editor system.

108. 108. The method of claim 105, wherein the deaminase is a cytidine deaminase. Base editor system.

109. 109. A method according to any one of claims 105 to 108, wherein the modification of a single nucleobase is an A to G modification. The base editor system described.

110. The modification of the nucleic acid base from A to G is The base editor system of any one of claims 105 to 109.

111. The splice site is a splice acceptor site located 5' of the exon of the SOD1 gene.

111. The base editor system of any one of claims 105 to 110, wherein

112. The exon of the SOD1 gene is exon 3 corresponding to SEQ ID NO: 3 or a variant thereof.

112. The base editor system of any one of claims 105 to 111.

113. The exon 3 of the SOD1 gene is identified by the numbering in SEQ ID NO:3 in the SOD1 polynucleotide sequence. adjacent to the splice acceptor AG or its variants at nucleotide position 6828 of the sequence 113. The base editor system of any one of claims 105-112, wherein

114. Alternative splicing of the SOD1 transcript results in exons 3 to 5 of the SOD1 gene corresponding to SEQ ID NO:

3. Any one of claims 105 to 113, wherein the transcript lacking 5 or a variant thereof is produced. The base editor system according to claim 1.

115. The exon of the SOD1 gene is exon 4 corresponding to SEQ ID NO: 3 or a variant thereof.

112. The base editor system of any one of claims 105 to 111.

116. The guide polynucleotide is a splice acceptor nucleic acid sequence of the SOD1 gene or 116. The method of any one of claims 105 to 115, comprising a nucleic acid sequence complementary to a splice donor nucleic acid sequence. The base editor system described above.

117. wherein the guide polynucleotide comprises a nucleic acid sequence selected from Table 19 or Table 23.

117. The base editor system of any one of paragraphs 105 to 116.

118. The guide polynucleotide is 5′-UUAAAGGAAAGUAAUGGACCAGU-3′, 5′-UAAAUAGGCUG UACCAGUGCAGG-3′, 5′-UUCAUUAUUAGGCAUGUUGGAGA-3′, 5′-AAAUAGGCUGUACCAGUGCAGGU-3 ', 5'-UAUUAGGCAUGUUGGAGACUUGG-3', and the complementary sequence thereof 118. The base editor system of any one of claims 105-117, comprising a nucleic acid sequence.

119. (i) a base editor or a nucleic acid sequence encoding the same; and (ii) a guide polynucleotide. and a nucleic acid sequence encoding the same, The base editor comprises a programmable DNA binding domain and a deaminase domain. Including the guide polynucleotide directs the adenosine base editor to produce androgen A base-editing gene that results in a single nucleobase modification at the splice site of the receptor (AR) gene Computer system.

120. (i) a base editor or a nucleic acid sequence encoding the same; and (ii) a guide polynucleotide. and a nucleic acid sequence encoding the same, The adenosine base editor comprises a programmable DNA binding domain and a deamina containing a lyase domain, the guide polynucleotide directs the adenosine base editor to produce an amplicon in the subject a single nucleic acid base modification in the steroid receptor (AR) gene, A base editor system in which modification of the nucleotide sequence results in a premature stop codon in the AR gene.

121. The deaminase is at amino acid position 82 or 166 according to the numbering in SEQ ID NO:

2.

121. The adenosine deaminase of claim 119 or 120, which is an adenosine deaminase containing an amino acid substitution. Editor system.

122. 122. The method of any one of claims 119 to 121, wherein the deaminase is a cytidine deaminase. Base editor system.

123. 123. The method of any one of claims 119 to 122, wherein the single nucleobase modification is a C to T modification. The base editor system described above.

124. 1. The method of claim 1, wherein the single nucleobase modification results in a CAG-TAG codon change in the AR gene. 19-123. A base editor system according to any one of claims 19-123.

125. The codon change is in exon 1 or exon 2 or or a variant thereof. Hmm.

126. 126. Any one of claims 119 to 125, wherein the single nucleobase modification is an A to G modification. Base editor system.

127. The A to G nucleic acid base modification is at the splice acceptor site of the AR gene. The base editor system of any one of claims 119 to 126,

128. The splice site is a splice acceptor site located 5' of an exon of the AR gene.

128. The base editor system of any one of claims 119-127, wherein

129. The exon of the AR gene is exon 2 corresponding to SEQ ID NO: 4 or a variant thereof.

129. The base editor system of any one of claims 119 to 128.

130. The splice site is a splice donor site located 3' of an exon of the AR gene.

128. The base editor system of any one of claims 119 to 127.

131. The exon of the AR gene is exon 1 corresponding to SEQ ID NO: 4 or a variant thereof.

131. The base editor system of any one of claims 119-127 and 130.

132. The guide polynucleotide is a splice acceptor nucleic acid sequence of the AR gene. Any one of claims 119 to 131, comprising a nucleic acid sequence complementary to a price donor nucleic acid sequence. Base editor system.

133. wherein the guide polynucleotide comprises a nucleic acid sequence selected from Table 41A or 41B. The base editor system of any one of claims 119 to 132.

134. The guide polynucleotide is 5'-ACUUACCGCAUGUCCCCGUAAGG-3', 5'- AGUGCAGUUA GGGCUGGGAAGGG-3′, 5′- AAGUGCAGUUAGGGCUGGGAAGG-3′, and their complementary strands The base editor of any one of Claims 119 to 133, comprising a nucleic acid sequence selected from system.

135. 135. Any one of claims 105 to 134, wherein the adenosine deaminase comprises TadA deaminase. The base editor system described in section .

136. 136. The base editor system of claim 135, wherein said adenosine deaminase is TadA7.

10. Tem.

137. The adenosine deaminase has a V28S mutation or a T166R mutation according to the numbering in SEQ ID NO:

2.

137. The salt of claim 135 or 136, which is TadA containing a mutation or a corresponding mutation. Base editor system.

138. The adenosine deaminase has the sequence Y147T, Y147R, Q154S, as numbered in SEQ ID NO: 2, Claims 135-13, comprising one or more of the mutations Y123H and Q154R or corresponding thereto.

8. A base editor system according to any one of claims 7 to 7.

139. The adenosine deaminase is selected from the group consisting of Y147T + Q154R; Y147T + Q154S; Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; Y 147R + Q154R +Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y123H + Y147R + Q154R + I76Y; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R or a combination of mutations corresponding thereto, The base editor system according to any one of claims 135 to 138.

140. The adenosine deaminase is selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156 and 157 Any of claims 135 to 139, comprising a C-terminal deletion beginning at a residue selected from the group consisting of: The base editor system of claim 1.

141. 141. The method of any one of claims 135 to 140, wherein the adenosine deaminase comprises a TadA dimer. The base editor system described above.

142. 135-1, wherein the adenosine deaminase comprises an adenosine deaminase monomer.

42. The base editor system of any one of claims 41.

143. the polynucleotide-programmable DNA-binding domain is a Cas9 domain 143. The base editor system of any one of claims 105 to 142.

144. 144. The base editor of Claim 143, wherein said Cas9 domain is a Cas9 nickase domain. -System.

145. 145. The base editor of Claim 143 or 144, wherein said Cas9 domain comprises an SpCas9 domain. -System.

146. the SpCas9 domain comprises D10A and / or H840A amino acids according to the numbering in SEQ ID NO: 1; Any one of claims 143 to 145, comprising an acid substitution or its corresponding amino acid substitution. Base editor system.

147. The base of any one of claims 143 to 146, wherein the Cas9 domain comprises a SaCas9 domain. Editor system.

148. Any of claims 105-147, wherein the Cas9 domain has specificity for an engineered PAM. The base editor system of any one of claims 1 to 4.

149. The Cas9 domain is selected from the group consisting of NGG, NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, and and NGC, where N is A, G, C, or or T, and R is A or G. -System.

150. The polynucleotide of any one of claims 105 to 149. a nucleic acid sequence encoding a nucleic acid-programmable DNA binding domain, said adenovirus and a nucleic acid sequence encoding a syndeaminase domain.

151. 151. The method of Claim 150, further comprising a nucleic acid sequence encoding said guide polynucleotide. vector.

152. 152. The vector of claim 150 or 151, wherein the vector is a viral vector.

153. The base editor system of any one of claims 105 to 149 or claims 150 to 152 A cell comprising the vector described in any one of the above.

154. 154. The method of claim 153, wherein the cell is a mammalian cell, a human cell, or a motor neuron. cells.

155. 155. The method of claim 153 or 154, wherein the cell is in vivo, ex vivo, or in vitro. cells.

156. 156. The method of any one of claims 153 to 155, wherein the cells are autologous cells isolated from the subject. cells.

157. The cell of any one of claims 153-156, wherein said cell is an allogeneic cell.

158. The base editor system of any one of claims 105 to 149 or claims 150 to 152 A population of cells comprising the vector of any one of claims 1 to 4.

159. 159. The population of cells of claim 158, which are mammalian cells, human cells, or motor neurons. 。

160. 160. The collection of cells of claim 158 or 159, which is in vivo, ex vivo, or in vitro. Group.

161. The cell of any one of claims 158-160, wherein the cell is an autologous cell isolated from the subject. A group of.

162. The base editor system of any one of claims 105 to 149, any one of claims 150 to 152 the vector of any one of claims 153 to 157, the cell of any one of claims 158 to 159, or the cell of claim 160 162. A pharmaceutical composition comprising a population of cells according to any one of claims 1 to 161 and a pharmaceutically acceptable carrier. thing.

163. 163. The pharmaceutical composition of claim 162, further comprising a lipid.

164. 163. The pharmaceutical composition of claim 162, further comprising a virus.

165. The base editor system of any one of claims 105 to 149 or claims 150 to 152. A kit comprising the vector described in any one of claims 1 to 4.

166. wherein at least one nucleotide of the guide polynucleotide comprises a non-natural modification.

10. The method according to any one of the preceding claims.

167. Claims 20, 36, 55, wherein at least one nucleotide of the nucleic acid sequence contains a non-natural modification. or 73, a method according to any one of claims 1 to 73.

168. 118 or 119, wherein at least one nucleotide of said nucleic acid sequence comprises a non-natural modification.

35. The base editor system of any one of claims 34.

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