Methods of editing disease-related genes using adenosine deaminase base editor, including treatment of genetic diseases

Abstract

The present invention relates to methods of editing disease-related genes using an adenosine deaminase base editor, including treatment of genetic diseases, and provides compositions comprising a novel programmable adenosine base editor system (e.g., ABE8) that provide for the treatment of genetic diseases by administering to a subject a programmable adenosine base editor system (e.g., ABE8) having improved efficiency. The present invention relates to adenosine deaminase variants (e.g., ABE8) for treating a disease or condition (e.g., Parkinson's disease, Seller's disease, Lutt's disease, or Stargart's disease) in a subject and methods of editing disease-related genes using these adenosine deaminase variants.

Description

[0001] This application is a divisional application of the PCT patent application entered into China with Chinese patent application number 202080028186.5, invention name “Methods for editing disease-related genes using adenosine deaminase base editors, including treatment of genetic diseases” and filing date February 13, 2020.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 805,271, filed on February 13, 2019; U.S. Provisional Application No. 62 / 852,228, filed on May 23, 2019; U.S. Provisional Application No. 62 / 852,224, filed on May 23, 2019; U.S. Provisional Application No. 62 / 873,138, filed on July 11, 2019; U.S. Provisional Application No. 62 / 888,867, filed on August 19, 2019; U.S. Provisional Application No. 62 / 931,722, filed on November 6, 2019; U.S. Provisional Application No. 62 / 941,569, filed on November 27, 2019; and U.S. Provisional Application No. 62 / 966,526, filed on January 27, 2020, the disclosures of which are hereby incorporated by reference in their entirety.

[0004] Incorporated by Reference

[0005] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. Unless otherwise indicated, the publications, patents and patent applications mentioned in this specification are incorporated herein by reference in their entirety. Background Art

[0006] Targeted editing of nucleic acid sequences, such as targeted cleavage or targeted modification of genomic DNA, is a promising method for studying gene function and has the potential to provide new treatments for human genetic diseases. Currently available base editors include cytidine base editors that convert target C·G base pairs to T·A (e.g., BE4) and adenine base editors that convert A·T to G·C (e.g., ABE7.10). The art needs improved base editors that can induce modifications within target sequences with greater specificity and efficiency. Summary of the invention

[0007] The present invention provides compositions including novel adenine base editors (e.g., ABE8) with improved efficiency and methods of using base editors including adenosine deaminase variants to edit target sequences.

[0008] In some aspects, provided herein is a method for treating a nervous system disorder in a subject, the method comprising: administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 or a corresponding position thereof numbered in SEQ ID NO: 2, and wherein the guide polynucleotide guide adenosine base editor effects an A to G nucleobase change in a target gene or a regulatory component thereof associated with the nervous system disorder in the subject, thereby treating the nervous system disorder in the subject. In another embodiment of the aspect, the target gene is an alpha-L-iduronidase (IDUA) gene and the nervous system disease is Hurler syndrome. In one embodiment of the aspect, the target gene is a leucine-rich repeat kinase-2 (leucine-rich repeat kinase-2, LRRK2) gene, and the nervous system disease is Parkinson's disease. In one embodiment of the aspect, the target gene is the methyl CpG binding protein 2 (MECP2) gene and the neurological disease is Rett syndrome. In another embodiment of the aspect, the target gene is the ATP binding cassette subfamily member 4 (ABCA4) gene and the neurological disease is Stargardt disease.

[0009] In some aspects, provided herein is a method for treating Hurler syndrome in a subject, the method comprising administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 numbered in SEQ ID NO: 2 or a position corresponding thereto, and wherein the guide polynucleotide guide adenosine base editor effects an A to G nucleobase change in an α-L-iduronidase (IDUA) gene or a regulatory component thereof in the subject, thereby treating Hurler syndrome in the subject.

[0010] In some embodiments, administration improves at least one symptom associated with Hurler's disease. In some embodiments, administration results in faster improvement of at least one symptom associated with Hurler's disease compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase.

[0011] In some embodiments, the IDUA gene or its regulatory element includes a SNP associated with Hurler's disease. In some embodiments, the A to G nucleobase change is at the SNP associated with Hurler's disease. In some embodiments, the SNP associated with Hurler's disease causes a W402X or W401X amino acid mutation in the IDUA polypeptide numbered in SEQ ID NO: 4 encoded by the IDUA gene or a variant thereof, wherein X is a stop codon. In some embodiments, the A to G nucleobase change changes the SNP associated with Hurler's disease to a wild-type nucleobase. In some embodiments, the A to G nucleobase change changes the SNP associated with Hurler's disease to a non-wild-type nucleobase, resulting in one or more improved symptoms of Hurler's disease. In some embodiments, the A to G change at the SNP associated with Hurler's disease changes the stop codon in the IDUA polypeptide encoded by the IDUA gene to tryptophan.

[0012] In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to an IDUA gene or its regulatory component comprising a SNP associated with Hurler's disease. In some embodiments, the adenosine base editor forms a complex with a single guide RNA (sgRNA), which comprises a nucleic acid sequence complementary to an IDUA gene or its regulatory component comprising a SNP associated with Hurler's disease. In some embodiments, the sgRNA comprises a nucleic acid sequence selected from the group consisting of: 5'-GACUCUAGGCAGAGGUCUCAA-3', 5'-ACUCUAGGCAGAGGUCUCAA-3', 5'-CUCUAGGCCGAAGUGUCGC-3', and 5'-GCUCUAGGCCGAAGUGUCGC-3'.

[0013] In some aspects, provided herein is a method for treating Parkinson's disease in a subject, the method comprising: administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 numbered in SEQ ID NO: 2 or a position corresponding thereto, and wherein the guide polynucleotide guides the adenosine base editor to effect an A to G nucleobase change in a leucine-rich repeat kinase-2 (LRRK2) gene or a regulatory component thereof in the subject, thereby treating Parkinson's disease in the subject.

[0014] In some embodiments, administration improves at least one symptom associated with Parkinson's disease. In some embodiments, administration results in faster improvement of at least one symptom associated with Parkinson's disease compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase.

[0015] In some embodiments, the LRRK2 gene or its regulatory element comprises a SNP associated with Parkinson's disease. In some embodiments, the A to G nucleobase change is at a SNP associated with Parkinson's disease. In some embodiments, the SNP associated with Parkinson's disease results in an A419V, R1441C, R1441H or G2019S amino acid mutation in the LRRK2 polypeptide numbered in SEQ ID NO: 3 encoded by the LRRK2 gene or a variant thereof.

[0016] In some embodiments, the A to G nucleobase change changes a SNP associated with Parkinson's disease to a wild-type nucleobase. In some embodiments, the A to G nucleobase change changes a SNP associated with Parkinson's disease to a non-wild-type nucleobase, thereby resulting in one or more improved symptoms of Parkinson's disease. In some embodiments, the A to G nucleobase change changes a cysteine ​​or histidine in a LRRK2 polypeptide encoded by a LRRK2 gene to an arginine. In some embodiments, the A to G change changes a serine in a LRRK2 polypeptide encoded by a LRRK2 gene to a glycine. In some embodiments, the A to G change replaces a cysteine ​​(C) or histidine (H) at position 144 of a LRRK2 polypeptide or variant thereof numbered in SEQ ID NO: 3 encoded by a LRRK2 gene with an arginine (R) or replaces a serine at position 144 with a glycine (G).

[0017] In some aspects, provided herein is a method for treating Parkinson's disease in a subject, the method comprising: administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, and wherein the guide polynucleotide guides the adenosine base editor to effect an A to G nucleobase change at a SNP in a LRRK2 gene associated with Parkinson's disease, wherein the SNP does not encode a G2019S mutation in the LRRK2 polypeptide numbered in SEQ ID NO: 3 or a variant thereof.

[0018] In some embodiments, the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 numbered in SEQ ID NO: 2 or a position corresponding thereto. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to an LRRK2 gene or a regulatory component thereof comprising a SNP associated with Parkinson's disease. In some embodiments, the adenosine base editor forms a complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to an LRRK2 gene or a regulatory component thereof comprising a SNP associated with Parkinson's disease. In some embodiments, the sgRNA comprises the nucleic acid sequence: 5'-AAGCGCAAGCCUGGAGGGAA-3'; or 5'-ACUACAGCAUUGCUCAGUAC-3'.

[0019] In some aspects, provided herein is a method for treating Rett syndrome in a subject, the method comprising administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 numbered in SEQ ID NO: 2 or a position corresponding thereto:, and wherein the guide polynucleotide guides the adenosine base editor to effect an A to G nucleobase change in a methyl CpG binding protein 2 (MECP2) gene or a regulatory component thereof in the subject, thereby treating Rett syndrome in the subject.

[0020] In some embodiments, administration improves at least one symptom associated with Rett's disease. In some embodiments, administration results in faster improvement of at least one symptom associated with Rett's disease compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase. In some embodiments, the MECP2 gene or its regulatory component includes a SNP associated with Rett's disease. In some embodiments, the A to G nucleobase change occurs at a SNP associated with Rett's disease. In some embodiments, the SNP associated with Rett's disease causes an R106W or T158M amino acid mutation in the MECP2 polypeptide numbered in SEQ ID NO: 5 or a variant thereof encoded by the MECP2 gene. In some embodiments, the SNP associated with Rett's disease causes an R255X or R270X amino acid mutation in the MECP2 polypeptide encoded by the MECP2 gene, wherein X is a stop codon.

[0021] In some embodiments, the A to G nucleobase change changes a SNP associated with Rett's disease to a wild-type nucleobase. In some embodiments, the A to G nucleobase change changes a SNP associated with Rett's disease to a non-wild-type nucleobase, resulting in improved symptoms of Rett's disease. In some embodiments, the A to G nucleobase change at a SNP associated with Rett's disease changes a stop codon in a MECP2 polypeptide to a tryptophan.

[0022] In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to a MECP2 gene or a regulatory component thereof comprising a SNP associated with Rett syndrome. In some embodiments, the adenosine base editor forms a complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to a MECP2 gene or a regulatory component thereof comprising a SNP associated with Rett syndrome. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of: 5'-CUUUUCACUUCCUGCCGGGG-3', 5'-AGCUUCCAUGUCCAGCCUUC-3', 5'-ACCAUGAAGUCAAAAUCAUU-3', and 5'-GCUUUCAGCCCCGUUUCUUG-3'.

[0023] In some aspects, provided herein is a method for treating Stargardt's disease in a subject, the method comprising administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 numbered in SEQ ID NO: 2, or a position corresponding thereto, and wherein the guide polynucleotide guide adenosine base editor effects an A to G nucleobase change in the subject's ATP-binding cassette subfamily member 4 (ABCA4) gene or a regulatory component thereof, thereby treating Stargardt's disease in the subject.

[0024] In some embodiments, administration improves at least one symptom associated with Stargardt's disease. In some embodiments, administration results in faster improvement of at least one symptom associated with Stargardt's disease compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase.

[0025] In some embodiments, the ABCA4 gene includes a SNP associated with Stargardt's disease. In some embodiments, the A to G nucleobase change is at a SNP associated with Stargardt's disease. In some embodiments, the SNP associated with Stargardt's disease results in an A1038V or G1961E amino acid mutation in an ABCA4 polypeptide numbered in SEQ ID NO: 6 or a variant thereof encoded by the ABCA4 gene. In some embodiments, the SNP associated with Stargardt's disease results in a G1961E amino acid mutation in an ABCA4 polypeptide numbered in SEQ ID NO: 6 or a variant thereof.

[0026] In some embodiments, the A to G nucleobase change changes a SNP associated with Stargardt's disease to a wild-type nucleobase. In some embodiments, the A to G nucleobase change changes a SNP associated with Stargardt's disease to a non-wild-type nucleobase, which results in one or more improved symptoms of Stargardt's disease. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence that is complementary to an ABCA4 gene or a regulatory component thereof that comprises a SNP associated with Stargardt's disease.

[0027] In some embodiments, the adenosine base editor forms a complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to an ABCA4 gene or a regulatory component thereof comprising a SNP associated with Stargardt's disease. In some embodiments, the sgRNA comprises the sequence 5'-CUCCAGGGCGAACUUCGACACACAGC-3'.

[0028] In various aspects, the treatments described herein result in improvement in symptoms of a nervous system disorder compared to treatment with a base editor comprising an adenosine deaminase domain without an amino acid substitution.

[0029] In some aspects, provided herein is a method for editing a target gene or a regulatory component thereof associated with a nervous system disorder, the method comprising making the target gene or its regulatory component with (i) an adenosine base editor and (ii) a guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 numbered in SEQ ID NO: 2 or a corresponding position thereof, wherein the guide polynucleotide guide adenosine base editor effects an A to G nucleobase change in a target gene or its regulatory component associated with a nervous system disorder. In one embodiment of the aspect, the target gene is a leucine-rich repeat kinase-2 (LRRK2) gene, and the nervous system disease is Parkinson's disease. In another embodiment of the aspect, the target gene is an alpha-L-iduronidase (IDUA) gene, and the nervous system disease is Hurler's disease. In one embodiment of the aspect, the target gene is a methyl CpG binding protein 2 (MECP2) gene, and the nervous system disease is Rett's disease. In another embodiment of the aspect, the target gene is the ATP-binding cassette subfamily member 4 (ABCA4) gene and the nervous system disease is Stargardt's disease.

[0030] In some aspects, the present invention provides a method for editing a leucine-rich repeat kinase-2 (LRRK2) gene or its regulatory components, the method comprising combining the LRRK2 gene or its regulatory components with (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide guides the adenosine base editor to effect an A to G nucleobase change in the LRRK gene or its regulatory components.

[0031] In some embodiments, the A to G nucleobase change is at a SNP associated with Parkinson's disease. In some embodiments, the SNP associated with Parkinson's disease results in an A419V, R1441C, R1441H, or G2019S amino acid mutation in an LRRK2 polypeptide numbered in SEQ ID NO: 3 encoded by the LRRK2 gene, or a variant thereof. In some embodiments, the A to G nucleobase change changes the SNP associated with Parkinson's disease to a wild-type nucleobase. In some embodiments, the A to G nucleobase change changes the SNP associated with Parkinson's disease to a non-wild-type nucleobase, resulting in one or more improved symptoms of Parkinson's disease.

[0032] In some embodiments, the A to G nucleobase change changes a cysteine ​​or histidine in a LRRK2 polypeptide encoded by a LRRK2 gene to an arginine. In some embodiments, the A to G change changes a serine in a LRRK2 polypeptide encoded by a LRRK2 gene to a glycine. In some embodiments, the A to G change replaces a cysteine ​​(C) or histidine (H) with an arginine (R) at position 144 of a LRRK2 polypeptide or variant thereof numbered in SEQ ID NO: 3 encoded by a LRRK2 gene with an arginine (R) or replaces a serine with a glycine (G) at position 2019.

[0033] In some aspects, the present invention provides a method for editing a leucine-rich repeat kinase-2 (LRRK2) gene or its regulatory components, the method comprising combining the LRRK gene or its regulatory components with (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, and wherein the guide polynucleotide guides the adenosine base editor to effect an A to G nucleobase change at a SNP in the LRRK2 gene, wherein the SNP does not encode a G2019S mutation in the LRRK2 polypeptide numbered in SEQ ID NO: 3 or a variant thereof.

[0034] In some embodiments, the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166, or a position corresponding thereto, numbered in SEQ ID NO: 2. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to a LRRK2 gene or a regulatory element thereof comprising a SNP associated with Parkinson's disease.

[0035] In some embodiments, the adenosine base editor forms a complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to a LRRK2 gene or a regulatory component thereof comprising a SNP associated with Parkinson's disease. In some embodiments, the sgRNA comprises the nucleic acid sequence: 5'-AAGCGCAAGCCUGGAGGGAA-3'; or 5'-ACUACAGCAUUGCUCAGUAC-3'.

[0036] In some aspects, the present invention provides a method for editing an α-L-iduronidase (IDUA) gene or a regulatory component thereof, the method comprising combining the IDUA gene or its regulatory component with (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide guides the adenosine base editor to effect an A to G nucleobase change in the IDUA gene or its regulatory component.

[0037] In some embodiments, the IDUA gene or its regulatory element comprises a SNP associated with Hurler's disease. In some embodiments, the A to G nucleobase change is at a SNP associated with Hurler's disease. In some embodiments, the SNP associated with Hurler's disease results in a W402X or W401X amino acid mutation in an IDUA polypeptide numbered in SEQ ID NO: 4 or a variant thereof encoded by the IDUA gene, wherein X is a stop codon.

[0038] In some embodiments, the A to G nucleobase change changes a SNP associated with Hurler's disease to a wild-type nucleobase. In some embodiments, the A to G nucleobase change changes a SNP associated with Hurler's disease to a non-wild-type nucleobase, resulting in one or more improved symptoms of Hurler's disease. In some embodiments, the A to G change at a SNP associated with Hurler's disease changes the stop codon in the IDUA polypeptide encoded by the IDUA gene to tryptophan.

[0039] In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to an IDUA gene or its regulatory component comprising a SNP associated with Hurler's disease. In some embodiments, the adenosine base editor forms a complex with a single guide RNA (sgRNA), which comprises a nucleic acid sequence complementary to an IDUA gene or its regulatory component comprising a SNP associated with Hurler's disease. In some embodiments, the sgRNA comprises a nucleic acid sequence selected from the group consisting of: 5'-GACUCUAGGCAGAGGUCUCAA-3', 5'-ACUCUAGGCAGAGGUCUCAA-3', 5'-CUCUAGGCCGAAGUGUCGC-3', and 5'-GCUCUAGGCCGAAGUGUCGC-3'.

[0040] In some aspects, the present invention provides a method for editing a methyl CpG binding protein 2 (MECP2) gene or a regulatory component thereof, the method comprising administering to a subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide guides the adenosine base editor to effect an A to G nucleobase change in the MECP2 gene or a regulatory component thereof.

[0041] In some embodiments, the MECP2 gene or its regulatory element comprises a SNP associated with Rett syndrome. In some embodiments, the A to G nucleobase change occurs at the SNP associated with Rett syndrome. In some embodiments, the SNP associated with Rett syndrome results in an R106W or T158M amino acid mutation in a MECP2 polypeptide numbered in SEQ ID NO: 5 encoded by the MECP2 gene or a variant thereof. In some embodiments, the SNP associated with Rett syndrome results in an R255X or R270X amino acid mutation in a MECP2 polypeptide encoded by the MECP2 gene, wherein X is a stop codon.

[0042] In some embodiments, the A to G nucleobase change changes a SNP associated with Rett's disease to a wild-type nucleobase. In some embodiments, the A to G nucleobase change changes a SNP associated with Rett's disease to a non-wild-type nucleobase, resulting in one or more improved symptoms of Rett's disease. In some embodiments, the A to G nucleobase change at a SNP associated with Rett's disease changes a stop codon in a MECP2 polypeptide to a tryptophan.

[0043] In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to a MECP2 gene or a regulatory component thereof comprising a SNP associated with Rett's disease. In some embodiments, the adenosine base editor forms a complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to a MECP2 gene or a regulatory component thereof comprising a SNP associated with Rett's disease. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of: 5'-CUUUUCACUUCCUGCCGGGG-3', 5'-AGCUUCCAUGUCCAGCCUUC-3', 5'-ACCAUGAAGUCAAAAUCAUU-3' and 5'-GCUUUCAGCCCCGUUUCUUG-3'.

[0044] In some aspects, provided herein is a method for editing an ATP-binding cassette subfamily member 4 (ABCA4) gene or a regulatory component thereof, the method comprising contacting the ABCA4 gene or its regulatory component with (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises a substitution of an amino acid at amino acid position 82 or 166 numbered in SEQ ID NO: 2 or a position corresponding thereto, and wherein the guide polynucleotide guides the adenosine base editor to effect an A to G nucleobase change in the ABCA4 gene or the regulatory component.

[0045] In some embodiments, administration improves at least one symptom associated with Stargardt's disease. In some embodiments, administration results in faster improvement of at least one symptom associated with Stargardt's disease compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase.

[0046] In some embodiments, the ABCA4 gene includes a SNP associated with Stargardt's disease. In some embodiments, the A to G nucleobase change is at the SNP associated with Stargardt's disease. In some embodiments, the SNP associated with Stargardt's disease results in an A1038V or G1961E amino acid mutation in the ABCA4 polypeptide numbered in SEQ ID NO:6 encoded by the ABCA4 gene, or a variant thereof. In some embodiments, the SNP associated with Stargardt's disease results in a G1961E amino acid mutation in the ABCA4 polypeptide numbered in SEQ ID NO:6, or a variant thereof.

[0047] In some embodiments, the A to G nucleobase change changes a SNP associated with Stargardt's disease to a wild-type nucleobase. In some embodiments, the A to G nucleobase change changes a SNP associated with Stargardt's disease to a non-wild-type nucleobase, resulting in one or more improved symptoms of Stargardt's disease. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence that is complementary to an ABCA4 gene or a regulatory component thereof that comprises a SNP associated with Stargardt's disease.

[0048] In some embodiments, the adenosine base editor forms a complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to an ABCA4 gene or a regulatory component thereof comprising a SNP associated with Stargardt's disease. In some embodiments, the sgRNA comprises the sequence 5'-CUCCAGGGCGAACUUCGACACACAGC-3'.

[0049] In various embodiments of the above aspects, contact is in a cell. In some embodiments, contact results in an indel less than 10% in the genome of the cell, wherein the indel rate is measured by the mismatch frequency between the sequence of the single nucleotide modified side and the unmodified sequence. In some embodiments, contact results in an indel less than 5% in the genome of the cell, wherein the indel rate is measured by the mismatch frequency between the sequence of the single nucleotide modified side and the unmodified sequence. In some embodiments, contact results in an indel less than 1% in the genome of the cell, wherein the indel rate is measured by the mismatch frequency between the sequence of the single nucleotide modified side and the unmodified sequence.

[0050] In various embodiments of the above aspects, the cell is a neuron. In some embodiments, the contact is in a cell population. In some embodiments, the contact causes an A to G nucleobase change in at least 40% of the cell population after the contact step. In some embodiments, the contact causes an A to G nucleobase change in at least 50% of the cell population after the contact step. In some embodiments, the contact causes an A to G nucleobase change in at least 70% of the cell population after the contact step. In some embodiments, at least 90% of the cells are viable after the contact step. In some embodiments, the cell population is not enriched after the contact step. In some embodiments, the cell population is a neuron. In some embodiments, the contact is in vivo or ex vivo.

[0051] In the above-mentioned various aspects and embodiments, the polynucleotide programmable DNA binding domain is Cas9. In some embodiments, Cas9 is SpCas9, SaCas9 or a variant thereof. In some embodiments, the polynucleotide programmable DNA binding domain includes a modified SpCas9 with a changed protospacer-adjacent motif (PAM) specificity. In some embodiments, Cas9 is specific to a PAM sequence selected from a group consisting of NGG, NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN and NGC; wherein N is A, G, C or T; and wherein R is A or G. In some embodiments, the polynucleotide programmable DNA binding domain is a nuclease inactivation variant. In some embodiments, the polynucleotide programmable DNA binding domain is a nickase variant. In some embodiments, the nickase variant includes an amino acid substitution D10A or its corresponding amino acid substitution. In various aspects and embodiments provided herein, the adenosine deaminase domain includes a TadA domain. In some embodiments, the adenosine deaminase comprises a TadA deaminase comprising a V82S change and / or a T166R change.

[0052] In the above various aspects and embodiments, the adenosine deaminase further comprises one or more of the following changes: Y147T, Y147R, Q154S, Y123H, Q154R or a combination thereof. In various aspects and embodiments provided herein, the adenosine deaminase comprises a combination of changes selected from the group consisting of: Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y147T+Q154R; Y147T+Q154S; and Y123H+Y147R+Q154R+I76Y. In various aspects and embodiments provided herein, the adenosine base editor domain comprises an adenosine deaminase monomer. In various aspects and embodiments provided herein, the adenosine base editor comprises an adenosine deaminase dimer. In some embodiments, the TadA deaminase is a TadA*8 variant. In some embodiments, the TadA*8 variant is selected from the group consisting of: TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, and TadA*8.13. In some embodiments, the adenosine base editor is an ABE8 base editor selected from the group consisting of: ABE8.1, ABE8.2, ABE8.3, ABE8.4, ABE8.5, ABE8.6, ABE8.7, ABE8.8, ABE8.9, ABE8.10, ABE8.11, ABE8.12, and ABE8.13.

[0053] In some aspects, provided herein are cells produced by the methods described in the various aspects and embodiments disclosed herein. In some aspects, provided herein are cell populations produced by the methods described in the various aspects and embodiments disclosed herein.

[0054] In some aspects, a base editor system is provided herein, comprising (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide guides the adenosine base editor to effect an A to G nucleobase change in a target gene or a regulatory component thereof associated with a nervous system disorder. In one embodiment of the above aspects, the target gene is a leucine-rich repeat kinase-2 (LRRK2) gene, and the nervous system disease is Parkinson's disease. In another embodiment of the aspects, the target gene is an alpha-L-iduronidase (IDUA) gene, and the nervous system disease is Hurler's disease. In one embodiment of the aspects, the target gene is a methyl CpG binding protein 2 (MECP2) gene, and the nervous system disease is Rett's disease. In another embodiment of the aspect, the target gene is the ATP-binding cassette subfamily member 4 (ABCA4) gene and the nervous system disease is Stargardt's disease.

[0055] In some aspects, the present invention provides a base editor system, which includes (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor includes a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain includes an amino acid substitution at amino acid position 82 or 166 numbered by SEQ ID NO: 2 or its corresponding position, and wherein the guide polynucleotide guides the adenosine base editor to achieve an A to G nucleobase change in the LRRK gene and its regulatory components.

[0056] In some embodiments, the A to G nucleobase change is located at a SNP associated with Parkinson's disease in the LRRK2 gene or its regulatory components. In some embodiments, the SNP associated with Parkinson's disease results in an A419V, R1441C, R1441H or G2019S amino acid mutation in the LRRK2 polypeptide numbered in SEQ ID NO: 3 encoded by the LRRK2 gene or a variant thereof.

[0057] In some embodiments, the A to G nucleobase change changes a SNP associated with Parkinson's disease to a wild-type nucleobase. In some embodiments, the A to G nucleobase change changes a SNP associated with Parkinson's disease to a non-wild-type nucleobase, resulting in an improvement in Parkinson's symptoms. In some embodiments, the A to G nucleobase change changes a cysteine ​​or histidine in a LRRK2 polypeptide encoded by the LRRK2 gene to an arginine. In some embodiments, the A to G change changes a serine in a LRRK2 polypeptide encoded by the LRRK2 gene to a glycine. In some embodiments, the A to G change replaces a cysteine ​​(C) or histidine (H) with an arginine (R) at position 144 of a LRRK2 polypeptide or a variant thereof numbered in SEQ ID NO: 3 encoded by the LRRK2 gene or a glycine (G) at position 2019. In some embodiments, the adenosine deaminase domain includes an amino acid substitution at amino acid position 82 or 166 numbered in SEQ ID NO: 2 or a corresponding position thereof.

[0058] In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to an LRRK2 gene or its regulatory component comprising a SNP associated with Parkinson's disease. In some embodiments, the adenosine base editor forms a complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to an LRRK2 gene or its regulatory component comprising a SNP associated with Parkinson's disease. In some embodiments, the sgRNA comprises the nucleic acid sequence: 5'-AAGCGCAAGCCUGGAGGGAA-3'; or 5'-ACUACAGCAUUGCUCAGUAC-3'.

[0059] In some aspects, the present invention provides a base editor system, which includes (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor includes a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain includes an amino acid substitution at amino acid position 82 or 166 numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide guide adenosine base editor achieves an A to G nucleobase change in an α-L-iduronidase (IDUA) gene or a regulatory component thereof.

[0060] In some embodiments, the IDUA gene or its regulatory element comprises a SNP associated with Hurler's disease. In some embodiments, the A to G nucleobase change is at the SNP associated with Hurler's disease. In some embodiments, the SNP associated with Hurler's disease results in a W402X or W401X amino acid mutation in an IDUA polypeptide numbered in SEQ ID NO: 4 encoded by the IDUA gene or a variant thereof, wherein X is a stop codon.

[0061] In some embodiments, the A to G nucleobase change changes a SNP associated with Hurler's disease to a wild-type nucleobase. In some embodiments, the A to G nucleobase change changes a SNP associated with Hurler's disease to a non-wild-type nucleobase, resulting in one or more improved symptoms of Hurler's disease. In some embodiments, the A to G change at a SNP associated with Hurler's disease changes the stop codon in the IDUA polypeptide encoded by the IDUA gene to tryptophan.

[0062] In some embodiments, the guide polynucleotide includes a nucleic acid sequence complementary to an IDUA gene or its regulatory component including a SNP associated with Hurler's disease. In some embodiments, the adenosine base editor forms a complex with a single guide RNA (sgRNA), which includes a nucleic acid sequence complementary to an IDUA gene or its regulatory component including a SNP associated with Hurler's disease. In some embodiments, the sgRNA includes a nucleic acid sequence selected from the group consisting of: 5'-GACUCUAGGCAGAGGUCUCAA-3', 5'-ACUCUAGGCAGAGGUCUCAA-3', 5'-CUCUAGGCCGAAGUGUCGC-3' and 5'-GCUCUAGGCCGAAGUGUCGC-3'.

[0063] In some aspects, the present invention provides a base editor system, which includes (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor includes a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain includes an amino acid substitution at amino acid position 82 or 166 numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide guides the adenosine base editor to achieve an A to G nucleobase change in the methyl CpG binding protein 2 (MECP2) gene or its regulatory component.

[0064] In some embodiments, the MECP2 gene or its regulatory element comprises a SNP associated with Rett syndrome. In some embodiments, the A to G nucleobase change occurs at the SNP associated with Rett syndrome. In some embodiments, the SNP associated with Rett syndrome results in an R106W or T158M amino acid mutation in a MECP2 polypeptide numbered in SEQ ID NO: 5 encoded by the MECP2 gene or a variant thereof. In some embodiments, the SNP associated with Rett syndrome results in an R255X or R270X amino acid mutation in a MECP2 polypeptide encoded by the MECP2 gene, wherein X is a stop codon.

[0065] In some embodiments, the A to G nucleobase change changes a SNP associated with Rett's disease to a wild-type nucleobase. In some embodiments, the A to G nucleobase change changes a SNP associated with Rett's disease to a non-wild-type nucleobase, resulting in one or more improved symptoms of Rett's disease. In some embodiments, the A to G nucleobase change at a SNP associated with Rett's disease changes a stop codon in a MECP2 polypeptide to a tryptophan.

[0066] In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to a MECP2 gene or its regulatory component comprising a SNP associated with Rett's disease. In some embodiments, the adenosine base editor forms a complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to a MECP2 gene or its regulatory component comprising a SNP associated with Rett's disease. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of: 5'-CUUUUCACUUCCUGCCGGGG-3', 5'-AGCUUCCAUGUCCAGCCUUC-3', 5'-ACCAUGAAGUCAAAAUCAUU-3', and 5'-GCUUUCAGCCCCGUUUCUUG-3'.

[0067] In some aspects, provided herein is a base editor system comprising contacting (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 numbered in SEQ ID NO: 2 or a position corresponding thereto, and wherein the guide polynucleotide guides the adenosine base editor to effect an A to G nucleobase change in the ATP-binding cassette subfamily member 4 (ABCA4) gene or a regulatory component thereof.

[0068] In some embodiments, administration improves at least one symptom associated with Stargardt's disease. In some embodiments, administration results in faster improvement of at least one symptom associated with Stargardt's disease compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase. In some embodiments, the ABCA4 gene includes a SNP associated with Stargardt's disease. In some embodiments, the A to G nucleobase change is at the SNP associated with Stargardt's disease. In some embodiments, the SNP associated with Stargardt's disease results in an A1038V or G1961E amino acid mutation in an ABCA4 polypeptide numbered in SEQ ID NO: 6 encoded by the ABCA4 gene, or a variant thereof. In some embodiments, the SNP associated with Stargardt's disease results in a G1961E amino acid mutation in an ABCA4 polypeptide numbered in SEQ ID NO: 6, or a variant thereof.

[0069] In some embodiments, the A to G nucleobase change changes a SNP associated with Stargardt's disease to a wild-type nucleobase. In some embodiments, the A to G nucleobase change changes a SNP associated with Stargardt's disease to a non-wild-type nucleobase, resulting in improved Stargardt's disease symptoms. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence that is complementary to an ABCA4 gene or a regulatory component thereof that comprises a SNP associated with Stargardt's disease.

[0070] In some embodiments, the adenosine base editor forms a complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to an ABCA4 gene or a regulatory component thereof comprising a SNP associated with Stargardt's disease. In some embodiments, the sgRNA comprises the sequence 5'-CUCCAGGGCGAACUUCGACACACAGC-3'.

[0071] In various aspects and embodiments provided herein, the polynucleotide programmable DNA binding domain is Cas9. In some embodiments, Cas9 is SpCas9, SaCas9 or a variant thereof. In some embodiments, the polynucleotide programmable DNA binding domain includes a modified SpCas9 with a changed pre-spaced adjacent motif (PAM) specificity. In some embodiments, Cas9 is specific to a PAM sequence selected from a group consisting of NGG, NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN and NGC, wherein N is A, G, C or T, and wherein R is A or G. In some embodiments, the polynucleotide programmable DNA binding domain is a nuclease inactivation variant. In some embodiments, the polynucleotide programmable DNA binding domain is a nickase variant. In some embodiments, the nickase variant includes an amino acid substitution D10A or its corresponding amino acid substitution.

[0072] In various aspects and embodiments provided herein, the adenosine deaminase domain comprises a TadA domain. In some embodiments, the adenosine deaminase comprises a TadA deaminase comprising a V82S change and / or a T166R change.

[0073] In various aspects and embodiments provided herein, the adenosine deaminase further comprises one or more of the following changes: Y147T, Y147R, Q154S, Y123H, Q154R, or a combination thereof. In various aspects and embodiments provided herein, the adenosine deaminase comprises a combination of changes selected from the group consisting of: Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y147T+Q154R; Y147T+Q154S; and Y123H+Y147R+Q154R+I76Y. In some embodiments, the adenosine base editor domain comprises an adenosine deaminase monomer. In some embodiments, the adenosine base editor comprises an adenosine deaminase dimer.

[0074] In various aspects and embodiments provided herein, the TadA deaminase is a TadA* 8 variant. In some embodiments, the TadA*8 variant is selected from the group consisting of TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, and TadA*8.13. In some embodiments, the adenosine base editor is an ABE8 base editor selected from the group consisting of ABE8.1, ABE8.2, ABE8.3, ABE8.4, ABE8.5, ABE8.6, ABE8.7, ABE8.8, ABE8.9, ABE8.10, ABE8.11, ABE8.12, and ABE8.13.

[0075] In some aspects, a vector comprising a nucleic acid sequence encoding an adenosine base editor described herein is provided herein. In some aspects, a vector comprising a nucleic acid sequence encoding an adenosine base editor described herein and a guide polynucleotide is provided herein. In some embodiments, the vector is a viral vector, a lentiviral vector, or an AAV vector.

[0076] In some aspects, provided herein is a cell comprising a base editor system or vector as described herein. In some embodiments, the cell is a central nervous system cell. In some embodiments, the cell is a neuron. In some embodiments, the cell is a photoreceptor. In some embodiments, the cell is in vitro, in vivo, or ex vivo.

[0077] In some aspects, a pharmaceutical composition is provided herein, comprising a base editor, a vector or a cell described herein, and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition described herein further comprises a lipid. In another embodiment, the pharmaceutical composition described herein further comprises a virus.

[0078] In some aspects, provided herein are kits comprising a base editor or a vector described herein.

[0079] In various embodiments of the methods described herein, at least one nucleotide of the guide polynucleotide includes a non-naturally occurring modification. In various embodiments of the methods described herein, at least one nucleotide of the nucleic acid sequence includes a non-naturally occurring modification. In various embodiments, at least one nucleotide of the nucleic acid sequence of the base editor system includes a non-naturally occurring modification. In some embodiments, the non-naturally occurring modification is a chemical modification. In some embodiments, the chemical modification is 2'-O-methylation. In some embodiments, the nucleic acid sequence includes a thiophosphate.

[0080] The description and examples herein describe the embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein and can therefore vary. Those skilled in the art will recognize that there are multiple variations and modifications in the present disclosure, and these variations and modifications are included within the scope thereof.

[0081] Unless otherwise indicated, the practice of some embodiments disclosed herein employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (FM Ausubel, et al. eds.); the series Methods In Enzymology (Academic 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 Specialized Applications, 6th Edition (RI Freshney, ed. (2010)).

[0082] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0083] Although various features of the present disclosure may be described in the context of a single embodiment, these features may also be provided individually or in any suitable combination. Conversely, although the present disclosure may be described herein in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0084] The features of the present disclosure are particularly set forth in the appended claims.A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description of illustrative embodiments, in which the principles of the present disclosure are utilized, and in view of the accompanying drawings as described below.

[0085] definition

[0086] The following definitions supplement those in the art and are specific to the current application and are not attributed to any related or unrelated cases, e.g., any commonly owned patents or applications. Although any methods and materials similar or equivalent to those described herein may be used to test the practice of the present disclosure, preferred materials and methods are described herein. Therefore, the terms used herein are only used for the purpose of describing specific embodiments and are not intended to be limiting.

[0087] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. The following references provide general definitions of many of the terms used in the present invention for those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0088] In this application, unless otherwise specifically stated, the use of the singular includes the plural. It must be noted that the singular forms "a", "an", and "the" used in the specification include plural references unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" and is understood to be inclusive unless otherwise stated. In addition, the use of the term "comprising" and other forms such as "including", "containing", and "included" are not restrictive.

[0089] As used in this specification and claims, the words "comprise" (and any form of including, such as "including" and "comprising"), "have" (and any form of having, such as "having" and "having"), "include" (and any form of including, such as "including" and "containing"), or "contain" (and any form of containing, such as "containing" and "containing") are inclusive or open-ended, and do not exclude additional, unrecited components or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or combination of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

[0090] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one skilled in the art, which will depend in part on how the value is measured or determined, i.e., the measurement system. For example, according to the practice in the art, "about" can mean within 1 standard deviation or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term can mean within an order of magnitude, such as within 5 times or 2 times the value. Where specific values ​​are described in the application and claims, the meaning of the term "about" should be assumed to be within an acceptable error range for the specific value unless otherwise indicated.

[0091] 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110

[0092] References in the specification to "some embodiments," "embodiments," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments of the present disclosure.

[0093] "Base-free editor" refers to an agent that can excise a nucleobase and insert a DNA nucleobase (A, T, C or G). Base-free editors include nucleic acid glycosylase polypeptides or fragments thereof. In one embodiment, the nucleic acid glycosylase is a mutant human uracil DNA glycosylase, which includes Asp at amino acid 204 in the following sequence (e.g., replacing Asn at amino acid 204), or a corresponding position in a uracil DNA glycosylase, and has cytosine-DNA glycosylase activity or an active fragment thereof. In one embodiment, the nucleic acid glycosylase is a mutant human uracil DNA glycosylase, which includes Ala, Gly, Cys or Ser at amino acid 147 in the following sequence (e.g., replacing Tyr at amino acid 147), or a corresponding position in a uracil DNA glycosylase, and has thymine-DNA glycosylase activity or an active fragment thereof. The sequence of the exemplary human uracil-DNA glycosylase isoform 1 is as follows:

[0094]

[0095] The sequence of human uracil-DNA glycosylase isoform 2 is as follows:

[0096]

[0097] In other embodiments, the base-free editor is any one of the base-free editors described in PCT / JP2015 / 080958 and US20170321210, which are incorporated herein by reference. In a specific embodiment, the base-free editor includes a mutation at a position shown in bold in the above sequence and has an underline, or at the corresponding amino acid in any other base-free editor or uracil deglycosylase known in the art. In one embodiment, the base-free editor includes mutations at Y147, N204, L272 and / or R276 or corresponding positions. In another embodiment, the base-free editor includes a Y147A or Y147G mutation or a corresponding mutation. In another embodiment, the base-free editor includes a N204D mutation or a corresponding mutation. In another embodiment, the base-free editor includes an L272A mutation or a corresponding mutation. In another embodiment, the base-free editor includes an R276E or R276C mutation or a corresponding mutation.

[0098] "Adenosine deaminase" refers to a polypeptide or fragment thereof that can catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to inosine or the hydrolytic deamination of deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminase provided herein (e.g., engineered adenosine deaminase, evolved adenosine deaminase) can be from any organism, such as bacteria.

[0099] In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is a TadA variant. In some embodiments, the TadA variant is TadA*8. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain does not exist in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, 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 99.7%, at least 99.8%, or at least 99.9% identical to a naturally occurring deaminase. For example, deaminase domains are described in International PCT Application No. PCT / 2017 / 045381 (WO 2018 / 027078) and International PCT Application No. PCT / US2016 / 058344 (WO 2017 / 070632), each of which is incorporated herein by reference in its entirety.See also Komor, AC, et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage" Nature 551, 464-471 (2017); Gaudelli, NM, et al. al., "Improved base excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A baseeditors with higher efficiency and product 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. doi: 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0100] The wild-type TadA (wt) adenosine deaminase has the following sequence (also referred to as the TadA reference sequence):

[0101]

[0102] In some embodiments, the adenosine deaminase comprises a change in the following sequence:

[0103]

[0104]

[0105] (also known as TadA*7.10).

[0106] In some embodiments, TadA*7.10 includes at least one change. In some embodiments, TadA*7.10 includes changes at amino acids 82 and / or 166. In specific embodiments, variants of the above sequences include one or more of the following changes: Y147T, Y147R, Q154S, Y123H, V82S, T166R and / or Q154R. Change Y123H is also referred to herein as H123H (change H123Y in TadA*7.10 reverts to Y123H (wild type)). In other embodiments, variants of the TadA*7.10 sequence include a combination of changes 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+Y 123H+Y147R;V82S+Y123H+Q154R;Y147R+Q154R+Y123H;Y147R+Q154R+I76Y;Y147R+Q154R+T166R;Y123H+Y147R+Q154R+I76Y;V82S+Y123H+Y147R+Q154R;and I76Y+V82S+Y123H+Y147R+Q154R.

[0107] In other embodiments, the invention provides adenosine deaminase variants comprising a deletion, such as TadA*8, including a deletion of the C-terminus starting from residues 149, 150, 151, 152, 153, 154, 155, 156 or 157. In other embodiments, the adenosine deaminase variant is a TadA (e.g., TadA*8) monomer comprising one or more of the following changes: Y147T, Y147R, Q154S, Y123H, V82S, T166R and / or Q154R. In other embodiments, the adenosine deaminase variant is TadA (e.g., TadA*8), a monomer comprising a combination of changes selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147 T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R +T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.

[0108] In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each of which has one or more of the following changes Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each of which has a combination of changes selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y12 3H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q 154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.

[0109] In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type TadA adenosine deaminase domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising one or more of the following changes Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type TadA adenosine deaminase domain and an adenosine deaminase variant domain (e.g., TadA*8), wherein the adenosine deaminase variant domain comprises a combination of changes selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V8 2S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.

[0110] In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8), wherein the adenosine deaminase variant domain comprises one or more of the following changes Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8), wherein the adenosine deaminase variant domain comprises a combination of one or more of the following changes: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V 82S+Y123H+Y147T;V82S+Y123H+Y147R;V82S+Y123H+Q154R;Y147R+Q154R+Y123H;Y147R+Q154R+I76Y;Y147R+Q154R+T166R;Y123H+Y147R+Q154R+I76Y;V82S+Y123H+Y147R+Q154R;or I76Y+V82S+Y123H+Y147R+Q154R.

[0111] In one embodiment, the adenosine deaminase is TadA*8, which comprises or consists essentially of the following sequence having adenosine deaminase activity or a fragment thereof:

[0112]

[0113] In some embodiments, TadA*8 is truncated. In some embodiments, the truncated TadA*8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 N-terminal amino acid residues relative to full-length TadA*8. In some embodiments, the truncated TadA*8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19 or 20 C-terminal amino acid residues relative to full-length TadA*8. In some embodiments, the adenosine deaminase variant is full-length TadA*8.

[0114] In certain embodiments, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from one of:

[0115] In certain embodiments, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from one of:

[0116] Escherichia coli TadA:

[0117]

[0118] Escherichia coli (E. coli) TadA (N-terminus is truncated):

[0119]

[0120] Staphylococcus aureus (S. aureus) TadA:

[0121]

[0122] Bacillus subtilis (B. subtilis) TadA:

[0123]

[0124] Salmonella typhimurium (S.typhimurium) TadA:

[0125]

[0126] Shewanella putrefaciens (S. putrefaciens) TadA:

[0127]

[0128] Haemophilus influenzae F3031 (H.influenzae) TadA:

[0129]

[0130] Caulobacter crescentus (C. crescentus) TadA:

[0131]

[0132] Geobacter sulfurreducens (G.sulfurreducens) TadA:

[0133]

[0134] Other TadA7.10 or TadA7.10 variants contemplated as components of heterodimers with TadA*8 include:

[0135]

[0136] TadA7.10 CP65

[0137]

[0138] TadA7.10 CP83

[0139]

[0140] TadA7.10 CP136

[0141]

[0142] TadA7.10 C-truncated

[0143]

[0144] TadA7.10 C-truncated 2

[0145]

[0146] TadA7.10Δ59-66+C-truncation

[0147]

[0148] TadA7.10Δ59-66

[0149]

[0150] In some embodiments, the adenosine deaminase variants include changes in TadA7.10. In some embodiments, TadA7.10 includes changes at amino acids 82 or 166. In specific embodiments, the variants in the above sequences include one or more of the following changes: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R. In other embodiments, the adenosine deaminase variants include a combination of changes selected from the group consisting of Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y147T+Q154R; Y147T+Q154S; and Y123H+Y147R+Q154R+I76Y.

[0151] In other embodiments, the invention provides adenosine deaminase variants comprising deletions, such as TadA7.10 comprising a C-terminal deletion starting at residues 149, 150, 151, 152, 153, 154, 155, 156 or 157. In some embodiments, the adenosine deaminase variant is a TadA monomer comprising one or more of the following changes: Y147T, Y147R, Q154S, Y123H, V82S, T166R, Q154R. In other embodiments, the adenosine deaminase variant is a monomer comprising the following changes: Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y147T+Q154R; Y147T+Q154S; and Y123H+Y147R+Q154R+I76Y. In yet other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains, each of which has one or more of the following changes Y147T, Y147R, Q154S, Y123H, V82S, T166R, Q154R. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type adenosine deaminase domain or a TadA7.10 domain and an adenosine deaminase variant domain comprising one or more of the following changes Y147T, Y147R, Q154S, Y123H, V82S, T166R, Q154R. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA7.10 domain and an adenosine deaminase variant of TadA7.10 comprising the following changes: Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y147T+Q154R; Y147T+Q154S; and Y123H+Y147R+Q154R+I76Y.

[0152] "Administration" in this article refers to providing one or more compositions described herein to a patient or subject. For example, but not limited to, the composition is administered (e.g., injected), which can be injected intravenously (iv), subcutaneously (sc), intradermally (id), intraperitoneally (ip), or intramuscularly (im). One or more such routes can be used. Parenteral administration can be, for example, by bolus or gradual infusion over time. In some embodiments, parenteral administration includes intravascular, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, intradermal, intraperitoneal, transtracheal, subcutaneous, subcolloidally, intraarticularly, subcapsularly, subarachnoidally, and intrasternal infusion or injection. Alternatively, or simultaneously, administration can be by oral route.

[0153] "Agent" refers to any small molecule compound, antibody, nucleic acid molecule or polypeptide, or a fragment thereof.

[0154] "Alteration" refers to a change (e.g., increase or decrease) in the structure, expression level, or activity of a gene or polypeptide, as detected by standard art-known methods, such as those described herein. As used herein, an alteration includes a change in a polynucleotide or polypeptide sequence or a change in expression level, such as a 10% change, a 25% change, a 40% change, a 50% change, or greater.

[0155] "Ameliorate" means to reduce, inhibit, attenuate, diminish, arrest or stabilize the development or progression of a disease.

[0156] "Analogs" refer to molecules that are not identical but have similar functional or structural features. For example, polynucleotide or polypeptide analogs retain the biological activity of the corresponding naturally occurring polynucleotide or polypeptide while having certain modifications that enhance the analog's function relative to the naturally occurring polynucleotide or polypeptide. Such modifications can increase the analog's affinity for DNA, efficiency, specificity, protease or nuclease resistance, membrane permeability, and / or half-life without altering, for example, ligand binding. Analogs can include non-natural nucleotides or amino acids.

[0157] "Base editor (BE)" or "nucleobase editor (NBE)" refers to an agent that binds to a polynucleotide and has nucleobase modification activity. In various embodiments, the base editor includes a nucleobase modification polypeptide (e.g., a deaminase) and a nucleic acid programmable nucleotide binding domain that binds to a guide polynucleotide (e.g., a guide RNA). In various embodiments, the agent is a biomolecular complex comprising a protein domain with base editing activity, i.e., a domain capable of modifying a base (e.g., A, T, C, G, or U) in a nucleic acid molecule (e.g., DNA). In some embodiments, the polynucleotide programmable DNA binding domain is fused or connected to a deaminase domain. In one embodiment, the agent is a fusion protein comprising a domain with base editing activity. In another embodiment, a protein domain with base editing activity is connected to a guide RNA (e.g., via an RNA binding motif on a guide RNA and an RNA binding domain fused to a deaminase). In some embodiments, a domain with base editing activity is capable of deaminating a base in a nucleic acid molecule. In some embodiments, a base editor is capable of deaminating one or more bases in a DNA molecule. In some embodiments, the base editor is capable of deaminating adenosine (A) within DNA. In some embodiments, the base editor is an adenosine base editor (ABE).

[0158] "Cytidine deaminase" refers to a polypeptide or fragment thereof that can catalyze a deamination reaction that converts an amino group to a carbonyl group. In some embodiments, the cytidine deaminase has at least about 85% identity to APOBEC or AID. In one embodiment, the cytidine deaminase converts cytosine to uracil or converts 5-methylcytosine to thymine. PmCDA1 (derived from Petromyzon marinus (sea lamprey cytosine deaminase 1, "PmCDA1"), AID (activation-induced cytidine deaminase; (activation-induced cytidine deaminase, AICDA)) (derived from mammals (e.g., humans, pigs, cattle, horses, monkeys, etc.) and APOBEC are exemplary cytidine deaminases.

[0159] In some embodiments, the base editor is a reprogrammable base editor fused to a deaminase (e.g., adenosine deaminase or cytidine deaminase). In some embodiments, the base editor is a Cas9 fused to a deaminase (e.g., adenosine deaminase or cytidine deaminase). In some embodiments, the base editor is a nuclease-free Cas9 (dCas9) fused to a deaminase (e.g., adenosine deaminase or cytidine deaminase). In some embodiments, Cas9 is a circularly arranged Cas9 (e.g., spCas9 or saCas9). Circularly arranged Cas9 is known in the art and is described in, for example, Oakes et al., Cell 176, 254-267, 2019. In some embodiments, the base editor is fused to a base excision repair inhibitor, for example, a UGI domain or a dISN domain. In some embodiments, the fusion protein includes a Cas9 nickase fused to a deaminase and a base excision repair inhibitor such as a UGI or dISN domain. In other embodiments, the base editor is a base-free base editor.

[0160] In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, adenosine deaminase is evolved from TadA. In some embodiments, the base editor of the present invention includes a napDNAbp domain with an internally fused catalytic (e.g., deaminase) domain. In some embodiments, napDNAbp is a Cas12a (Cpf1) with an internally fused deaminase domain. In some embodiments, napDNAbp is a Cas12b (c2c1) with an internally fused deaminase domain. In some embodiments, napDNAbp is a Cas12c (c2c3) with an internally fused deaminase domain. In some embodiments, napDNAbp is a Cas12d (CasX) with an internally fused deaminase domain. In some embodiments, napDNAbp is a Cas12e (CasY) with an internally fused deaminase domain. In some embodiments, napDNAbp is a Cas12g with an internally fused deaminase domain. In some embodiments, the napDNAbp is a Cas12h with an internally fused deaminase domain. In some embodiments, the napDNAbp is a Cas12i with an internally fused deaminase domain. In some embodiments, the base editor is a catalytically dead Cas12 (dCas12) fused to a deaminase domain. In some embodiments, the base editor is a Cas12 nickase (nCas12) fused to a deaminase domain.

[0161] In some embodiments, the base editor is produced by cloning an adenosine deaminase variant (e.g., TadA*8) into a scaffold comprising a circularly arranged Cas9 (e.g., spCAS9 or saCAS9) and a bipartite nuclear localization sequence (e.g., ABE8). Circularly arranged Cas9 is known in the art and is described in, for example, Oakes et al., Cell 176, 254-267, 2019. The exemplary circular arrangement is as follows, where the bold sequence represents a sequence derived from Cas9, the italic sequence represents a linker sequence, and the bottom line sequence represents a bipartite nuclear localization sequence.

[0162] CP5 (with MSP "NGC = Pam variant conventional Cas9 with mutation like NGG" PID = Protein Interacting Domain and "D10A" nickase):

[0163]

[0164] In some embodiments, ABE8 is a base editor selected from Tables 6 to 9, 13 or 14 below. In some embodiments, ABE8 contains an adenosine deaminase variant evolved from TadA. In some embodiments, the adenosine deaminase variant of ABE8 is a TadA*8 variant as described in Tables 7, 9, 13 or 14 below. In some embodiments, the adenosine deaminase variant is a TadA*7.10 variant (e.g., TadA*8) comprising one or more changes selected from Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In various embodiments, ABE8 comprises a polypeptide 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; TadA*7.10 variants (e.g., TadA*8) of the altered combinations of the group consisting of Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. In some embodiments, ABE8 is a monomer construct. In some embodiments, ABE8 is a heterodimer construct. In some embodiments, the ABE8 base editor comprises the following sequence:

[0165]

[0166] In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR-related (e.g., Cas or Cpf1) enzyme. In some embodiments, the base editor is a catalytically dead Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a deaminase domain. In some embodiments, the base editor is fused to a base excision repair (BER) inhibitor. In some embodiments, the base excision repair inhibitor is a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the base excision repair inhibitor is an inosine base excision repair inhibitor.

[0167] Details of base editors are described in International PCT Application No. PCT / 2017 / 045381 (WO 2018 / 027078) and International PCT Application No. PCT / US2016 / 058344 (WO 2017 / 070632), each of which is incorporated herein by reference in its entirety. See also Komor, AC et al., "Programmable base editing of a target base ingenomic 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 genomicDNA without DNA cleavage" Nature 551, 464-471 (2017); Komor, 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 product purity" Science Advances 3:eaao4774 (2017), and Rees, HA, et al., "Base editing: precisionchemistry on the genome and transcriptome of living cells.”Nat Rev Genet. 2018 Dec; 19(12): 770-788. doi: 10.1038 / s41576-018-0059-1, the entire contents of which are hereby incorporated by reference.

[0168] For example, the cytidine base editor used in the base editing compositions, systems and methods described herein has 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.aao4774) as follows. Also included are polynucleotide sequences having at least 95% or higher identity to the BE4 nucleic acid sequence.

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178] BE4 amino acid sequence:

[0179]

[0180] For example, the adenine base editor (ABE) used in the base editing compositions, systems and methods described herein has a nucleic acid sequence (8877 base pairs), (Addgene, 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): 843-846. doi: 10.1038 / nbt.4172.) as follows. Also included are polynucleotide sequences having at least 95% or higher identity to the ABE nucleic acid sequence.

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188] "Base editing activity" refers to the activity used to chemically change a base within a polynucleotide. In one embodiment, the first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, such as converting the target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, such as converting A·T to G·C. In another embodiment, the base editing activity is cytidine deaminase activity, such as converting the target C·G to T·A and adenosine or adenine deaminase activity, such as converting A·T to G·C. In some embodiments, base editing activity is evaluated by editing efficiency. Base editing efficiency can be measured in any suitable manner, for example, by Sanger sequencing or next-generation sequencing. In some embodiments, base editing efficiency is measured by the percentage of total sequencing reads with nuclear base conversions affected by the base editor, for example, the percentage of total sequencing reads with target AT base pairs converted to GC base pairs. In some embodiments, when base editing is performed in a population of cells, the base editing efficiency is measured by the percentage of total cells that have a nucleobase transition effected by the base editor.

[0189] The term "base editor system" refers to a system for editing the nucleobase of a target nucleotide sequence. In various embodiments, the base editor system includes (1) a polynucleotide programmable nucleotide binding domain (e.g., Cas9); (2) a deaminase domain (e.g., adenosine deaminase or cytidine deaminase) for deaminating the nucleobase; (3) one or more guide polynucleotides (e.g., guide RNA). In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor system is ABE8.

[0190] In some embodiments, the base editor system may include more than one base editing component. For example, the base editor system may include more than one deaminase. In some embodiments, the base editor system may include one or more adenosine deaminases. In some embodiments, a single guide polynucleotide may be used to target different deaminases to a target nucleic acid sequence. In some embodiments, a single pair of guide polynucleotides may be used to target different deaminases to a target nucleic acid sequence.

[0191] The deaminase domain and the polynucleotide programmable nucleotide binding component of the base editor system can be covalently or non-covalently linked to each other, or any combination of their links and interactions. For example, in some embodiments, the deaminase domain can target the target nucleotide sequence through the polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or connected to the deaminase domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the deaminase domain to the target nucleotide sequence by non-covalent interaction or connection with the deaminase domain. For example, in some embodiments, the deaminase domain may include an additional heterologous portion or domain that is capable of interacting, linking or forming a complex with an additional heterologous portion or domain that is part of the polynucleotide programmable nucleotide binding domain. In some embodiments, the additional heterologous portion may be able to bind to, interact with, link to or form a complex with a polypeptide. In some embodiments, the additional heterologous portion may be able to bind to, interact with, link to or form a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be able to bind to a guide polynucleotide. In some embodiments, the additional heterologous portion may be able to bind to a polypeptide linker. In some embodiments, the additional heterologous moiety is capable of binding to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a Kappa homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0192] The base editor system may further include a guide polynucleotide component. It should be understood that the components of the base editor system can be associated with each other via covalent bonds, non-covalent interactions, or any combination of their association and interaction. In some embodiments, the deaminase domain can target the target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the deaminase domain may include additional heterologous parts or domains (e.g., polynucleotide binding domains, such as RNA or DNA binding proteins) that can interact with the following substances, connect or form a complex with a part or segment of the guide polynucleotide (e.g., a polynucleotide motif). In some embodiments, additional heterologous parts or domains (e.g., polynucleotide binding domains, such as RNA or DNA binding proteins) can be fused or connected to the deaminase domain. In some embodiments, additional heterologous parts may be able to bind to, interact with, connect to, or form a complex with a polypeptide. In some embodiments, additional heterologous parts may be able to bind to, interact with, connect to, or form a complex with a polynucleotide. In some embodiments, additional heterologous parts may be able to bind to guide polynucleotides. In some embodiments, additional heterologous parts may be able to bind to polypeptide linkers. In some embodiments, the additional heterologous moiety is capable of binding to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a Kappa homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0193] In some embodiments, the base editor system may further include an inhibitor of a base excision repair (BER) component. It should be understood that the components of the base editor system can be associated with each other via covalent bonds, non-covalent interactions, or any combination of their association and interaction. The inhibitor of the BER component may include a BER inhibitor. In some embodiments, the BER inhibitor may be a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the BER inhibitor may be an inosine BER inhibitor. In some embodiments, the BER inhibitor may target the target nucleotide sequence through a polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain may be fused or connected to a BER inhibitor. In some embodiments, the polynucleotide programmable nucleotide binding domain may be fused or connected to a deaminase domain and a BER inhibitor. In some embodiments, the polynucleotide programmable nucleotide binding domain may target the BER inhibitor to a target nucleotide sequence by non-covalent interaction or connection with the BER inhibitor. For example, in some embodiments, the inhibitor of the BER component may include an additional heterologous portion or domain that is capable of interacting, connecting, or forming a complex with an additional heterologous portion or domain that is part of a programmable polynucleotide binding domain.

[0194] In some embodiments, a BER inhibitor can target a target nucleotide sequence via a guide polynucleotide. For example, in some embodiments, a BER inhibitor can include an additional heterologous portion or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) that can interact, bind, or form a complex with a portion or segment (e.g., a polynucleotide motif) of a guide polynucleotide. In some embodiments, an additional heterologous portion or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) of a guide polynucleotide can be fused or linked to a BER inhibitor. In some embodiments, an additional heterologous portion may be able to bind, interact, bind, or form a complex with a polynucleotide. In some embodiments, an additional heterologous portion may be able to bind to a guide polynucleotide. In some embodiments, an additional heterologous portion may be able to bind to a polypeptide linker. In some embodiments, an additional heterologous portion may be able to bind to a polynucleotide linker. An additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMuCom coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0195] The term "Cas9" or "Cas9 domain" refers to a binding domain of an RNA-guided nuclease Cas9 comprising a Cas9 protein or a fragment thereof (e.g., a protein comprising an active, inactive or partially active DNA cleavage domain of Cas9, and / or gRNA). Cas9 nucleases are sometimes also referred to as Casn1 nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)-related nucleases. CRISPR is an adaptive immune system that provides protection against mobile genetic components (viruses, transposons, and conjugative plasmids). The CRISPR cluster includes a spacer, a sequence complementary to the preceding mobile component, and a target invading nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In a class II CRISPR system, transcoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein are required for the correct processing of pre-crRNA. TracrRNA serves as a guide for ribonuclease 3 to assist in the processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cuts a linear or circular dsDNA target complementary to the spacer. The target strand that is not complementary to crRNA is first cut by endonucleolysis and then trimmed by 3'-5' exonucleolysis. In nature, DNA binding and cutting usually require proteins and two RNAs. However, a single guide RNA ("sgRNA", or "gNRA" for short) can be modified to integrate various aspects of crRNA and tracrRNA into a single RNA species. See, for example, Jinek M., et al., Science 337: 816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes short motifs in CRISPR repeat sequences (PAM or pre-spacer adjacent motifs) to help distinguish self from non-self.Cas9 nuclease sequence and structure are well known to those skilled in the art (see, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., Proc. 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 adaptive bacterial immunity." Jinek M., et al., Science 337:816-821 (2012), each of which is incorporated herein by reference in its entirety). Cas9 orthologs have been described in various species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus. Other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences include those from the organisms and sites disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737; which is incorporated herein by reference in its entirety.

[0196] An exemplary Cas9 is Streptococcus pyogenes Cas9 (spCas9), the amino acid sequence of which is provided below:

[0197]

[0198] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0199] Nuclease-inactivated Cas9 proteins are interchangeably referred to as "dCas9" proteins (Cas9 "dead" for nucleases) or catalytically inactivated Cas9. Methods for producing Cas9 proteins (or fragments thereof) with inactivated DNA cleavage domains are known (see, e.g., Jinek et al., Science. 337: 816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28; 152 (5): 1173-83, each incorporated herein by reference in its entirety). For example, it is known that the DNA cleavage domain of Cas9 includes two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cuts the strand complementary to the gRNA, while the RuvC1 subdomain cuts the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas (Jinek et al., Science. 337: 816-821 (2012); Qi et al., Cell. 28; 152 (5): 1173-83 (2013)). In some embodiments, the Cas9 nuclease has an inactivated (e.g., inactivated) DNA cleavage domain, that is, Cas9 is a nickase, referred to as a "nCas9" protein (for "nickase" Cas9). In some embodiments, a protein comprising a Cas9 fragment is provided. For example, in some embodiments, the protein comprises one of two Cas9 domains: (1) a gRNA binding domain of Cas9; or (2) a DNA cleavage domain of Cas9. In some embodiments, a protein comprising Cas9 or a fragment thereof is referred to as a "Cas9 variant". Cas9 variants have homology with Cas9 or a fragment thereof. For example, the Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9.In some embodiments, the Cas9 variant can have 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, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant includes a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the corresponding wild-type Cas9 amino acid length.

[0200] In some embodiments, the length of the fragment is at least 100 amino acids. In some embodiments, the length of the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1050, 1100, 1150, 1200, 1250 or at least 1300 amino acids.

[0201] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, nucleotide and amino acid sequences are as follows).

[0202]

[0203]

[0204]

[0205]

[0206] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0207] In some embodiments, the wild-type Cas9 corresponds to or includes the following nucleotide and / or amino acid sequence:

[0208]

[0209]

[0210]

[0211]

[0212] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0213] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2 (nucleotide sequence below); and Uniprot Reference Sequence: Q99ZW2 (amino acid sequence below).

[0214]

[0215]

[0216]

[0217] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0218] In some embodiments, Cas9 refers to Cas9 from: Corynebacterium ulcerans (NCBI Reference Sequences: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Reference Sequences: NC_016782.1, NC_016786.1); Spiroplasma yrphidicola (NCBI Reference Sequence: NC_021284.1); Prevotella intermedia (NCBI Reference Sequence: NC_017861.1); Spiroplasma taiwanense (NCBI Reference Sequence: NC_021846.1); Streptococcus iniae (NCBI Reference Sequence: NC_021314.1); Belliella baltica) (NCBI Reference Sequence: NC_018010.1); Psychroflexus Torquisi (NCBI Reference Sequence: NC_018721.1); Streptococcus thermophilus (NCBI Reference Sequence: YP_820832.1), Listeria innocua (NCBI Reference Sequence: NP_472073.1), Campylobacter jejuni (NCBI Reference Sequence: YP_002344900.1), or Neisseria meningitidis (NCBI Reference Sequence: YP_002342100.1), or Cas9 from any other organism.

[0219] In some embodiments, dCas9 corresponds to or includes, in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain includes mutations numbered D10A and H840A in SEQ ID NO: 1 or corresponding mutations in another Cas9. In some embodiments, dCas9 includes the amino acid sequence of dCas9 (D10A and H840A):

[0220]

[0221]

[0222] (Single underline: HNH domain; double underline: RuvC domain).

[0223] In some embodiments, the Cas9 domain includes a D10A mutation, while the residue at position 840 remains a histidine in the amino acid sequence provided above, or at the corresponding position in any of the amino acid sequences provided herein.

[0224] In other embodiments, dCas9 variants with mutations other than D10A and H840A are provided, which, for example, result in nuclease-inactivated Cas9 (dCas9). For example, such mutations include other amino acid substitutions at D10 and H840, or other substitutions within the Cas9 nuclease domain (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs of dCas9 are provided, which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants with short or long about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids or more are provided.

[0225] In some embodiments, the Cas9 fusion protein provided herein includes the full-length amino acid sequence of the Cas9 protein, such as one of the Cas9 sequences provided herein. However, in other embodiments, the fusion protein provided herein does not include the full-length Cas9 sequence, but only includes one or more fragments thereof. Suitable Cas9 domains and exemplary amino acid sequences of Cas9 fragments are provided herein, and other suitable sequences of Cas9 domains and fragments are apparent to those skilled in the art.

[0226] It should be understood that additional Cas9 proteins (e.g., nuclease dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease active Cas9), including variants and homologs thereof, are within the scope of the present disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is a nuclease dead Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease active Cas9.

[0227] Example catalytically inactivated Cas9 (dCas9):

[0228]

[0229] Example catalyzed Cas9 nickase (nCas9):

[0230]

[0231]

[0232] Example catalytically active Cas9:

[0233]

[0234] In some embodiments, Cas9 refers to Cas9 from archaea (e.g., nanoarchaea), which constitutes the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, Cas9 refers to CasX or CasY, which has been described in, for example, Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21, which is hereby incorporated as a whole by introduction. Using genome-analyzed metagenomics, many CRISPR-Cas systems have been identified, including Cas9 reported for the first time in the field of archaea. This divergent Cas9 protein was found in nanoarchaea, which have rarely been studied, as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, were found, which are one of the most compact systems discovered so far. In some embodiments, Cas9 refers to a variant of CasX or CasX. In some embodiments, Cas9 refers to a variant of CasY or CasY. It will be appreciated that other RNA-guided DNA binding proteins may be used as nucleic acid-programmable DNA binding proteins (napDNAbps) and are within the scope of the present disclosure.

[0235] In certain embodiments, napDNAbps useful in the methods of the invention include circular arrays known in the art and described, for example, by Oakes et al., Cell 176, 254–267, 2019. Exemplary circular arrays are as follows: bold sequences represent sequences derived from Cas9, italic sequences represent linker sequences, underline sequences represent bipartite nuclear localization sequences, CP5 (with MSP "NGC = conventional Cas9 with mutation Pam variant like NGG" PID = protein interaction domain and "D10A" nickase):

[0236]

[0237]

[0238] Non-limiting examples of polynucleotide programmable nucleotide binding domains that can be incorporated into base editors include those derived from CRISPR protein domains, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs).

[0239] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein 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. In some embodiments, the amino acid sequence included in the napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% protein identical to naturally occurring CasX or CasY. In some embodiments, the napDNAbp is a naturally occurring CasX or CasY protein. In some embodiments, the amino acid sequence included in the napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to any CasX or CasY protein described herein. It will be appreciated that Cas12b / C2c1, CasX and CasY from other bacterial species may also be used in accordance with the present disclosure.

[0240] Cas12b / C2c1(uniprot.org / uniprot / T0D7A2#2)

[0241] sp|T0D7A2|C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acido-terrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) GN=c2c1 PE=1 SV=1

[0242]

[0243]

[0244] CasX(uniprot.org / uniprot / F0NN87;uniprot.org / uniprot / F0NH53)

[0245] >tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx proteins OS=Sulfolobus islandicus (strain HVE10 / 4) GN=SiH_0402 PE=4 SV=1

[0246]

[0247] >tr|F0NH53|F0NH53_SULIR CRISPR-associated proteins, Casx OS=Sulfolobus sulphophilus (strain REY15A) GN=SiRe_0771 PE=4SV=1

[0248]

[0249] Proteus CasX

[0250]

[0251]

[0252] CasY(ncbi.nlm.nih.gov / protein / APG80656.1)

[0253] >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria bacteria]

[0254]

[0255] The term "Cas12" or "Cas12 domain" refers to an RNA-guided nuclease comprising a Cas12 protein or a fragment thereof (e.g., a protein comprising an active, inactivated or partially active DNA cleavage domain of Cas12 and / or a gRNA binding domain of Cas12). Cas12 belongs to a Class 2 V-type CRISPR / Cas system. Cas12 nucleases are sometimes also referred to as CRISPR (clustered regularly interspaced short palindromic repeats)-related nucleases. The sequence of an exemplary Bacillus hisashii Cas 12b (BhCas12b) Cas 12 domain is provided below:

[0256]

[0257] Amino acid sequences having at least 85% or higher identity to the BhCas12b amino acid sequence can also be used in the methods of the invention.

[0258] "Cytidine deaminase" refers to a polypeptide or fragment thereof that can catalyze a deamination reaction that converts an amino group to a carbonyl group. In one embodiment, a cytidine deaminase converts cytosine to uracil or converts 5-methylcytosine to thymine. PmCDA1 (derived from sea lamprey (sea lamprey cytosine deaminase 1, "PmCDA1")), AID (activation-induced cytidine deaminase; AICDA) (derived from mammals (e.g., humans, pigs, cattle, horses, monkeys, etc.)) and APOBEC are exemplary cytidine deaminases.

[0259] The term "conservative amino acid substitution" or "conservative mutation" refers to the substitution of one amino acid by another amino acid with common properties. A functional method to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Based on such an analysis, groups of amino acids can be defined as those in which the amino acids within the group are preferentially interchangeable with each other and are therefore most similar to each other in terms of their effect on the overall protein structure (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include amino acid substitutions of amino acids, such as substitutions of arginine with lysine and vice versa, so that a positive charge can be maintained; substitutions of aspartic acid with glutamic acid and vice versa, so that a negative charge can be maintained; substitutions of threonine with serine, so that a free -OH can be maintained; and substitutions of asparagine with glutamine, so that a free -NH 2 .

[0260] The terms "coding sequence" or "protein coding sequence" as used interchangeably herein refer to a polynucleotide segment that encodes a protein. The region or sequence has a start codon near the 5' end and a stop codon near the 3' end. A coding sequence may also be referred to as an open reading frame.

[0261] As used herein, the term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenine to hypoxanthine. In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine or adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA). The adenosine deaminase provided herein (e.g., engineered adenosine deaminase, evolved adenosine deaminase) can be from any organism, such as bacteria. In some embodiments, the adenosine deaminase is from bacteria, such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, or Bacillus crescentus.

[0262] In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is a TadA variant. In some embodiments, the TadA variant is TadA*8. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain does not exist in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, 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 99.7%, at least 99.8%, or at least 99.9% identical to a naturally occurring deaminase. For example, deaminase domains are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is incorporated herein by reference in its entirety.See also Komor, AC, et al., "Programmable base editing of a 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 excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A baseeditors with higher efficiency and product 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. doi: 10.1038 / s41576-018-0059-1, which is hereby incorporated by reference in its entirety.

[0263] "Detection" refers to identifying the presence, absence or amount of an analyte to be detected. In one embodiment, sequence changes in a polynucleotide or polypeptide are detected. In another embodiment, the presence of an indel is detected.

[0264] "Detectable label" refers to a composition that, when attached to a molecule of interest, renders the latter detectable via spectroscopic, photochemical, biochemical, immunochemical or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., commonly used in enzyme-linked immunosorbent assays (ELISA)), biotin, digoxigenin, or haptens.

[0265] "Disease" refers to any condition or disorder that damages or interferes with the normal function of cells, tissues, or organs.

[0266] As used herein, the term "effective amount" refers to the amount of a bioactive agent sufficient to induce a desired biological response. The effective amount of the active agent used to implement the present invention to treat a disease varies depending on the mode of administration, the age, weight and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. This amount is referred to as an "effective" amount. In one embodiment, the effective amount is sufficient to introduce a base editor of the present invention (e.g., a fusion protein including a programmable DNA binding protein, a nuclear base editor and a gRNA) that is changed in a gene of interest in a cell (e.g., in vitro or in vivo). In some embodiments, the fusion protein provided herein, such as an effective amount of a nuclear base editor including an nCas9 domain and a deaminase domain (e.g., adenosine deaminase or cytidine deaminase) may refer to a target site that is sufficient to induce editing to be specifically bound and edited by a nuclear base editor. In one embodiment, the effective amount is the amount of the base editor required to achieve a therapeutic effect (e.g., to alleviate or control a disease or its symptoms or condition). Such therapeutic effects need not be sufficient to alter the gene of interest in all cells of a subject, tissue or organ, but rather only alter approximately 1%, 5%, 10%, 25%, 50%, 75% or more of the gene of interest present in a subject, tissue or organ.

[0267] In some embodiments, the effective amount of a fusion protein provided herein, such as a nucleobase editor comprising an nCas9 domain and a deaminase domain (e.g., an adenosine deaminase or a cytidine deaminase), refers to an amount sufficient to induce a target site specifically bound and edited by a nucleobase editor as described herein. It will be understood by those skilled in the art that an effective amount of an agent, such as a fusion protein, a nuclease, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide, may vary depending on various factors, such as the desired biological response, such as at a specific allele, genome, or target site to be edited, the cell or tissue to be targeted, and / or the agent used.

[0268] "Fragment" refers to a portion of a polypeptide or nucleic acid molecule. The portion includes at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of a reference nucleic acid molecule or polypeptide. A fragment may include 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0269] "Guide RNA" or "gRNA" refers to a polynucleotide that can be specific to a target sequence and can form a complex with a polynucleotide-programmable nucleotide binding domain protein (e.g., Cas9 or Cpf1). In one embodiment, the guide polynucleotide is a guide RNA (gRNA). The gRNA 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 may be referred to as a single guide RNA (sgRNA), but "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or a complex of two or more molecules. Typically, a gRNA that exists as a single RNA species includes two domains: (1) a domain that has homology with the target nucleic acid (e.g., the binding of the guide Cas9 complex to the target); (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence called tracrRNA and includes a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to the tracrRNA provided in Jinek et al., Science 337: 816-821 (2012), which is incorporated herein by reference in its entirety. Other embodiments of gRNAs (e.g., those including domain 2) can be found in U.S. Provisional Patent Application USSN 61 / 874,682 filed on September 6, 2013 (entitled "Switchable Cas9 Nucleases and Uses Thereof") and U.S. Provisional Patent Application USSN 61 / 874,746 filed on September 6, 2013 (entitled "Delivery System For Functional Nucleases"), each of which is hereby incorporated herein by reference in its entirety. In some embodiments, the gRNA includes two or more of domains (1) and (2), and can be referred to as an "extended gRNA". As described herein, the extended gRNA will bind to two or more Cas9 proteins and bind to the target nucleic acid at two or more different regions. The gRNA includes a nucleotide sequence complementary to the target site, which mediates the binding of the nuclease / RNA complex to the target site, providing sequence specificity of the nuclease: RNA complex.

[0270] "Hybridization" refers to hydrogen bonding between complementary nucleobases, which may be Watson-Crick, Hoogstein or reversed Hoogstein hydrogen bonding. For example, adenine and thymine are complementary nucleobases that pair by forming hydrogen bonds.

[0271] The term "base repair inhibitor" or "IBR" refers to a protein that can inhibit the activity of a nucleic acid repair enzyme, such as a base excision repair (BER) enzyme. In some embodiments, the IBR is an inhibitor of inosine base excision repair. Exemplary inhibitors of base repair include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGG1, hNEIL1, T7 Endol, T4PDG, UDG, hSMUG1, and hAAG. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is a catalytically inactive EndoV or a catalytically inactive hAAG. In some embodiments, the base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, the base repair inhibitor is a catalytically inactive EndoV or a catalytically inactive hAAG.

[0272] In some embodiments, the base repair inhibitor is a uracil glycosylase inhibitor (UGI). UGI refers to a protein that can inhibit uracil-DNA glycosylase base-excision repair enzymes. In some embodiments, the UGI domain includes a fragment of wild-type UGI or a fragment of wild-type UGI. In some embodiments, the UGI protein provided herein includes a fragment of UGI and a protein homologous to UGI or a fragment of UGI. In some embodiments, the base repair inhibitor is an inosine base excision repair inhibitor. In some embodiments, the base repair inhibitor is a "catalytically inactivated inosine-specific nuclease" or a "dead inosine-specific nuclease". Without wishing to be bound by any particular theory, catalytically inactivated inosine glycosylases (e.g., alkyladenine glycosylases (AAG)) can bind to inosine, but cannot produce abasic sites or remove inosine, thereby spatially blocking the newly formed inosine portion from DNA damage / repair mechanisms. In some embodiments, catalytically inactivated inosine-specific nucleases can bind to inosine in nucleic acids, but do not cut nucleic acids. Non-limiting exemplary catalytically inactive inosine-specific nucleases include catalytically inactive alkyl adenyl glycosylases (AAG nucleases), such as from humans, and catalytically inactive endonucleases V (EndoV nucleases), such as from E. coli. In some embodiments, the catalytically inactive AAG nuclease includes an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0273] By "increase" is meant a positive change of at least 10%, 25%, 50%, 75% or 100%.

[0274] "Intein" is a protein fragment that can self-excise and connect the remaining fragment (extein) with a peptide bond in a process called protein splicing. Introns are also called "protein introns". The process in which the intein excises itself and connects the remaining part of the protein is referred to as "protein splicing" or "intein-mediated protein splicing" in this article. In some embodiments, the intein of the precursor protein (the protein containing the intein before the intein-mediated protein splicing) comes from two genes. Such inteins are referred to as split inteins (e.g., split intein-N and split intein-C) in this article. For example, in cyanobacteria, DnaE of the catalytic subunit a of DNA polymerase III is encoded by two independent genes dnaE-n and dnaE-c. The intein encoded by the dnaE-n gene may be referred to as "intein-N" in this article. The intein encoded by the dnaE-c gene may be referred to as "intein-C" in this article.

[0275] Other intein systems may also be used. For example, synthetic inteins based on the dnaE intein, Cfa-N (e.g., split intein-N), and Cfa-C (e.g., split intein-C) intein pairs have been described (e.g., in Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5, incorporated herein by reference). Non-limiting examples of intein pairs that may be used in accordance with the present disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, and Cne Prp8 intein (e.g., described in U.S. Pat. No. 8,394,604, incorporated herein by reference).

[0276] Exemplary nucleotide and amino acid sequences of inteins are provided.

[0277] DnaE Intein-N DNA:

[0278]

[0279] DnaE Intein-N Protein:

[0280]

[0281] DnaE intein-C DNA:

[0282]

[0283] Intein-C:

[0284]

[0285] Cfa-N DNA:

[0286]

[0287] Cfa-N protein:

[0288]

[0289] Cfa-C DNA:

[0290]

[0291] Cfa-C protein:

[0292]

[0293] Intein-N and intein-C can be fused to the N-terminal portion of the split Cas9 and the C-terminal portion of the split Cas9, respectively, for connecting the N-terminal portion of the split Cas9 and the C-terminal portion of the split Cas9. For example, in some embodiments, intein-N is fused to the C-terminus of the N-terminal portion of the split Cas9, i.e., forming N--[structure of the N-terminal portion of the split Cas9]-[intein-N]--C. In some embodiments, intein-C is fused to the N-terminus of the C-terminal portion of the split Cas9, i.e., forming a portion of N-[intein-C]--[structure of the C-terminal Cas9]-C. The mechanism of intein-mediated protein splicing for connecting proteins fused to intein (e.g., split Cas9) is known in the art, for example, as described in Shah et al., Chem Sci. 2014; 5(1):446-461, which is incorporated herein by reference. Methods for designing and using inteins are known in the art and are described, for example, in WO2014004336, WO2017132580, US20150344549, and US20180127780, each of which is herein incorporated by reference in its entirety.

[0294] The terms "isolated", "purified" or "biologically pure" refer to materials that are free to varying degrees from components that normally accompany them in their native state. "Isolated" means a degree of separation from the original source or surrounding environment. "Purified" means a degree of separation above the degree of isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials so that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, if the nucleic acid or peptide of the present invention is substantially free of cellular material, viral material or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized, the nucleic acid or peptide is purified. Purity and homogeneity are generally determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that a nucleic acid or protein produces essentially one band in an electrophoretic gel. For proteins that can be modified (e.g., phosphorylated or glycosylated), different modifications may produce different isolated proteins that can be purified separately.

[0295] "Isolated polynucleotide" refers to a nucleic acid (e.g., DNA) that does not contain genes in the naturally occurring genome of the organism from which the nucleic acid molecule of the present invention is derived, and the genes are on the sides of the genes. Therefore, the term includes, for example, recombinant DNA integrated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or as an individual molecule unrelated to other sequences (e.g., cDNA or genomic or cDNA fragments produced by PCR or restriction endonuclease digestion). In addition, the term includes RNA molecules transcribed from DNA molecules, and recombinant DNA as part of a hybrid gene encoding an additional polypeptide sequence.

[0296] "Isolated polypeptide" refers to a polypeptide of the present invention that has been separated from naturally associated components. Generally, a polypeptide is isolated when it is at least 60% (by weight) free of proteins and naturally occurring organic molecules. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the polypeptide of the present invention. An isolated polypeptide of the present invention can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0297] As used herein, the term "linker" may refer to a covalent linker (e.g., a covalent bond), a non-covalent linker, a chemical group, or a molecule that connects two molecules or parts (e.g., two components of a protein complex or a ribonucleic acid complex or two domains of a fusion protein), such as a polynucleotide programmable DNA binding domain (e.g., dCas9) and a deaminase domain (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) or a napDNAbp domain (e.g., Cas12b) and a deaminase domain (e.g., adenosine deaminase or cytidine deaminase). In certain embodiments, the linker is on the side of the deaminase domain inserted into the Cas protein or its fragment. The linker can connect different components or different parts of the components of the base editor system. For example, in some embodiments, the linker can connect the guide polynucleotide binding domain of the polynucleotide programmable nucleotide binding domain and the catalytic domain of the deaminase. In some embodiments, the linker can connect a CRISPR polypeptide and a deaminase. In some embodiments, the linker can connect Cas9 and a deaminase. In some embodiments, the linker can connect dCas9 and a deaminase. In some embodiments, the linker can connect nCas9 and a deaminase. For example, in some embodiments, the linker can connect Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h or Cas12i and a deaminase. In some embodiments, the linker can connect a guide polynucleotide and a deaminase. In some embodiments, the linker can connect the deamination component of the base editor system and the polynucleotide programmable nucleotide binding component. In some embodiments, the linker can connect the deamination component of the base editor system and the RNA binding portion of the napDNAbp component. In some embodiments, the linker can connect the deamination component of the base editor system and the RNA binding portion of the polynucleotide programmable nucleotide binding component. In some embodiments, the linker can connect the RNA binding portion of the deamination component of the base editor system and the RNA binding portion of the polynucleotide programmable nucleotide binding component. In some embodiments, the linker can connect the RNA binding portion of the deamination component of the base editor system and the RNA binding portion of the polynucleotide programmable nucleotide binding component. The linker can be located between or on the sides of two groups, molecules or other parts and connected to each via a covalent bond or non-covalent interaction, thus connecting the two. In some embodiments, the linker can be an organic molecule, group, polymer or chemical part. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA linker. In some embodiments, the linker can be an RNA linker. In some embodiments, the linker can include an aptamer capable of binding a ligand. In some embodiments, the ligand can be a carbohydrate, a peptide, a protein or a nucleic acid. In some embodiments, the linker can include an aptamer that can be derived from a riboswitch.The riboswitch derived from the aptamer can be selected from theophylline riboswitch, thiamine pyrophosphate (TPP) riboswitch, adenosylcobalamin (AdoCbl) riboswitch, S-adenosylmethionine (SAM) riboswitch, SAH riboswitch, flavin mononucleotide (FMN) riboswitch, tetrahydrofolate riboswitch, lysine riboswitch, glycine riboswitch, purine riboswitch, GlmS riboswitch or Q riboswitch 1 (pre-queosine 1, PreQ1) riboswitch. In some embodiments, the linker may include an aptamer that binds to a polypeptide or protein domain such as a polypeptide ligand. In some embodiments, the polypeptide ligand can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif. In some embodiments, the polypeptide ligand can be part of a base editor system component. For example, a nucleobase editing component can include a deaminase domain and an RNA recognition motif.

[0298] In some embodiments, the connexon can be an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the connexon can be about 5 to 100 amino acids in length, such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 100 amino acids in length. In some embodiments, the connexon can be about 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, or 450 to 500 amino acids in length. Longer or shorter connexons can also be considered.

[0299] In some embodiments, the linker connects the gRNA binding domain of the RNA programmable nuclease, including the Cas9 nuclease domain and the catalytic domain of the nucleic acid editing protein (e.g., cytidine or adenosine deaminase). In some embodiments, the linker connects dCas9 and the nucleic acid editing protein. For example, the linker is located between or on the side of two groups, molecules or other parts, and is connected to each via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, a group, a polymer or a chemical part. In some embodiments, the length of the linker is 5 to 200 amino acids, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 70, 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 in length. Longer or shorter linkers are also contemplated.

[0300] In some embodiments, the domain of the nucleobase editor is fused via a linker comprising an amino acid sequence of SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGG. In some embodiments, the domain of the nucleobase editor is fused via a linker comprising 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. In some embodiments, the linker comprises (SGGS) n 、(GGGS) n 、(GGGGS) n , (G) n , (EAAAK) n 、(GGS) n , SGSETPGTSESATPES, or (XP) n motif, or any combination of these, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0301] In some embodiments, the length of the linker is 24 amino acids. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the length of the linker is 40 amino acids. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the length of the linker is 64 amino acids. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS. In some embodiments, the length of the linker is 92 amino acids. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0302] "Marker" refers to any protein or polynucleotide having an alteration in expression level or activity associated with a disease or disorder.

[0303] As used herein, the term "mutation" refers to the replacement of a residue in a sequence (e.g., a nucleic acid or amino acid sequence) by another residue, or the deletion or insertion of one or more residues in a sequence. Mutations are generally described herein by identifying the original residue followed by the position of the residue in the sequence and by the identity of the newly replaced residue. Various methods for performing amino acid substitutions (mutations) provided herein are well known in the art and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)). In some embodiments, the currently disclosed base editor can effectively produce "expected mutations" such as point mutations in nucleic acids (e.g., nucleic acids in the subject's genome) without producing a large number of unexpected mutations, such as unexpected point mutations. In some embodiments, the expected mutation is a mutation generated by the combination of a specific base editor (e.g., a cytidine base editor or an adenosine base editor) with a guide polynucleotide (e.g., gRNA), specifically designed to produce expected mutations.

[0304] Typically, mutations generated or identified in a sequence (e.g., an amino acid sequence as described herein) are numbered relative to a reference (or wild-type) sequence (i.e., a sequence that does not include the mutation). Those skilled in the art will readily understand how to determine the position of a mutation in an amino acid and nucleic acid sequence relative to a reference sequence.

[0305] The term "non-conservative mutation" refers to amino acid substitutions between different groups, for example, substitutions of tryptophan with lysine, or substitutions of phenylalanine with serine, etc. In this case, the non-conservative amino acid substitutions preferably do not interfere with, or inhibit the biological activity of, the functional variant. The non-conservative amino acid substitutions can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the wild-type protein.

[0306] The term "nuclear localization sequence", "nuclear localization signal" or "NLS" refers to an amino acid sequence that promotes protein import into the nucleus. Nuclear localization sequences are known in the art and are described, for example, in International PCT Application PCT / EP2000 / 011690 by Plank et al., filed on November 23, 2000, which was published as WO / 2001 / 038547 on May 31, 2001, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS, such as described by Koblan et al., Nature Biotech.2018doi:10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC. \

[0307] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to compounds including nucleobases and acidic moieties, such as polymers of nucleosides, nucleotides or nucleotides. Typically, polymeric nucleic acids, such as nucleic acid molecules including three or more nucleotides, are linear molecules in which adjacent nucleotides are interconnected by phosphodiester bonds. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain including three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" are used interchangeably to refer to polymers of nucleotides (e.g., strings of at least three nucleotides). In some embodiments, "nucleic acid" includes RNA and single-stranded and / or double-stranded DNA. Nucleic acids can be naturally occurring, such as in genomes, transcripts, mRNA, tRNA, rRNA, siRNA, snRNA, plasmids, cosmids, chromosomes, chromatids or other naturally occurring nucleic acid molecules. On the other hand, nucleic acid molecules can be non-naturally occurring molecules, such as recombinant DNA or RNA, artificial chromosomes, engineered genomes or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids or nucleotides or nucleosides that comprise non-naturally occurring. In addition, term " nucleic acid ", " DNA ", " RNA " and / or similar terms include nucleic acid analogs, such as analogs with other skeletons except the phosphodiester skeleton. Nucleic acid can be purified from natural sources, produced using a recombinant expression system and optionally purified, chemically synthesized, etc. In suitable cases, such as in the case of chemically synthesized molecules, nucleic acid can include nucleoside analogs, such as analogs or sugar and skeleton modifications with chemically modified bases. Unless otherwise indicated, nucleotide sequence is presented in 5' to 3' directions. In some embodiments, the nucleic acid is or includes natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, 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-oxoguanosine, O(6)-methylguanosine and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (2'-e.g., fluororibose, ribose, 2'-deoxyribose, arabinose and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite bonds).

[0308] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" can be used interchangeably with "polynucleotide programmable nucleotide binding domain" to refer to a protein linked to a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA) that guides the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 protein. The Cas9 protein can be linked to a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, such as a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactivated Cas9 (dCas9). Non-limiting examples of nucleic acid-programmable DNA-binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i.Non-limiting examples of Cas enzymes 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 / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc 2. Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, class II Cas effector protein, type V Cas effector protein, class VI Cas effector protein, CARF, DinG, homologs thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, although they may not be specifically listed in the present disclosure. See, for example, Makarova et al. "Classification 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 type VCRISPR-Cas systems" Science. 2019 Jan 4; 363 (6422): 88-91. doi: 10.1126 / science.aav7271, each of which is hereby incorporated by reference.

[0309] The terms "nucleobase", "nitrogenous base" or "base" are used interchangeably herein and refer to nitrogenous biological compounds that form nucleosides, which are components of nucleotides. The ability of nucleobases to form base pairs and stack on each other directly leads to long-chain helical structures, such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Five nucleobases—adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U)—are called primary or canonical. Adenine and guanine are derived from purines, while cytosine, uracil and thymine are derived from pyrimidines. DNA and RNA may also include other (non-primary) modified bases. Non-limiting exemplary modified nucleobases may include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C) and 5-hydromethylcytosine. Hypoxanthine and xanthine can be produced by the presence of mutagens, both of which are produced by deamination (replacing an amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can be produced by deamination of cytosine. "Nucleoside" is composed of a nucleobase and a pentose (ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. Examples of nucleosides with modified nucleobases include inosine (I), xanthine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C) and pseudouridine (Ψ). "Nucleotide" is composed of a nucleobase, a pentose (ribose or deoxyribose) and at least one phosphate group.

[0310] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" refers to a protein linked to a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid that guides napDNAbp to a specific nucleic acid sequence. For example, the Cas12 protein can be connected to a guide RNA of a specific DNA sequence complementary to the guide RNA with a guide Cas12 protein. In some embodiments, the napDNAbp is a Cas12 domain, such as a nuclease active Cas12 domain. Examples of napDNAbps include Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. Other napDNAbps are also within the scope of the present disclosure, although they may not be specifically listed in the present disclosure. See, e.g., Makarova et al. "Classification 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 type V CRISPR-Cas systems" Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, each of which is hereby incorporated by reference in its entirety.

[0311] As used herein, the term "nucleobase editing domain" or "nucleobase editing protein" refers to a protein or enzyme that can catalyze the modification of nucleobases in RNA or DNA, such as deamination of cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine) and adenine (or adenosine) to hypoxanthine (or inosine), as well as non-templated nucleotide addition and insertion. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., an adenine deaminase or adenosine deaminase; or a cytidine deaminase or a cytosine deaminase). In some embodiments, the nucleobase editing domain is more than one deaminase domain (e.g., an adenine deaminase or adenosine deaminase and a cytidine or cytosine deaminase). In some embodiments, the nucleobase editing domain may be a naturally occurring nucleobase editing domain. In some embodiments, the nucleobase editing domain may be an engineered or evolved nucleobase editing domain from a naturally occurring nucleobase editing domain. The nucleobase editing domain can be from any organism, such as bacteria, humans, chimpanzees, gorillas, monkeys, cows, dogs, rats, or mice. For example, nucleobase editing proteins are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is incorporated herein by reference in its entirety. See also Komor, AC, et al., “Programmable editing of a target base in genomic DNAwithout 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); and Komor, 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 product purity” Science Advances 3:eaao4774 (2017), the entire contents of which are hereby incorporated by reference.

[0312] As used herein, "obtaining" in "obtaining an agent" includes synthesizing, purchasing or otherwise obtaining the agent.

[0313] As used herein, "patient" or "subject" refers to a mammalian subject or individual diagnosed with, at risk of or developing, or suspected of having or developing a disease or condition. In some embodiments, the term "patient" refers to a mammalian subject with a higher than average likelihood of developing a disease or condition. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cattle, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs), and other therapies disclosed herein. Exemplary human patients can be male and / or female.

[0314] A "patient in need thereof" or "subject in need thereof" refers herein to a patient diagnosed with, at risk of or having, scheduled to have, or suspected of having a disease or condition.

[0315] The terms "pathogenic mutation," "disease-causing variant," "disease envelope mutation," "pathogenic variant," "disease-causing mutation," or "susceptibility mutation" refer to a mutation that increases an individual's susceptibility or predisposition to a disease or condition. In some embodiments, the pathogenic mutation comprises a substitution of at least one wild-type amino acid with at least one pathogenic amino acid in the protein encoded by the gene.

[0316] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or excipient, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate or stearic acid) or solvent encapsulating material, which is involved in carrying or transporting a compound from one part of the body (e.g., a delivery site) to another part (e.g., an organ, tissue or part of the body). A pharmaceutically acceptable carrier is "acceptable" (e.g., physiologically compatible, sterile, physiological pH, etc.) in the sense that it is compatible with the other ingredients of the formulation and is not harmful to the tissues of the subject. Terms such as "excipient", "carrier", "pharmaceutically acceptable carrier", "carrier" and the like are used interchangeably herein.

[0317] The term "pharmaceutical composition" may refer to a composition formulated for pharmaceutical use.

[0318] The terms "protein", "peptide", "polypeptide" and their grammatical equivalents are used interchangeably herein and refer to polymers of amino acid residues linked together by peptide (amide) bonds. These terms refer to proteins, peptides or polypeptides of any size, structure or function. Typically, a protein, peptide or polypeptide is at least three amino acids long. A protein, peptide or polypeptide may refer to an individual protein or a collection of proteins. One or more amino acids in a protein, peptide or polypeptide may be modified, for example, by adding chemical entities such as carbohydrate groups, hydroxyls, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization or other modifications, etc. A protein, peptide or polypeptide may also be a single molecule or may be a multimolecular complex. A protein, peptide or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide or polypeptide may be naturally occurring, recombinant or synthetic, or any combination thereof. As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. A protein can be located at the amino terminal (N-terminal) portion or the carboxyl terminal (C-terminal) protein of the fusion protein, thereby forming an amino-terminal fusion protein or a carboxyl-terminal fusion protein, respectively. The protein can include different domains, for example, a nucleic acid binding domain (for example, a gRNA binding domain of Cas9 that guides the protein to bind to the target site) and a nucleic acid cleavage domain, or a catalytic domain of a nucleic acid editing protein. In some embodiments, the protein includes a protein portion (for example, an amino acid sequence constituting a nucleic acid binding domain) and an organic compound (for example, a compound that can act as a nucleic acid cleavage agent). In some embodiments, the protein forms a complex or connection with a nucleic acid such as RNA or DNA. Any protein provided herein can be produced by any method known in the art. For example, the protein provided herein can be produced via recombinant protein expression and purification, which is particularly suitable for fusion proteins including peptide linkers. Methods for recombinant protein expression and purification are well known, including Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), the entire contents of which are incorporated herein by reference.

[0319] The polypeptides and proteins disclosed herein (including functional parts and functional variants thereof) may include 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, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, dihydroindole ... -2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine. Polypeptides and proteins may be associated with post-translational modifications of one or more amino acids of the polypeptide construct. Non-limiting examples of post-translational modifications include phosphorylation, acylation (including acetylation and formylation), glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation (including methylation and ethylation), ubiquitination, addition of pyrrolidone carboxylic acid, disulfide bond formation, sulfation, myristoylation, palmitoylation, prenylation, farnesylation, geranylation, glycosylphosphatidylinositolization, fatty acylation, and iodination.

[0320] The term "polynucleotide programmable nucleotide binding domain" or "nucleic acid programmable DNA binding protein (napDNAbp)" refers to a protein linked to a nucleic acid (e.g., DNA or RNA), such as a guide polynucleotide (e.g., guide RNA) that guides the polynucleotide programmable nucleotide binding domain to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas12 protein.

[0321] The term "recombinant" as used herein in the context of a protein or nucleic acid refers to a protein or nucleic acid that does not occur in nature but is a product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to any naturally occurring sequence.

[0322] "Decrease" refers to a negative change of at least 10%, 25%, 50%, 75% or 100%.

[0323] "Reference" refers to a standard or control condition. In one embodiment, the reference is a wild-type or healthy cell. In other embodiments and without limitation, the reference is an untreated cell that has not been subjected to the test condition or has been subjected to a placebo or saline, culture medium, buffer, and / or a control vector that does not include the polynucleotide of interest.

[0324] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or all of a particular sequence; for example, a fragment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will typically be 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 the reference nucleic acid sequence is typically at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides, or about 300 nucleotides, or any integer near or between them. In some embodiments, the reference sequence is the wild-type sequence of the protein of interest. In other embodiments, the reference sequence is a polynucleotide sequence encoding a wild-type protein.

[0325] The terms "RNA programmable nuclease" and "RNA guided nuclease" are used with (e.g., bound to or linked to) one or more RNAs that are not targets for cleavage. In some embodiments, when a complex is formed with an RNA, the RNA programmable nuclease may be referred to as a nuclease: RNA complex. Typically, the bound RNA is referred to as a guide RNA (gRNA). A gRNA 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 may be referred to as a single guide RNA (sgRNA), although "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or a complex of two or more molecules. Typically, a gRNA that exists as a single RNA species includes two domains: (1) a domain that has homology to a target nucleic acid (e.g., guides binding of the Cas9 complex to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and includes a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337: 816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNAs (e.g., those comprising domain 2) can be found in U.S. provisional patent application USSN 61 / 874,682 filed on September 6, 2013 (entitled "Switchable Cas9 Nucleases and Uses Thereof") and U.S. provisional patent application USSN 61 / 874,746 filed on September 6, 2013 (entitled "Delivery System For Functional Nucleases"), the entire contents of each of which are incorporated herein by reference. In some embodiments, the gRNA includes two or more of domains (1) and (2), and can be referred to as an "extended gRNA". For example, as described herein, the extended gRNA will, for example, bind to two or more Cas9 proteins and bind to the target nucleic acid at two or more different regions. The gRNA includes a nucleotide sequence complementary to the target site, which mediates the binding of the nuclease / RNA complex to the target site, providing the sequence specificity of the nuclease: RNA complex.

[0326] In some embodiments, the RNA programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, such as Cas9 from Streptococcus pyogenes (Casn1) (see, e.g., "Complete genome sequence of an Mlstrain of Streptococcus pyogenes." Ferretti JJ, et al., Proc. 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).

[0327] Because RNA-programmable nucleases such as Cas9 use RNA:DNA hybrids to target DNA cleavage sites, these proteins can in principle target any sequence specified by the guide RNA. Methods for site-specific cleavage (e.g., modifying genomes) using RNA-programmable nucleases such as Cas9 are known in the art (see, e.g., Cong, L. et al., Multiplex genome 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-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, JE et al., Genome 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. Nature biotechnology 31, 233-239 (2013); the entire contents of which are incorporated herein by reference).

[0328] The term "single nucleotide polymorphism (SNP)" is a variation of a single nucleotide occurring at a specific position in a genome, wherein each variation exists to a certain extent (e.g., >1%) in a population. For example, at a specific base position in the human genome, a C nucleotide may occur in most individuals, but in a few individuals, the position is occupied by an A. This means that there is a SNP at the specific position, and two possible nucleotide variations, C or A, are referred to as alleles at the position. SNP is the basis for differences in disease susceptibility. The severity of the disease and the way our bodies respond to treatment are also manifestations of genetic variation. SNP can fall into the coding region of a gene, the non-coding region of a gene, or the intergenic region (region between genes). In some embodiments, due to the degeneracy of the genetic code, the SNP in the coding sequence does not necessarily change the amino acid sequence of the protein produced. There are two types of SNPs in the coding region: synonymous SNPs and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, while non-synonymous SNPs change the amino acid sequence of the protein. There are two types of non-synonymous SNPs: missense and nonsense. SNPs that are not in protein coding regions can still affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNA. Gene expression affected by such SNPs is called eSNP (expression SNP) and can be located upstream or downstream of the gene. Single nucleotide variants (SNVs) are variations of a single nucleotide without any frequency restrictions and can occur in somatic cells. Somatic single nucleotide variants can also be called single nucleotide changes.

[0329] "Specific binding" refers to a nucleic acid molecule, a polypeptide or a complex thereof (e.g., a nucleic acid-programmable DNA binding domain and a guide nucleic acid), a compound or a molecule of the present invention that recognizes and binds to a polypeptide and / or nucleic acid molecule of the present invention, but does not substantially recognize and bind to other molecules in a sample, such as a biological sample.

[0330] Nucleic acid molecules that can be used in the methods of the present invention include any nucleic acid molecules encoding polypeptides of the present invention or fragments thereof. Such nucleic acid molecules do not need to be 100% identical to endogenous nucleic acid sequences, but will generally exhibit basic identity. Polynucleotides having "substantial identity" to endogenous sequences are generally capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules that can be used in the methods of the present invention include any nucleic acid molecules encoding polypeptides of the present invention or fragments thereof. Such nucleic acid molecules do not need to be 100% identical to endogenous nucleic acid sequences, but will generally exhibit basic identity. Polynucleotides having "substantial identity" to endogenous sequences are generally capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. "Hybridization" refers to pairing between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof to form double-stranded molecules under various stringent conditions. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152: 399; Kimmel, AR (1987) Methods Enzymol. 152: 507).

[0331] For example, stringent salt concentrations are typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of an organic solvent such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions typically include a temperature of at least about 30° C., more preferably at least about 37° C., most preferably at least about 42° C. It is well known to those skilled in the art to vary additional parameters, such as hybridization time, detergent concentration, for example, sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA. Different degrees of stringency are achieved by combining these different conditions as needed. In one embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization will occur in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C. In another embodiment, hybridization will occur in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations of these conditions will be apparent to those skilled in the art.

[0332] For most applications, the washing step after hybridization will also vary in stringency. Washing stringency conditions can be defined by salt concentration and temperature. As above, washing stringency can be increased by reducing salt concentration or increasing temperature. For example, the stringent salt concentration of the washing step is preferably less than about 30mM NaCl and 3mM trisodium citrate, and most preferably less than about 15mM NaCl and 1.5mM trisodium citrate. The stringent temperature conditions of the washing step generally include a temperature of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In one embodiment, the washing step will occur at 25°C in 30mM NaCl, 3mM trisodium citrate and 0.1% SDS. In a more preferred embodiment, the washing step will be carried out at 42°C in 15mM NaCl, 1.5mM trisodium citrate and 0.1% SDS. In a more preferred embodiment, the washing step will be carried out at 68°C in 15mM NaCl, 1.5mM trisodium citrate and 0.1% SDS. Other variations of these conditions will be 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 and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0333] "Split" means to separate into two or more fragments.

[0334] "Split Cas9 protein" or "split Cas9" refers to a Cas9 protein provided as an N-terminal fragment and a C-terminal fragment encoded by two separate nucleotide sequences. The polypeptides corresponding to the N-terminal portion and the C-terminal portion of the Cas9 protein can be spliced ​​to form a "reconstructed" Cas9 protein. In a specific embodiment, the Cas9 protein is divided into two fragments within the disordered region of the protein, for example, as described in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014, or as described in Jiang et al. (2016) Science 351: 867-871. PDB file: 5F9R, each of which is incorporated herein by reference. In some embodiments, the protein is divided into two fragments at any C, T, A or S between about amino acids A292 to G364, F445 to K483, or E565 to T637 within the SpCas9 region, or at any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other napDNAbp. In some embodiments, the protein is divided into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, the process of dividing a protein into two fragments is referred to as "splitting" the protein.

[0335] In other embodiments, the N-terminal portion of the Cas9 protein includes amino acids 1 to 573 or 1 to 637 of Streptococcus pyogenes Cas9 wild type (SpCas9) (NCBI reference sequence: NC_002737.2, Uniprot reference sequence: Q99ZW2) and the C-terminal portion of the Cas9 protein includes a portion of amino acids 574 to 1368 or 638 to 1368 of the SpCas9 wild type.

[0336] The C-terminal portion of the split Cas9 can be connected to the N-terminal portion of the split Cas9 to form a complete Cas9 protein. In some embodiments, the C-terminal portion of the Cas9 protein starts where the N-terminal portion of the Cas9 protein ends. Therefore, in some embodiments, the C-terminal portion of the split Cas9 includes a portion of amino acids (551-651)-1368 of spCas9. "(551-651)-1368" refers to amino acids starting between amino acids 551 to 651 (inclusive) and ending at amino acid 1368.For example, the C-terminal portion of the split Cas9 may include any one of amino acids 551 to 1368, 552 to 1368, 553 to 1368, 554 to 1368, 555 to 1368, 556 to 1368, 557 to 1368, 558 to 1368, 559 to 1368, 560 to 1368, 561 to 1368, 562 to 1368, 563 to 1368, 564 to 1368, 565 to 1368, 566 to 1368, 567 to 1368, 568 to 1368, 569 to 1368, 570 to 1368, 571 to 1368, 572 to 1368, 573 to 1 368, 574 to 1368, 575 to 1368, 576 to 1368, 577 to 1368, 578 to 1368, 579 to 1368, 580 to 1368, 581 to 1368, 582 to 1368, 583 to 1368, 584 to 1368, 585 to 1368, 586 to 1368, 587 to 1368, 588 to 1368, 589 to 1368, 590 to 1368, 591 to 1368, 592 to 1368, 593 to 1368, 594 to 1368, 595 to 1368, 596 to 1368, 597 to 1368, 598 to 1368, 599 to 1368 , 600 to 1368, 601 to 1368, 602 to 1368, 603 to 1368, 604 to 1368, 605 to 1368, 606 to 1368, 607 to 1368, 608 to 1368, 609 to 1368, 610 to 1368, 611 to 1368, 612 to 1368, 613 to 1368, 614 to 1368, 615 to 1368, 616 to 1368, 617 to 1368, 618 to 1368, 619 to 1368, 620 to 1368, 621 to 1368, 622 to 1368, 623 to 1368, 624 to 1368, 625 to 1368, 6 to 1368, 627 to 1368, 628 to 1368, 629 to 1368, 630 to 1368, 631 to 1368, 632 to 1368, 633 to 1368, 634 to 1368, 635 to 1368, 636 to 1368, 637 to 1368, 638 to 1368, 639 to 1368, 640 to 1368, 641 to 1368, 642 to 1368, 643 to 1368, 644 to 1368, 645 to 1368, 646 to 1368, 647 to 1368, 648 to 1368, 649 to 1368, 650 to 1368, or a portion of 651 to 1368. In some embodiments, the C-terminal portion of the split Cas9 protein includes amino acids 574 to 1368 or 638 to 1368 of SpCas9.

[0337] "Subject" refers to a mammal, including but not limited to a human or non-human mammal, such as a cow, horse, dog, sheep or cat. Subjects include livestock, domesticated animals raised to produce labor and provide commodities such as food, including but not limited to cattle, goats, chickens, horses, pigs, rabbits and sheep.

[0338] "Substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any of the amino acid sequences set forth herein) or a nucleic acid sequence (e.g., any of the amino acid sequences set forth herein). In one embodiment, such a sequence is at least 60%, 80% or 85%, 90%, 95% or even 99% identical to the sequence used for comparison at the amino acid level or nucleic acid.

[0339] Sequence identity is typically determined using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, sequence similarity search, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method of determining the degree of identity, a sequence similarity search program may be used, wherein e -3 and e -100 Probability scores between and indicate closely related sequences.

[0340] For example, COBALT is used with the following parameters:

[0341] a) Alignment parameters: gap penalty -11, -1 and end gap penalty -5, -1,

[0342] b) CDD parameters: use RPS sequence similarity search (BLAST); sequence similarity search E-value 0.003; find conserved columns and recalculate, and

[0343] c) Query clustering parameters: Use query clustering; font size 4; maximum cluster distance 0.8; regular letters.

[0344] For example, when using EMBOSS Needle, the following parameters are used:

[0345] a) Matrix: BLOSUM62;

[0346] b) Gap opening: 10;

[0347] c) Notch extension: 0.5;

[0348] d) Output format: Yes;

[0349] e) End gap penalty: False;

[0350] f) terminal nick opening: 10; and

[0351] g) Terminal notch extension: 0.5.

[0352] 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 deaminated by a deaminase or a fusion protein comprising a deaminase (e.g., cytidine or adenine deaminase).

[0353] As used herein, the terms "treat", "treating", "treatment" and the like refer to reducing or ameliorating a condition and / or symptoms associated therewith or obtaining a desired pharmacological and / or physiological effect. It will be understood that although not excluded, treating a condition or disorder does not require the complete elimination of the condition, the condition or symptoms associated therewith. In some embodiments, the effect is therapeutic, i.e., not limited to the effect partially or completely reducing, attenuating, eliminating, slowing down, alleviating, reducing the intensity of the disease and / or adverse symptoms attributable to the disease, or curing the disease and / or adverse symptoms. In some embodiments, the effect is preventive, i.e., the effect protects or prevents the occurrence or recurrence of a disease or condition. To this end, the presently disclosed method comprises administering a therapeutically effective amount of a composition as described herein.

[0354] "Uracil glycosylase inhibitor" or "UGI" refers to an agent that inhibits the uracil-excision repair system. In one embodiment, the agent is a protein or fragment thereof that binds to the host uracil-DNA glycosylase and prevents the removal of uracil residues from DNA. In one embodiment, UGI is a protein, fragment or domain capable of inhibiting uracil-DNA glycosylase base excision repair enzyme. In some embodiments, the UGI domain includes a wild-type UGI or a modified form thereof. In some embodiments, the UGI domain includes a fragment of the exemplary amino acid sequence set forth below. In some embodiments, the UGI fragment includes an amino acid sequence that includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of at least the exemplary UGI sequence provided below. In some embodiments, UGI includes the following amino acid sequence homologous to the exemplary UGI amino acid sequence or a fragment thereof. In some embodiments, the UGI or a portion thereof is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a wild-type UGI or a UGI sequence or a portion thereof.

[0355] Exemplary UGIs include the following amino acid sequences:

[0356] >splP14739IUNGI_BPPB2 uracil-DNA glycosylase inhibitorMTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDES TDENVMLLTSD APEYKPWALVIQDSNGENKIKML.

[0357] The term "vector" refers to a means for introducing a nucleic acid sequence into a cell to produce a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, and episomes. An "expression vector" is a nucleic acid sequence that includes a nucleotide sequence to be expressed in a recipient cell. An expression vector may include additional nucleic acid sequences to facilitate and / or promote expression of the introduced sequence, such as initiation, termination, enhancer, promoter, and secretion sequences.

[0358] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0359] DNA editing has emerged as a viable means to alter disease states by correcting pathogenic mutations at the genetic level. Until recently, all DNA editing platforms functioned by inducing DNA double-strand breaks (DSBs) at specific genomic loci and relying on endogenous DNA repair pathways to determine product outcomes in a semi-stochastic manner, generating complex populations of genetic products. While precise, user-defined repair outcomes can be achieved via homology directed repair (HDR) pathways, a number of challenges have hampered the use of HDR for efficient repair in therapeutically relevant cell types. In practice, the pathway is inefficient relative to competing, error-prone non-homologous end joining pathways. Furthermore, HDR is strictly restricted to the G1 and S phases of the cell cycle, preventing precise repair of DSBs in post-mitotic cells. As a result, it has proven difficult or impossible to efficiently alter genomic sequences in these populations in a user-defined, programmable manner.

[0360] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0361] DNA editing has emerged as a viable means to alter disease states by correcting pathogenic mutations at the genetic level. Until recently, all DNA editing platforms functioned by inducing DNA double-strand breaks (DSBs) at specific genomic loci and relying on endogenous DNA repair pathways to determine product outcomes in a semi-stochastic manner, generating complex populations of genetic products. While precise, user-defined repair outcomes can be achieved via homology-directed repair (HDR) pathways, a number of challenges have hampered the use of HDR for efficient repair in therapeutically relevant cell types. In practice, the pathway is inefficient relative to competing, error-prone non-homologous end joining pathways. Furthermore, HDR is strictly restricted to the G1 and S phases of the cell cycle, preventing precise repair of DSBs in post-mitotic cells. As a result, it has proven difficult or impossible to efficiently alter genomic sequences in these populations in a user-defined, programmable manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0362] Figures 1A-1C Plasmids are depicted. Figure 1A It is an expression vector encoding the TadA7.10-dCas9 base editor. Figure 1B The plasmid comprises a nucleic acid molecule encoding a protein conferring chloramphenicol resistance (CamR) and spectinomycin resistance (SpectR). The plasmid also comprises a kanamycin resistance gene which is inactivated by two point mutations. Figure 1C The plasmid comprises a nucleic acid molecule encoding a protein conferring chloramphenicol resistance (CamR) and spectinomycin resistance (SpectR). The plasmid also comprises a kanamycin resistance gene which is inactivated by three point mutations.

[0363] Figure 2 is an image of a bacterial colony transduced with the expression vector depicted in Figure 1, which includes a defective kanamycin resistance gene. The vector includes ABE7.10 variants generated using error-prone PCR. Using increasing concentrations of kanamycin, bacterial cells expressing these "evolved" ABE7.10 variants are selected to obtain kanamycin resistance. Bacteria expressing ABE7.10 variants with adenosine deaminase activity are able to correct the mutation introduced into the kanamycin resistance gene, thereby restoring kanamycin resistance. Kanamycin-resistant cells are selected for further analysis.

[0364] Figure 3A and 3B Illustration of editing of the regulatory region of the hemoglobin subunit gamma (HGB1) locus, a therapeutically relevant site for upregulation of fetal hemoglobin. Figure 3A A diagram of a portion of the regulatory region of the HGB1 gene. Figure 3B The efficiency and specificity of adenosine deaminase variants were quantified. Editing was detected at the hemoglobin subunit gamma 1 (HGB1) site in HEK293T cells, a therapeutically relevant site for upregulation of fetal hemoglobin. The figure above depicts the nucleotide residues in the target region of the HGB1 gene regulatory sequence. A5, A8, A9, and A11 represent edited adenosine residues in HGB1.

[0365] Figure 4 Illustrated are the relative effectiveness of adenosine base editors including dCas9 that recognize non-canonical PAM sequences. The top panel depicts the coding sequence for a hemoglobin subunit. The bottom panel shows the efficiency of adenosine deaminase variant base editors with guide RNAs of varying lengths.

[0366] Figure 5 Figure 2 is a graph illustrating the efficiency and specificity of the ABE8 base editor. The percentage of editing of the intended target nucleotide and the unintended target nucleotide (bystander) is quantified.

[0367] Figure 6 Figure 2 is a graph illustrating the efficiency and specificity of the ABE8 base editor. The percentage of editing of the intended target nucleotide and the unintended target nucleotide (bystander) is quantified.

[0368] Figures 7A-7D Depicted is an eighth-generation adenine base editor mediating remarkable A·T to G·C conversion in human cells. Fig. 7AAn overview of adenine base editing is illustrated: i) ABE8 creates an R-loop at the sgRNA targeting site in the genome; ii) TadA* deaminase chemically converts adenine to inosine via hydrolytic deamination of the ss-DNA portion of the R-loop; iii) the D10A nickase of Cas9 cleaves the strand opposite to the strand containing inosine; iv) the strand containing inosine can be used as a template during DNA replication; v) inosine preferentially base pairs with cytosine in the context of DNA polymerases; and vi) after replication, inosine may be replaced by guanosine. Figure 7B The diagram shows the architecture of ABE8.xm and ABE8.xd. Figure 7C Shown are three views of the E. coli TadA deaminase (PDB 1Z3A) aligned with S. aureus TadA (not shown) (PDB 2B3J) in complex with tRNAArg2. Mutations identified in the eighth round of evolution are highlighted. Fig.7D is a graph depicting the A·T to G·C base editing efficiency of the core ABE8 construct relative to the ABE7.10 construct in Hek293T cells across eight genomic loci. Values ​​and error bars reflect the mean and standard deviation of three independent biological replicates performed on different days.

[0369] Figures 8A to 8C Figure 5. Cas9 PAM-mutant ABE8 and catalytically dead Cas9 ABE8 variants mediate higher A·T to G·C conversions than the corresponding ABE7.10 variants in human cells. Values ​​and error bars reflect the mean and standard deviation of three independent biological replicates performed on different days. Fig. 8A is a graph depicting the conversion of A·T to G·C in Hek293T cells with NG-Cas9 ABE8 (-NG PAM). Figure 8B is a graph depicting the conversion of A·T to G·C in Hek293T cells with Sa-Cas9 ABE8 (-NNGRRT PAM). Figure 8C Graph depicting A·T to G·C conversion in Hek293T cells with catalytically inactivated dCas9-ABE8 (D10A, H840A in S. pyogenes Cas9).

[0370] Figures 9A to 9E Depicted is a comparison of on-target and off-target editing frequencies between ABE7.10, ABEmax, and ABEmax with a BPNLS in Hek293T cells. Individual data points for n=3 independent biological replicates performed on different days are shown, and error bars represent standard deviations. Fig. 9A and 9B is a graph depicting the frequency of on-target DNA editing. Fig. 9B and 9Cis a graph depicting the frequency of off-target DNA editing guided by sgRNA. Fig.9E is a graph depicting the frequency of RNA off-target editing.

[0371] Figures 10A to 10B Depicted are the median A·T to G·C transitions and corresponding INDEL formation for TadA, C-terminal α-helical truncation ABE constructs in HEK293T cells. Fig. 10A is a heat map depicting the median A·T to G·C editing transitions across eight genomic sites. Fig. 10B is a heat map depicting INDEL formation. The Δ residue values ​​correspond to the deletion positions in TadA. The median values ​​were generated from n=3 biological replicates.

[0372] Fig.11 is a heat map depicting the median A·T to G·C transitions of 40 ABE8 constructs in HEK293T cells across 8 genomic loci. The median was determined from two or more biological replicates.

[0373] Fig.12 is a heat map depicting the median INDEL% of 40 ABE8 constructs in HEK293T cells across 8 genomic loci. The median was determined from two or more biological replicates.

[0374] Fig.13 Figure 2 is a graph depicting the fold change in editing of ABE8:ABE7. Representation of the average ABE8:ABE7 A·T to G·C editing of all A positions within the target of eight different genomic loci in HEK293T cells. Positions 2 to 12 represent the location of the target adenine within the 20-nucleotide pre-spacer, with position 20 being directly 5' of the -NGG PAM.

[0375] Fig.14 Dendrogram depicting ABE8. The core ABE8 construct selected for further study is highlighted in black.

[0376] Fig.15 is a heat map depicting the median A·T to G·C transitions for the core 8 ABE8 constructs in HEK293T cells across 8 genomic loci. The median was determined from three or more biological replicates.

[0377] Fig.16 is a heatmap depicting the median INDEL frequencies of the core 8 ABE8s tested at 8 genomic loci in HEK293T cells.

[0378] Fig.17 is a heat map depicting the median A·T to G·C conversion of core NG-ABE8 construct 9 (-NGPAM) at six genomic sites in HEK293T cells. Median values ​​were generated from n=3 biological replicates.

[0379] Fig.18 is a heatmap depicting the median INDEL frequencies of core NG-ABE8 at six genomic loci tested in HEK293T cells. Median values ​​generated from n=3 biological replicates.

[0380] Fig.19 is a heat map depicting the median A·T to G·C transitions of the core Sa-ABE8 construct (-NNGRRT PAM) at six genomic sites in HEK293T cells. Site positions are numbered -2 ​​to 20 (5' to 3') in the 22-nucleotide pre-interval. Position 20 is 5' to the NNGRRT PAM. Medians were generated from n=3 biological replicates.

[0381] Fig. 20 is a heatmap depicting the median INDEL frequency of core Sa-ABE8 at 8 genomic loci tested in HEK293T cells. Median values ​​were generated from n=3 biological replicates.

[0382] Fig.21 is a heat map depicting median A·T to G·C transitions of the core dC9-ABE8-m construct at eight genomic sites in HEK293T cells. Dead Cas9 (dC9) was defined as the D10A and H840A mutations in S. pyogenes Cas9. Median values ​​were generated from n≥3 biological replicates.

[0383] Fig. 22 is a heat map depicting the median A·T to G·C transitions of the core dC9-ABE8-d construct at eight genomic sites in HEK293T cells. Dead Cas9 (dC9) was defined as the D10A and H840A mutations in S. pyogenes Cas9. Median values ​​were generated from n≥3 biological replicates.

[0384] Fig.23A and 23B Depicted are the median INDEL frequencies of core dC9-ABE8 at eight genomic loci tested in HEK293T cells. Median values ​​were generated from n ≥ 3 biological replicates. Fig.23A is a heat map depicting the indel frequency of the dC9-ABE8-m variant relative to ABE7.10. Fig. 23B is a heat map depicting the indel frequency of the dC9-ABE8-d variant relative to ABE7.10.

[0385] Fig.24 Figure 2 is a graph depicting C·G to T·A editing in Hek293T cells treated with ABE8 and ABE7.10. The editing frequency for each site is averaged across all C positions within the target. Cytosines in the protospacer are shaded.

[0386] Figures 25A to 25H Depicts on-target DNA and sgRNA-dependent off-target DNA editing by ABE8 constructs and ABE8 constructs with TadA mutations that improve specificity for DNA. Single data points are shown for n=3 independent runs on different days, with error bars representing standard deviations for biological replicates. Fig.25A and 25B is a graph depicting the on-target DNA editing frequency of the core ABE8 construct compared to ABE7. Fig.25C and 25D is a graph depicting the on-target DNA editing frequency of ABE8 with mutations that improve RNA off-target editing. Fig.25E and 25F is a graph depicting the frequency of off-target DNA editing guided by sgRNAs of the core ABE 8 construct compared to ABE7.

[0387] Figure 25G and 25H is a graph depicting the frequency of DNA off-target editing guided by sgRNAs of ABE 8 constructs with mutations that improve RNA off-target editing.

[0388] Fig.26 is a graph depicting indel frequencies at 12 previously identified sgRNA-dependent Cas9 off-target loci in human cells. Individual data points for n=3 independent biological replicates performed on different days are shown and error bars represent standard deviations. ,.

[0389] Fig.27A and 27B Depicting the A·T to G·C conversion and phenotypic consequences in primary cells. Fig.27A Figure 2 is a graph depicting the conversion of A·T to G·C at the -198HBG1 / 2 locus in CD34+ cells treated with ABE from two different donors. NGS analysis was performed 48 and 144 hours after treatment. -198HBG1 / 2 target sequence with A7 highlighted. The percentage of A·T to G·C of A7 is plotted. Fig.27B is a graph depicting the percentage of γ-globulin formed as a fraction of α-globulin. Shown are values ​​from two different donors, after ABE treatment and erythroid differentiation.

[0390] Fig.28A and 28B Depicted is the switch from A·T to G·C in CD34+ cells treated with ABE8 at the -198 promoter site upstream of HBG1 / 2. Fig.28A is a heat map depicting the frequency of A to G editing of ABE8 in CD34+ cells from two donors at 48 and 144 hours after editor treatment, where donor 2 is heterozygous for sickle cell disease. Fig.28B is a graphical representation of the distribution of total sequencing reads containing either A7-only or combined (A7+A8) edits.

[0391] Fig.29 is a heat map depicting the frequency of INDELs in CD34+ cells treated with ABE8 at the -198 site of the γ-globulin promoter. Frequencies from two donors are shown at the 48 hour and 144 hour time points.

[0392] Fig.30 Depicted is the UHPLC UV-Vis trace (220 nm) of untreated differentiated CD34+ cells (Donor 1) and integration of globin chain levels.

[0393] Fig.31 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells treated with ABE7.10-m (Donor 1) and integration of globin chain levels.

[0394] Fig.32 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells treated with ABE7.10-d (Donor 1) and integration of globin chain levels.

[0395] Fig.33 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells treated with ABE8.8-m (Donor 1) and integration of globin chain levels.

[0396] Fig.34 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells treated with ABE8.8-d (Donor 1) and integration of globin chain levels.

[0397] Fig.35 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells treated with ABE8.13-m (Donor 1) and integration of globin chain levels.

[0398] Fig.36 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells treated with ABE8.13-d (Donor 1) and integration of globin chain levels.

[0399] Fig.37 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells treated with ABE8.17-m (Donor 1) and integration of globin chain levels.

[0400] Fig.38Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells treated with ABE8.17-d (Donor 1) and integration of globin chain levels.

[0401] Fig.39 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells treated with ABE8.20-m (Donor 1) and integration of globin chain levels.

[0402] Fig.40 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.20-d and integration of globin chain levels.

[0403] Fig.41 Depicted is the UHPLC UV-Vis trace (220 nm) of untreated differentiated CD34+ cells (Donor 2) and integration of globin chain levels. Note: Donor 2 is heterozygous for sickle cell disease.

[0404] Fig.42 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE7.10-m and integration of globin chain levels. Note: Donor 2 is heterozygous for sickle cell disease.

[0405] Fig.43 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE7.10-d and integration of globin chain levels. Note: Donor 2 is heterozygous for sickle cell disease.

[0406] Fig.44 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE8.8-m and integration of globin chain levels. Note: Donor 2 is heterozygous for sickle cell disease.

[0407] Fig.45 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE8.8-d and integration of globin chain levels. Note: Donor 2 is heterozygous for sickle cell disease.

[0408] Fig.46 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE8.13-m and integration of globin chain levels. Note: Donor 2 is heterozygous for sickle cell disease.

[0409] Fig.47 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE8.13-d and integration of globin chain levels. Note: Donor 2 is heterozygous for sickle cell disease.

[0410] Fig.48 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE8.17-m and integration of globin chain levels. Note: Donor 2 is heterozygous for sickle cell disease.

[0411] Fig.49 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE8.17-d and integration of globin chain levels. Note: Donor 2 is heterozygous for sickle cell disease.

[0412] Fig.50 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE8.20-m and integration of globin chain levels. Note: Donor 2 is heterozygous for sickle cell disease.

[0413] Figures 51A to 51E Depicted editing with ABE8.8 at two independent sites prior to enucleation reached over 90% editing by day 11 after erythroid differentiation, with approximately 60% of gamma globulins over alpha globulins or total beta family globulins by day 18 after erythroid differentiation. Fig.51A is a graph depicting the average of ABE8.8 editing of 2 healthy donors in 2 independent experiments. Editing efficiency was measured using primers that distinguish between HBG1 and HBG2. Fig.51B is a graph depicting the average of 1 healthy donor from 2 independent experiments. Editing efficiency was measured using primers recognizing HBG1 and HBG2. Fig.51C is a diagram depicting editing of ABE8.8 in a donor with a heterozygous E6V mutation. Fig.51D and 51E is a graph depicting the increase in gamma globulin in ABE8.8 edited cells.

[0414] Fig.52A and 52B Depicted are the percentage editing used to correct the sickle cell mutation using the ABE variants. Fig.52A is a diagram depicting a screen of different editor variants with approximately 70% editing in SCD patient fibroblasts. Fig.52BFigure 2 is a graph depicting CD34 cells from a healthy donor edited with a lead ABE variant to target a synonymous mutation A13 in an adjacent proline located within the editing window and as a surrogate for editing SCD mutations. The average editing frequency of the ABE8 variant on surrogate A13 was approximately 40%.

[0415] Fig.53A and 53B Depicts RNA amplicon sequencing to detect cellular A to I editing in RNA associated with ABE treatment. Individual data points are shown for n = 3 independent biological replicates performed on different days, and error bars represent standard deviations. ,. Fig.53A is a graph depicting the frequency of A to I editing in the targeted RNA amplicon of the core ABE 8 construct compared to ABE7 and Cas9(D10A) nickase controls. Fig.53B is a graph depicting the frequency of A to I editing in targeted RNA amplicons of ABE8 with mutations that have been reported to improve RNA off-target editing.

[0416] Fig.54 is a schematic diagram illustrating the loss of dopamine resulting from the loss of dopaminergic neurons in Parkinson's disease.

[0417] Fig.55 is a schematic diagram showing guide RNA and target sequences for correcting R1441C and R1441H mutations in LRRK2 associated with Parkinson's disease.

[0418] Fig.56 is a schematic diagram showing target sequences for correction of Y1699C, G2019S and I2020 mutations in LRRK2 associated with Parkinson's disease.

[0419] Figures 57A to 57C Provides charts, diagrams and tables. Fig.57A Quantification of the percentage of A to G conversion at nucleic acid position 7 of the LRRK2 target sequence. The editors used are designated PV1 to PV14, a description of which is provided below. pCMV indicates CMV promoter; bpNLS indicates bipartite nuclear localization signal; monoABE8.1 indicates the monomeric form of the ABE8.1 base editor. Fig.57B Depicts target sequence and guide RNA for correction of the R1441C mutation in LRRK2 associated with Parkinson's disease. Fig.57C The percentage of A to G conversion at nucleic acid position 7 of the LRRK2 target sequence is shown. Editors PV1 to 14 were used to edit LRRKR1441C. Editors (15 to 28) were used to edit G2109. Editors (PV1-28) used to correct LRRK mutations are as follows:

[0420] PV1 (also known as PV15).pCMV_monoABE8.1_bpNLS+Y147T

[0421]

[0422] PV2 (also known as PV16).pCMV_monoABE8.1_bpNLS+Y147R

[0423]

[0424] PV3 (also known as PV17).pCMV_monoABE8.1_bpNLS+Q154S

[0425]

[0426] PV4 (also known as PV18).pCMV_monoABE8.1_bpNLS+Y123H

[0427]

[0428] PV5 (also known as PV19).pCMV_monoABE8.1_bpNLS+V82S

[0429]

[0430] PV6 (also known as PV20).pCMV_monoABE8.1_bpNLS+T166R

[0431]

[0432] PV7 (also known as PV21).pCMV_monoABE8.1_bpNLS+Q154R

[0433]

[0434] PV8 (also known as PV22).pCMV_monoABE8.1_bpNLS+Y147R_Q154R_Y123H

[0435]

[0436] PV9 (also known as PV23).pCMV_monoABE8.1_bpNLS+Y147R_Q154R_I76Y

[0437]

[0438] PV10 (also known as PV24).pCMV_monoABE8.1_bpNLS+Y147R_Q154R_T166R

[0439]

[0440] PV11 (also known as PV25).pCMV_monoABE8.1_bpNLS+Y147T_Q154R

[0441]

[0442] PV12 (also known as PV26).pCMV_monoABE8.1_bpNLS+Y147T_Q154S

[0443]

[0444] PV13 (also known as PV27).pCMV_monoABE8.1_bpNLS+H123Y123H_Y147R_Q154R_I76Y

[0445]

[0446] PV14 (also known as PV28).pCMV_monoABE8.1_bpNLS+V82S+Q154R

[0447]

[0448] Figures 58A to 58C Provides charts, diagrams and tables. Fig.58A Quantification of the percentage of A to G conversion at nucleic acid position 6 of the LRRK2 target sequence. The editors used are designated PV15 to PV28, a description of which is provided above. pCMV indicates CMV promoter; bpNLS indicates bipartite nuclear localization signal; monoABE8.1 indicates the monomeric form of the ABE8.1 base editor. Fig.58B Depicts target sequence and guide RNA for correction of the G2019S mutation in LRRK2 associated with Parkinson's disease. Fig.58C The percentage of A to G transitions at nucleic acid positions 4 and 6 of the LRRK2 target sequence is shown. The A to G transition at position 4 is a bystander effect.

[0449] Figures 59A to 59L Sequence reads depicting the A to G transition at position 7 of the LRRK2 target sequence encoding R1441C (see Figures 57A to 57C ). The editor (PV1 to 14) is displayed. Fig.56 Descriptions of PV1 to 28 are provided in.

[0450] Figures 60A to 60W Sequence reads depicting the A to G transitions at positions 4 and 6 of the LRRK2 target sequence encoding G2019S (see Figures 58A to 58C ).

[0451] Fig.61A A schematic diagram is provided depicting the target sequence for correction of the pathogenic mutation A419V in LRRK2, which is encoded by an antisense strand G>A mutation. The mutation was corrected using an ABE to position 12 with a SpCas9 variant specific for the TGG PAM.

[0452] Fig.61B A schematic diagram is provided depicting a target sequence for correction of the pathogenic mutation L1114L in LRRK2 associated with Parkinson's disease. The mutation is an antisense strand T>C corrected using a base editor with cytidine deaminase activity (CBE).

[0453] Fig.61C A schematic diagram is provided depicting a target sequence for correction of the pathogenic mutation I1122V in LRRK2 associated with Parkinson's disease. The mutation is an antisense strand T>C corrected using a base editor with cytidine deaminase activity (CBE).

[0454] Fig.61D A schematic diagram is provided describing the target sequence for correction of the pathogenic mutation M1869V in LRRK2 associated with Parkinson's disease. The mutation is an antisense strand T>C corrected using a base editor with cytidine deaminase activity (CBE).

[0455] Fig.62A and 62B Depicts precise base editing correction of the Mus musculus IDUA W401X mutation in HEK293T cells. Fig.62A Graph depicting the percentage of base editing of the Mus musculus IDUA W401X mutation using ABE8 base editor variants with 21 nucleotide guide RNAs. Fig.62B is a graph depicting the indel percentages of ABE8 base editor variants using 21 nucleotide guide RNAs.

[0456] Fig.63 Graph depicting the percentage of base editing of the Mus musculus IDUA W401X mutation using ABE8 base editor variants with either a 20-nucleotide guide RNA or a 21-nucleotide guide RNA.

[0457] Fig.64Illustration depicting the nucleic acid and amino acid sequence of the Homo sapiens IDUA genome as a target for A to G nucleotide base editing to correct the W402X mutation. Also shown is the nucleic acid sequence of the corresponding guide RNA (gRNA). Indicated in the figure is the target adenosine (A) nucleobase in the IDUA nucleic acid sequence (boxed).

[0458] Fig.65A and 65B Depicting precise base editing correction of the Homo sapiens IDUA W402X mutation in HEK293T cells. Fig.65A is a graph depicting the percentage of base editing of the Homo sapiens IDUA W402X mutation using ABE8 base editor variants using a 20-nucleotide guide RNA. Fig.65B is a graph depicting the indel percentages of ABE8 base editor variants using a 20-nucleotide guide RNA.

[0459] Figures 66A to 66O is a table describing the efficiency of the percentage of A to G nucleotide changes in IDUA nucleic acid sequences using ABE8 base editor variants as detected by deep sequencing (MySeq) following PCR of genomic DNA in cells in which base editing has occurred. Figures 66A to 66M Depicted are the percentages of A to G base editing at position 6 in the IDUA nucleic acid target site for three samples using each ABE8 base editor variant ABE8.1 to ABE8.13. Figure 66N Depicted are the percentages of A to G base editing at position 6 in the IDUA nucleic acid target site for three samples using the positive control base editor ABE7.10. Fig.66O Depicted are the percentages of A to G base editing at position 6 in the IDUA nucleic acid target site using two negative control samples.

[0460] Fig.67 Diagram showing Rett / MECP2: mutation correction. MECP2 loss of function – may be caused by many different de novo mutations. X-linked: XX patients are mosaic for loss of MECP2; XY usually results in infant lethality.

[0461] Fig.68 Figure 3. Correction of the Rett syndrome R106W mutation by the first three guide sequences.

[0462] Fig.69 Illustration of correction of the Rett syndrome R255X mutation using an editor with NGTT PAM optimization.

[0463] Figures 70A to 70C : Hurler / IDUA mutation correction. Fig.70ASchematic representation of the experimental design for correction of the IDUA W402X mutation. Fig.70B Plots the edit percentage for each editor construct. Fig.70C Specific activities (nmol / mg / h) of edited and non-edited constructs are shown.

[0464] Fig.71 Depicts in vivo base editing using ABE 8.8. Example samples are shown from left to right: Guide 11 (AAV9), Guide 12 (AAV9), Guide 11 (PHP.eB), Guide 12 (PHP.eB), and control.

[0465] Figures 72A to 72B . A·T to G·C transitions of the ABE7.10 and ABE8 variants on the ABCA4 G1961E allele in model cell lines. Fig.72A Figure 2: Integrated disease allele and A·T to G·C conversion at the wobble base of the ABCA4 G1961E codon in HEK293T cells after lipofection of plasmids containing 21 nucleotide-spaced sgRNAs and base editor variants. Cells were incubated for 5 days after lipofection before editing assessment. Fig.72B : DNA sequence of the locus of interest containing the ABCA4 G1961E disease allele, the wobble base of the codon, and the -NGG PAM used by the 21 nucleotide spacer sgRNA. Error bars represent the standard deviation of three replicates. In each data set, the disease allele is on the left and the wobble base is on the right.

[0466] Fig.73 .sgRNA spacer length variants for the ABCA4 G1961E allele in a model cell line convert A·T to G·C. Integration of the disease allele and the A·T to G·C conversion at the wobble base of the ABCA4 G1961E codon in HEK293T cells following lipofection of plasmids for sgRNAs of varying spacer lengths and ABE7.10. Cells were incubated for 5 days post-lipofection prior to assessment of editing. hRz = inclusion of a self-cleaving hammerhead ribozyme at the 5' end of the sgRNA. Error bars represent standard deviations of three replicates. In each data set, the disease allele is on the left and the wobble base is on the right.

[0467] Fig.74 Schematic diagram of dual AAV delivery of split base editors reconstructed using split inteins. Two AAV particles are packaged separately with the components required for base editing. One virus encodes the base editor C-terminal region fused to the N-terminal split intein, and the complementary virus encodes the base editor N-terminal region fused to the C-terminal split intein and the sgRNA. After co-transduction of the complementary viruses, the sgRNA is transcribed, and each half of the base editor is expressed and recombined by protein trans-splicing via the split intein.

[0468] Figures 75A to 75B A·T to G·C conversion was achieved by dual AAV delivery of the split ABE variant at ABCA4 G1961 in wild-type cells. Fig.75A : A·T to G·C and C·G to T·A conversions of the wild-type ABCA4 G1961 target site in wild-type ARPE-19 cells, with editing of position 8A as an alternative target for editing in these cells. Cells were infected at an MOI of 5E+4 viral genomes per virus per cell. Cells were incubated for 2 weeks post-infection and then assessed for editing. Error bars represent the standard deviation of six replicates. For each data point, samples treated with the alternative site at position 8 (A>G) are shown on the left, and samples treated with the alternative site at position 5 (C>T) are shown on the right.

[0469] Fig.75B : DNA sequence of the wild-type target site containing the -NGG PAM used by the ABCA4 G1961 allele and the 21 nucleotide interval sgRNA targeting the wild-type sequence.

[0470] Figures 76A to 76B Off-target base editing in wild-type ARPE-19 cells infected with AAV2 expressing split ABE7.10 and a sgRNA targeting the ABCA4 G1961E disease allele. Fig.76A : Maximum A·T to G·C conversion of the on-target or off-target pre-intervals 2 weeks after co-infection with dual AAV (cyan) compared with untreated controls (grey). Fig.76B : Maximum non-A·T to G·C conversion of the target or off-target pre-interval 2 weeks after co-infection with dual AAV (cyan) compared to untreated controls (grey). For each data point, samples treated with wild-type (wt) ARPE-19 cells are shown on the left, while untreated wt ARPE-19 cells are shown on the right.

[0471] like Fig.77 Indels formed as a result of base editing in wild-type ARPE-19 cells dually infected with AAV2 expressing split ABE7.10 and an sgRNA targeting the disease allele of ABCA4 G1961E. Percentage of indels formed within or near the on-target or off-target pre-intervals 2 weeks after co-infection with dual AAV (cyan) compared to untreated controls (grey). For each data point, samples treated with wild-type (wt) ARPE-19 cells are shown on the left, while untreated wt ARPE-19 cells are shown on the right.

[0472] Fig.78: Primate retinal integrity and GFP expression at day 22 in culture. Sections were immunolabeled overnight at 4°C with anti-rhodopsin, anti-GFP, and biotinylated peanut agglutinin antibodies. Anc80L65.hGRK.eGFP shows that GFP is only observed in the outer nuclear layer (ONL) containing photoreceptors, confirming the photoreceptor-specific activity of the GRK promoter. Top row is day 0, untransduced. Second row is day 22, untransduced. Third row is day 22, GRK. Fourth row is day 22, CMB. Columns are unstained (column 1), DAPI (column 2), GFP (column 3), PNA (column 4), and rhodopsin (column 5).

[0473] Fig.79 : Cas9 expression in NHP. Cas9 expression was detected in primate retina as early as day 6 after culture. Results are shown for ABE7.10 (columns 1 and 2), ABE8.5 (columns 2 and 3), and ABE8.9 (columns 3 and 4). Top row: day 6 after culture. Bottom row: day 17 after culture. Results represent AA system delivery of split intein expressing Cas9. Scale bar: 100 μm.

[0474] Main component symbols

[0475] none. DETAILED DESCRIPTION

[0476] The present invention provides compositions including novel adenine base editors (e.g., ABE8) with improved efficiency and methods of using them to generate modifications in target nucleobase sequences.

[0477] Nucleobase editors

[0478] Disclosed herein is a base editor or a nuclear base editor for editing, modifying or changing a target nucleotide sequence of a polynucleotide.Described herein is a nuclear base editor or a base editor including a polynucleotide programmable nucleotide binding domain (e.g., Cas9) and a nuclear base editing domain (e.g., adenosine deaminase).When a polynucleotide programmable nucleotide binding domain (e.g., Cas9) is combined with a bound guide polynucleotide (e.g., gRNA), the target polynucleotide sequence can be specifically bound (i.e., via complementary base pairing of the bound guide nucleic acid and the base of the target polynucleotide sequence), and thereby the base editor is positioned to the target nucleic acid sequence that needs to be edited.In some embodiments, the target polynucleotide sequence includes single-stranded DNA or double-stranded DNA.In some embodiments, the target polynucleotide sequence includes RNA.In some embodiments, the target polynucleotide sequence includes a DNA-RNA hybrid.

[0479] Polynucleotide programmable nucleotide binding domain

[0480] It should be understood that the polynucleotide programmable nucleotide binding domain can also include nucleic acid programmable proteins that bind RNA. For example, the polynucleotide programmable nucleotide binding domain can be associated with a nucleic acid that guides the polynucleotide programmable nucleotide binding domain to RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, although they are not specifically listed in the present disclosure.

[0481] The polynucleotide programmable nucleotide binding domain of the base editor itself may include one or more domains. For example, the polynucleotide programmable nucleotide binding domain may include one or more nuclease domains. In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain may include an endonuclease or an exonuclease. In this article, the term "exonuclease" refers to a protein or polypeptide that can cleave nucleic acid (e.g., RNA or DNA) from a free end, and the term "endonuclease" refers to a protein or polypeptide that can catalyze (e.g., cut) an internal region. In some embodiments, an endonuclease can cut a single strand of a double-stranded nucleic acid. In some embodiments, an endonuclease can cut two chains of a double-stranded nucleic acid molecule. In some embodiments, the polynucleotide programmable nucleotide binding domain may be a deoxyribonuclease. In some embodiments, the polynucleotide programmable nucleotide binding domain may be a ribonuclease.

[0482] In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain can cut zero, one or two chains of the target polynucleotide. In some embodiments, the polynucleotide programmable nucleotide binding domain may include a nickase domain. In this article, the term "nickase" refers to a polynucleotide programmable nucleotide binding domain including a nuclease domain, which includes being able to cut only one of the two chains in a double-stranded nucleic acid molecule (e.g., DNA). In some embodiments, a nickase can be derived from a polynucleotide programmable nucleotide binding domain in a fully catalytically active (e.g., natural) form by introducing one or more mutations into an active polynucleotide programmable nucleotide binding domain. For example, when a polynucleotide programmable nucleotide binding domain includes a nickase domain derived from Cas9, the nickase domain derived from Cas9 may include a D10A mutation and histidine at position 840. In this type of embodiment, residue H840 retains catalytic activity and can thereby cut a single strand of a nucleic acid duplex. In another embodiment, a nickase domain derived from Cas9 may include an H840A mutation, while the amino acid residue at position 10 is still D. In some embodiments, the nickase can be derived from a fully catalytically active (e.g., native) form of a polynucleotide programmable nucleotide binding domain by removing all or part of the nuclease domain not required for nickase activity. For example, in the case where the polynucleotide programmable nucleotide binding domain includes a nickase domain derived from Cas9, the nickase domain derived from Cas9 may include a RuvC domain or a deletion of all or part of the HNH domain.

[0483] The amino acid sequence of an exemplary catalytically active Cas9 is as follows:

[0484]

[0485]

[0486] A base editor including a polynucleotide programmable nucleotide binding domain comprising a nickase domain is therefore able to produce a single-stranded DNA break (nick) at a specific polynucleotide target sequence (e.g., determined by the complementary sequence of the bound guide nucleic acid). In some embodiments, the chain of the nucleic acid duplex target polynucleotide sequence cut by a base editor including a nickase domain (e.g., a nickase domain derived from Cas9) is a chain that is not edited by a base editor (i.e., the chain cut by the base editor is opposite to the chain including the base to be edited). In other embodiments, a base editor including a nickase domain (e.g., a nickase domain derived from Cas9) can cut the chain of a DNA molecule that is targeted for editing. In this type of embodiment, the non-targeted chain is not cut.

[0487] Base editors are also provided herein, which include catalytically dead polynucleotide programmable nucleotide binding domains (i.e., target polynucleotide sequences cannot be cut). In this article, the terms "catalytically dead" and "nuclease dead" are used interchangeably to refer to one or more mutations and / or deletions of polynucleotide programmable nucleotide binding domains with chains that cannot cut nucleic acids. In some embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain base editor may lack nuclease activity due to specific point mutations in one or more nuclease domains. For example, in the case where the base editor includes a Cas9 domain, Cas9 may include a D10A mutation and an H840A mutation. Such mutations inactivate two nuclease domains, resulting in loss of nuclease activity. In other embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain may include one or more deletions of all or part of a catalytic domain (e.g., RuvC1 and / or HNH domain). In a further embodiment, the catalytically dead polynucleotide programmable nucleotide binding domain includes a point mutation (e.g., D10A or H840A) and a deletion of all or part of a nuclease domain.

[0488] Mutations of polynucleotide-programmable nucleotide binding domains that are capable of generating catalytic death from a previously functional version of the polynucleotide-programmable nucleotide binding domain are also contemplated herein. For example, in the case of a catalytically dead Cas9 ("dCas9"), variants with mutations other than D10A and H840A of Cas9 that cause nuclease inactivation are provided. For example, such mutations include other amino acid replacements at D10 and H840 or other substitutions within the Cas9 nuclease domain (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). Based on the present disclosure and the knowledge in the art, other suitable dCas9 domains without nuclease activity are apparent to those skilled in the art and are within the scope of the present disclosure. Such additional exemplary suitable nuclease-inactivating Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, Prashant et al., CAS9 transcriptional activators for target specificity 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).

[0489] Non-limiting examples of polynucleotide programmable nucleotide binding domains that can be incorporated into base editors include domains derived from CRISPR proteins, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some embodiments, the base editor includes a polynucleotide programmable nucleotide binding domain, which includes a natural or modified protein or portion thereof, which can bind to nucleic acid sequence-mediated nucleic acid modifications during CRISPR (i.e., clustered regularly spaced short palindromic repeats) via a bound guide nucleic acid. Such proteins are referred to herein as "CRISPR proteins". Therefore, disclosed herein are base editors (i.e., base editors including all or part of a CRISPR protein as a domain) comprising a polynucleotide programmable nucleotide binding domain of all or part of a CRISPR protein, also referred to as "domains derived from CRISPR proteins"). The CRISPR protein source domain incorporated into the base editor can be modified compared to the wild-type or natural version of the CRISPR protein. For example, as described below, a domain derived from a CRISPR protein can include one or more mutations, insertions, deletions, rearrangements, and / or recombination relative to a wild-type or native form of the CRISPR protein.

[0490] CRISPR is an adaptive immune system that provides protection against mobile genetic components (viruses, transposons, and conjugative plasmids). The CRISPR cluster contains a spacer, a sequence complementary to the preceding mobile component, and a target invading nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In the class II CRISPR system, the correct processing of pre-crRNA requires transcoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. TracrRNA serves as a guide for pre-crRNA assisted by ribonuclease 3. Subsequently, Cas9 / crRNA / tracrRNA endonucleases a linear or circular dsDNA target that is complementary to the spacer. The target strand that is not complementary to crRNA is first endonucleased and then 3'-5' exonucleases. In nature, DNA binding and cleavage generally require proteins and two RNAs. However, single guide RNA ("sgRNA" or simply "gNRA") can be engineered to integrate various aspects of crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., et al., Science 337:816-821 (2012), which is incorporated herein by reference in its entirety.Cas9 recognizes a short motif in the CRISPR repeat sequence (PAM or protospacer adjacent motif) to help distinguish self from non-self.

[0491] In some embodiments, the methods described herein can utilize engineered Cas proteins. A guide RNA (gRNA) is a short synthetic RNA consisting of a scaffold sequence required for Cas binding and a user-defined ~20 nucleotide interval that defines the genomic target to be modified. Thus, one skilled in the art can alter the genomic target specificity of a Cas protein depending in part on the specificity of the gRNA targeting sequence for the genomic target compared to the rest of the genome.

[0492] In some embodiments, the gRNA scaffold sequence is as follows: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAUAAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU.

[0493] In some embodiments, the domain derived from CRISPR protein incorporated into the base editor is an endonuclease (e.g., a deoxyribonuclease or a ribonuclease) that can bind to a target polynucleotide when bound to a bound guide nucleic acid. In some embodiments, the domain derived from CRISPR protein incorporated into the base editor is a nickase that can bind to a target polynucleotide when bound to a bound guide nucleic acid. In some embodiments, the domain derived from CRISPR protein incorporated into the base editor is a catalytic death domain that can bind to a target polynucleotide when bound to a bound guide nucleic acid. In some embodiments, the target polynucleotide bound by the domain derived from CRISPR protein of the base editor is DNA. In some embodiments, the target polynucleotide bound by the domain derived from CRISPR protein of the base editor is RNA.

[0494] Cas proteins useful herein include Class 1 and Class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Cmr3, Cmr4, Cmr5, Cmr6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsfO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h and Cas12i, CARF, DinG, homologs thereof, or modified versions thereof. Unmodified CRISPR enzymes can have DNA cleavage activity, such as Cas9, which has two functional endonuclease domains: RuvC and HNH. The CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within the target sequence and / or within the complement of the target sequence. For example, the CRISPR enzyme can direct cleavage of one or both strands about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence.

[0495] A vector encoding a CRISPR enzyme may be used that is mutated relative to the corresponding wild-type enzyme so that the mutated CRISPR enzyme lacks the ability to cut one or both chains of a target polynucleotide containing a target sequence. Cas9 may refer to a sequence identity and / or sequence homology with a wild-type embodiment Cas9 polypeptide (e.g., Cas9 from Streptococcus pyogenes) of at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Cas9 may refer to a sequence identity and / or sequence homology with a wild-type embodiment Cas9 polypeptide (e.g., from Streptococcus pyogenes) of at most or at most about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Cas9 may refer to a wild-type or modified form of a Cas9 protein, which may include amino acid changes, such as deletions, insertions, substitutions, variations, mutations, fusions, chimeras, or any combination thereof.

[0496] In some embodiments, the CRISPR protein-derived domain of the base editor may include Cas9 from Corynebacterium ulcerans (NCBI Reference Sequences: NC_015683.1, NC_017317.1); Corynebacterium diphtheriae (NCBI Reference Sequences: NC_016782.1, NC_016786.1); Syrphidae (NCBI Reference Sequence: NC_021284.1); Prevotella intermedia (NCBI Reference Sequence: NC_017861.1); Spiroplasma taiwanense (NCBI Reference Sequence: NC_021846.1); pisciformis Streptococcus (NCBI Reference Sequence: NC_021314.1); Bellella balticum (NCBI Reference Sequence: NC_018010.1); Campylobacter pyogenes (NCBI Reference Sequence: NC_018721.1); Streptococcus thermophilus (NCBI Reference Sequence: YP_820832.1), Listeria innocua (NCBI Reference Sequence: NP_472073.1), Campylobacter jejuni (NCBI Reference Sequence: YP_002344900.1) or Neisseria meningitidis (NCBI Reference Sequence: YP_002342100.1), Streptococcus pyogenes or Staphylococcus aureus All or part of Cas9.

[0497] Cas9 domains of nucleobase editors

[0498] Cas9 nuclease sequence and structure are well known to those skilled in the art (see, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., Proc. 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 adaptive bacterial immunity." Jinek M., et al., Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus. Other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0499] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting exemplary Cas9 domains are provided herein. The Cas9 domain can be a nuclease activity Cas9 domain, a nuclease inactivation Cas9 domain (dCas9) or a Cas9 nickase (nCas9). In some embodiments, the Cas9 domain is a nuclease activity domain. For example, the Cas9 domain can be a Cas9 domain that cuts two chains of a double-stranded nucleic acid (e.g., two chains of a double-stranded DNA molecule). In some embodiments, the Cas9 domain includes any one amino acid sequence as described herein. In some embodiments, the Cas9 domain includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to any one amino acid sequence described herein. In some embodiments, the Cas9 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any one of the amino acid sequences set forth herein. In some embodiments, the Cas9 domain comprises a polypeptide having at least 10, 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 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical consecutive amino acid residues as compared to any one of the amino acid sequences set forth herein.

[0500] In some embodiments, a protein comprising a Cas9 fragment is provided. For example, in some embodiments, the protein comprises one of two Cas9 domains: (1) a gRNA binding domain of Cas9; or (2) a DNA cleavage domain of Cas9. In some embodiments, a protein comprising Cas9 or a fragment thereof is referred to as a "Cas9 variant". A Cas9 variant has homology to Cas9 or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, the Cas9 variant can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 4546, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant includes a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical to the corresponding fragment of wild-type Cas9, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% of the length of the corresponding wild-type Cas9 amino acids. In some embodiments, the length of the fragment is at least 100 amino acids. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0501] In some embodiments, the Cas9 fusion protein provided herein includes the full-length amino acid sequence of the Cas9 protein, such as one of the Cas9 sequences provided herein. However, in other embodiments, the fusion protein provided herein does not include the full-length Cas9 sequence, but only includes one or more fragments thereof. Suitable Cas9 domains and exemplary amino acid sequences of Cas9 fragments are provided herein, and other suitable sequences of Cas9 domains and fragments are apparent to those skilled in the art.

[0502] The Cas9 protein can be associated with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 domain, such as a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Examples of nucleic acid-programmable DNA-binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, Cas12b / C2C1, and Cas12c / C2C3.

[0503] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, nucleotide and amino acid sequences are as follows).

[0504]

[0505]

[0506]

[0507]

[0508] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0509] In some embodiments, the wild-type Cas9 corresponds to or includes the following nucleotide and / or amino acid sequence:

[0510]

[0511]

[0512]

[0513] (Single underline: HNH domain; double underline: RuvC domain).

[0514] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI reference sequence: NC_002737.2 (nucleotide sequence as follows); and Uniprot reference sequence: Q99ZW2 (amino acid sequence as follows):

[0515]

[0516]

[0517]

[0518] In some embodiments, Cas9 refers to Cas9 from: Cas9 from Corynebacterium ulcerans Corynebacterium ulcerans (NCBI Reference Sequences: NC_015683.1, NC_017317.1); Corynebacterium diphtheriae (NCBI Reference Sequences: NC_016782.1, NC_016786.1); Syrphidae (NCBI Reference Sequence: NC_021284.1); Prevotella intermedia (NCBI Reference Sequence: NC_017861.1); Spiroplasma taiwanense (NCBI Reference Sequence: NC_021846.1); Streptococcus pisciformis (NCBI Reference Sequence: NC_021314.1); Bellella balticola (NCBI Reference Sequence: NC_018010.1); Campylobacter psychrophilus (NCBI Reference Sequence: NC_018721.1); Streptococcus thermophilus (NCBI Reference Sequence: YP_820832.1), Listeria innocua (NCBI Reference Sequence: NP_472073.1), Campylobacter jejuni (NCBI Reference Sequence: YP_002344900.1) or Neisseria meningitidis (NCBI Reference Sequence: YP_002342100.1), Streptococcus pyogenes or Staphylococcus aureus.

[0519] It should be understood that additional Cas9 proteins (e.g., nuclease-dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9) (including variants and homologs thereof) are within the scope of the present disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is a nuclease-dead Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease-active Cas9.

[0520] In some embodiments, the Cas9 domain is a Cas9 domain (dCas9) without nuclease activity. For example, the dCas9 domain can bind to a double-stranded nucleic acid molecule (e.g., via a gRNA molecule) without cutting any one chain of the double-stranded nucleic acid molecule. In some embodiments, the dCas9 domain of nuclease inactivation includes D10X mutations and H840X mutations of the amino acid sequences listed herein, or the corresponding mutations in any amino acid sequence provided herein, wherein X is any amino acid change. In some embodiments, the dCas9 domain of nuclease inactivation includes D10A mutations and H840A mutations of the amino acid sequences described herein, or the corresponding mutations in any amino acid sequence provided herein. As an example, the Cas9 domain without nuclease activity includes the amino acid sequence listed in the cloning vector pPlatTET-gRNA2 (accession number BAV54124).

[0521] The amino acid sequence of an exemplary catalytically inactivated Cas9 (dCas9) is as follows:

[0522]

[0523]

[0524] (See, e.g., Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.” Cell. 2013; 152(5): 1173-83, the entire contents of which are incorporated herein by reference).

[0525] Based on the present disclosure and the knowledge in the art, other suitable nuclease-inactivated dCas9 domains will be apparent to those skilled in the art and are within the scope of the present disclosure. Such additional exemplary suitable nuclease-inactivated Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, e.g., Prashant et al., CAS9 transcriptional activators for target specificity 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).

[0526] In some embodiments, the Cas9 nuclease has an inactivated (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase, referred to as a "nCas9" protein (for "nickase" Cas9). Nuclease-inactivated Cas9 proteins are interchangeably referred to as "dCas9" proteins (for nuclease-"dead" Cas9) or catalytically inactivated Cas9. Methods for producing Cas9 proteins (or fragments thereof) with inactivated DNA cleavage domains are known (see, e.g., Jinek et al., Science. 337: 816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28; 152 (5): 1173-83, the entire contents of each of which are incorporated herein by reference). For example, it is known that the DNA cleavage domain of Cas9 includes two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cuts the complementary strand to the gRNA, while the RuvC1 subdomain cuts the non-complementary strand. Mutations in these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337: 816-821 (2012); Qi et al., Cell. 28; 152 (5): 1173-83 (2013)).

[0527] In some embodiments, the dCas9 domain includes any one of the dCas9 domains provided herein that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical. In some embodiments, the Cas9 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any of the amino acid sequences set forth herein. In some embodiments, the Cas9 domain comprises at least 10, 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 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical consecutive amino acid residues compared to any of the amino acid sequences set forth herein.

[0528] In some embodiments, the dCas9 corresponds to or includes, in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain includes D10A and H840A mutations or corresponding mutations in another Cas9.

[0529] In some embodiments, the dCas9 comprises the amino acid sequence of dCas9(D10A and H840A):

[0530]

[0531]

[0532] In some embodiments, the Cas9 domain includes a D10A mutation, while the residue at position 840 remains a histidine in the amino acid sequence provided above, or at the corresponding position in any of the amino acid sequences provided herein.

[0533] In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, which, for example, result in nuclease-inactive Cas9 (dCas9). For example, such mutations include other amino acid substitutions at D10 and H840, or other substitutions within the Cas9 nuclease domain (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs of dCas9 are provided that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, dCas9 variants are provided having amino acid sequences that are shorter or longer than about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or more.

[0534] In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase can be a Cas9 protein that can only cut one chain of a double-stranded nucleic acid molecule (e.g., a double-stranded DNA molecule). In some embodiments, the Cas9 nickase cuts the target chain of the double-stranded nucleic acid molecule, indicating that the Cas9 nickase cuts the chain that is base-paired (complementary) with the gRNA (e.g., sgRNA) that binds Cas9. In some embodiments, the Cas9 nickase includes a D10A mutation and has histidine at position 840. In some embodiments, the Cas9 nickase cuts the non-target, non-base editing chain of the double-stranded nucleic acid molecule, indicating that the Cas9 nickase cuts the chain that is not base-paired with the gRNA (e.g., sgRNA) that binds Cas9. In some embodiments, the Cas9 nickase includes an H840A mutation and has an aspartic acid residue at position 10, or a corresponding mutation. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any Cas9 nickase provided herein. Based on the present disclosure and knowledge in the art, other suitable Cas9 nickases will be apparent to those skilled in the art and are within the scope of the present disclosure.

[0535] The amino acid sequence of an exemplary catalytic Cas9 nickase (nCas9) is as follows:

[0536]

[0537] In some embodiments, Cas9 refers to Cas9 from archaea (e.g., nanoarchaea), which constitutes the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, the programmable nucleotide binding protein may be a CasX or CasY protein, which has been described in, for example, Burstein et al., "New CRISPR-Cas systems from uncultivatedmicrobes." Cell Res. 2017Feb 21.doi: 10.1038 / cr.2017.21, the entire contents of which are hereby incorporated by reference. Using genome-analyzed metagenomics, many CRISPR-Cas systems are identified, including Cas9, which was first reported in the field of archaea. This divergent Cas9 protein was found in the little-known nanoarchaea that is part of an active CRISPR-Cas system. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, were found, which are one of the most compact systems discovered so far. In some embodiments, in the base editor system described herein, Cas9 is replaced by a variant of CasX or CasX. In some embodiments, in the base editor system described herein, Cas9 is replaced by CasY or a variant of CasY. It should be understood that other RNA-guided DNA binding proteins can be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0538] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein 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. In some embodiments, the napDNAbp includes an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to a naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide binding protein is a naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide binding protein includes at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% of the amino acid sequence, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to any CasX or CasY protein described herein. It should be understood that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure.

[0539] In some embodiments, Cas9 is Neisseria meningitidis Cas9 (NmeCas9) or a variant thereof. The NmeCas9 features and PAM sequences described in Edraki et al. Mol. Cell. (2019) 73(4): 714-726 are incorporated herein by reference in their entirety.

[0540] An exemplary amino acid sequence of Nme1Cas9 is provided below:

[0541] Class II CRISPR RNA-guided endonuclease Cas9 [Neisseria meningitidis] WP_002235162.1

[0542]

[0543] An exemplary amino acid sequence of Nme2Cas9 is provided below:

[0544] Class II CRISPR RNA-guided endonuclease Cas9 [Neisseria meningitidis] WP_002230835.1

[0545]

[0546] In some embodiments, the Cas protein is CasX or CasY. A canonical CasX ((uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53)tr|F0NN87|F0NN87_SULIHCRISPR-associated Casx protein OS=Icelandic Sulfolobus (strain HVE10 / 4) GN=SiH_0402PE=4SV=1) amino acid sequence is as follows:

[0547]

[0548] The amino acid sequence of the exemplary CasX (>tr|F0NH53|F0NH53_SULIR CRISPR-associated protein Casx OS=Icelandic Sulfolobus (strain REY15A) GN=SiRe_0771 PE=4SV=1) is as follows:

[0549]

[0550] Proteus CasX

[0551]

[0552] The amino acid sequence of an exemplary CasY ((ncbi.nlm.nih.gov / protein / APG80656.1)>APG80656.1 CRISPR-associated protein CasY [uncultured Thresholdia]) is as follows:

[0553]

[0554] The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Cas9 undergoes a conformational change upon target binding, positioning the nuclease domains to cleave the opposite strand of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (approximately 3 to 4 nucleotides upstream of the PAM sequence). The resulting DSB is then repaired via one of two general repair pathways: (1) the efficient but error-prone non-homologous end-joining (NHEJ) pathway; or (2) the less efficient but high-fidelity homology-directed repair (HDR) pathway.

[0555] The "efficiency" of non-homologous end joining (NHEJ) and / or homology-directed repair (HDR) can be calculated by any convenient method. For example, in some embodiments, efficiency can be expressed as a percentage of successful HDR. For example, a surveyor nuclease assay can be used to produce a cleavage product, and the ratio of product to substrate can be used to calculate a percentage. For example, a surveyor nuclease can be used to directly cleave DNA containing a newly integrated restriction sequence as a result of successful HDR. More cleaved substrates represent a higher HDR percentage (higher HDR efficiency). As an illustrative example, the following equation [(cleavage product) / (substrate plus cleavage product)] can be used to calculate the score (percentage) of HDR (e.g., (b+c) / (a+b+c), where "a" is the band intensity of the DNA substrate, and "b" and "c" are cleavage products).

[0556] In some embodiments, efficiency can be expressed as a percentage of successful NHEJ. For example, the T7 endonuclease I assay can be used to produce cleavage products, and the ratio of product to substrate can be used to calculate the percentage of NHEJ. T7 endonuclease I cuts the mismatched heteroduplex DNA produced by hybridization of wild-type and mutant DNA chains (NHEJ produces small random insertions or deletions (indels) at the original break site). More cutting indicates a higher NHEJ percentage (higher NHEJ efficiency). As an illustrative example, the score (percentage) of NHEJ can be calculated using the following equation: (1-(1-(b+c) / (a+b+c)) 1 / 2)×100, where “a” is the band intensity of the DNA substrate, and “b” and “c” are the cleavage products (Ran et al., Cell. 2013 Sep. 12; 154(6): 1380-9; and Ran et al., Nat Protoc. 2013 Nov.; 8(11): 2281–2308).

[0557] The NHEJ repair pathway is the most active repair mechanism, which often results in small nucleotide insertions or deletions (indels) at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical significance because cell populations expressing Cas9 and gRNA or guide polynucleotides can lead to a variety of mutations. In most embodiments, NHEJ produces small indels in the target DNA, resulting in amino acid deletions, insertions, or frameshift mutations, leading to premature stop codons within the open reading frame (ORF) of the target gene. The ideal end result is a loss-of-function mutation within the target gene.

[0558] While NHEJ-mediated DSB repair typically disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to generate specific nucleotide changes ranging from single nucleotide changes to large insertions such as the addition of a fluorophore or label.

[0559] To utilize HDR for gene editing, a DNA repair template including the desired sequence can be delivered to the cell type of interest using one or more gRNAs and Cas9 or Cas9 nickase. The repair template can include the desired edit as well as additional homologous sequences immediately upstream and downstream of the target (called left and right homology arms). The length of each homology arm depends on the size of the introduced change, with larger insertions requiring longer homology arms. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. Even in cells expressing Cas9, gRNA, and an exogenous repair template, the efficiency of HDR is often low (<10% modified alleles). The efficiency of HDR can be improved by synchronizing cells, as HDR occurs during the S and G2 phases of the cell cycle. Chemical or genetic inhibitors of genes involved in NHEJ can also increase HDR frequency.

[0560] In some embodiments, Cas9 is a modified Cas9. A given gRNA targeting sequence can have other sites with partial homology throughout the genome. These sites are called off-target sites and need to be considered when designing gRNAs. In addition to optimizing gRNA design, the specificity of CRISPR can also be improved by modifying Cas9. Cas9 produces double-strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. Cas9 nickase is a D10A mutant of SpCas9 that retains one nuclease domain and produces DNA nicks instead of DSBs. The nickase system can also be combined with HDR-mediated gene editing for specific gene editing.

[0561] In some embodiments, Cas9 is a variant Cas9 protein. The variant Cas9 polypeptide has an amino acid sequence that differs by one amino acid compared to the amino acid sequence of the wild-type Cas9 protein (e.g., with deletion, insertion, substitution, fusion). In some cases, the variant Cas9 polypeptide has an amino acid change (e.g., deletion, insertion or substitution) that reduces the nuclease activity of the Cas9 polypeptide. For example, in some cases, the variant Cas9 polypeptide has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% of the activity of the wild-type Cas9 protein corresponding to the nuclease. In some embodiments, the variant Cas9 protein has no substantial nuclease activity. When the subject Cas9 protein is a variant Cas9 protein without substantial nuclease activity, it can be referred to as "dCas9".

[0562] In some embodiments, the variant Cas9 protein has reduced nuclease activity. For example, the variant Cas9 protein exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the endonuclease activity of a wild-type Cas9 protein (e.g., a wild-type Cas9 protein).

[0563] In some embodiments, the variant Cas9 protein can cut the complementary strand of the guide target sequence, but the ability to cut the non-complementary strand of the double-stranded guide target sequence is reduced. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, the variant Cas9 protein has D10A (aspartic acid to alanine at amino acid position 10), and thus can cut the complementary strand of the double-stranded guide target sequence, but cut the complementary strand of the non-double-stranded guide target sequence (so when the variant Cas9 protein cuts the double-stranded target nucleic acid, it causes a single-strand break (SSB) instead of a double-strand break (DSB)) (see, e.g., Jinek et al., Science. 2012 Aug. 17; 337 (6096): 816-21).

[0564] In some embodiments, the variant Cas9 protein can cut the non-complementary strand of the double-stranded guide target sequence, but the ability to cut the complementary strand of the guide target sequence is reduced. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC / HNH / RuvC domain motif). As a non-limiting example, in some embodiments, the variant Cas9 protein has an H840A (histidine to alanine at amino acid position 840) mutation, so it can cut the non-complementary strand of the guide target sequence, but the ability to cut the complementary strand of the guide target sequence is reduced (thus, when the variant Cas9 protein cuts the double-stranded guide target sequence, SSB is generated instead of DSB). Such Cas9 proteins have a reduced ability to cut a guide target sequence (e.g., a single-stranded guide target sequence), but retain the ability to bind to a guide target sequence (e.g., a single-stranded guide target sequence).

[0565] In some embodiments, the variant Cas9 protein has a reduced ability to cut the complementary and non-complementary strands of a double-stranded target DNA. As a non-limiting example, in some embodiments, the variant Cas9 protein includes both D10A and H840A mutations, so that the ability of the polypeptide to cut the complementary and non-complementary strands of a double-stranded target DNA is reduced. Such Cas9 proteins have a reduced ability to cut target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0566] As another non-limiting example, in some embodiments, the variant Cas9 protein contains W476A and W1126A mutations, so that the ability of the polypeptide to cut the target DNA is reduced. Such Cas9 proteins have a reduced ability to cut the target DNA (e.g., single-stranded target DNA), but retain the ability to bind to the target DNA (e.g., single-stranded target DNA).

[0567] As another non-limiting example, in some embodiments, the variant Cas9 protein contains P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, so that the polypeptide has a reduced ability to cut the target DNA. Such Cas9 proteins have a reduced ability to cut the target DNA (e.g., single-stranded target DNA), but retain the ability to bind to the target DNA (e.g., single-stranded target DNA).

[0568] As another non-limiting example, in some embodiments, variant Cas9 protein includes H840A, W476A and W1126A mutations, so that the ability of polypeptide to cut target DNA is reduced. Such Cas9 protein cuts target DNA (e.g., single-stranded target DNA) ability to reduce, but retains the ability to bind target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some embodiments, variant Cas9 protein contains H840A, D10A, W476A and W1126A mutations, so that polypeptide has the ability to cut target DNA reduced. Such Cas9 protein has the ability to cut target DNA (e.g., single-stranded target DNA) reduced, but retains the ability to bind target DNA (e.g., single-stranded target DNA). In some embodiments, variant Cas9 restores catalytic His residues at position 840 in Cas9HNH domain (A840H).

[0569] As another non-limiting example, in some embodiments, variant Cas9 protein includes H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, so that the ability of polypeptide to cut target DNA is reduced. Such Cas9 protein has the ability to cut target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind target DNA (e.g., single-stranded target DNA) is retained. As another non-limiting example, in some embodiments, variant Cas9 protein contains D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, so that the ability of polypeptide to cut target DNA is reduced. Such Cas9 protein has the ability to cut target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind target DNA (e.g., single-stranded target DNA) is retained. In some embodiments, when the variant Cas9 protein includes W476A and W1126A mutations or when the variant Cas9 protein includes P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, the variant Cas9 protein does not effectively bind to the PAM sequence. Therefore, in some such embodiments, when such variant Cas9 proteins are used in the binding method, the method does not require a PAM sequence. In other words, in some embodiments, when such variant Cas9 proteins are used in the binding method, the method may include a guide RNA, but the method may be performed in the absence of a PAM sequence (and the specificity of the binding is therefore provided by the targeting fragment of the guide RNA). Other residues may be mutated to achieve the above effects (even if one or other nuclease is partially inactivated). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 may be altered (i.e., substituted). In addition, mutations other than alanine substitutions are also suitable.

[0570] In some embodiments, a variant Cas9 protein with reduced catalytic activity (e.g., when the Cas9 protein has D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 mutations, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A), the variant Cas9 protein can still bind to the target DNA in a site-specific manner (because it is still guided to the target DNA sequence by the guide RNA) as long as it retains the ability to interact with the guide RNA.

[0571] In some embodiments, the variant Cas protein can be spCas9, spCas9-VRQR, spCas9-VRER, xCas9(sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.

[0572] In some embodiments, a modified SpCas9 comprising amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and having specificity for an altered PAM 5'-NGC is used.

[0573] Alternatives to S. pyogenes Cas9 may include RNA-guided endonucleases from the Cpf1 family that show cleavage activity in mammalian cells. CRISPR of Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease of the class II CRISPR / Cas system. This acquired immunity mechanism is present in Prevotella and Francisella. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses a guide RNA to seek out and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the limitations of the CRISPR / Cas9 system. Unlike the Cas9 nuclease, the result of Cpf1-mediated DNA cleavage is a double-stranded break with a short 3' overhang. The staggered cleavage pattern of Cpf1 can open up the possibility of targeted gene transfer, which is similar to traditional restriction enzyme cloning and can improve the efficiency of gene editing. Like the above Cas9 variants and orthologs, Cpf1 can also expand the number of CRISPR targetable sites to AT-rich regions or AT-rich genomes that lack NGG PAM sites favored by SpCas9. The Cpf1 locus includes a mixed α / β domain, RuvC-I, followed by a helical region, RuvC-II, and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. In addition, Cpf1 does not have a HNH endonuclease domain, and the N-terminus of Cpf1 does not have the α-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain architecture shows that Cpf1 is functionally unique and is classified as a Class 2 type V CRISPR system. The Cas1, Cas2, and Cas4 proteins encoded by the Cpf1 locus are more similar to class I and class III than to those from class II systems. Functional Cpf1 does not require a trans-activating CRISPR RNA (tracrRNA), and therefore, only CRISPR (crRNA) is required. This facilitates genome editing because not only is Cpf1 smaller than Cas9, but its sgRNA molecules are also smaller (approximately half the nucleotides of Cas9). The Cpf1-crRNA complex cleaves the target DNA or RNA by recognizing the protospacer adjacent motif 5'-YTN-3', compared to the G-rich PAM targeted by Cas9. Upon recognition of the PAM, Cpf1 introduces a sticky-end-like DNA double-strand break with a 4 or 5 nucleotide overhang.

[0574] Cas12 domains of nucleobase editors

[0575] Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multi-subunit effector complexes, while Class 2 systems have single protein effectors. For example, Cas9 and Cpf1 are Class 2 effectors, although of different types (Class II and Type V, respectively). In addition to Cpf1, Class 2 Type V CRISPR-Cas systems also include Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i). See, e.g., Shmakov et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems," Mol. Cell, 2015 Nov. 5; 60(3): 385-397; Makarova et al., "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR Journal, 2018, 1(5): 325-336; and Yan et al., "Functionally Diverse Type V CRISPR-Cas Systems," Science, 2019 Jan. 4; 363: 88-91; the entire contents of each of which are hereby incorporated by reference. Type V Cas proteins contain a RuvC (or RuvC-like) nuclease domain. Although the production of mature CRISPR RNA (crRNA) is generally independent of tracrRNA, for example, Cas12b / C2c1 requires tracrRNA to produce crRNA. Cas12b / C2c1 relies on crRNA and tracrRNA for DNA cleavage.

[0576] The nucleic acid programmable DNA binding proteins considered in the present invention include Cas proteins classified as Class 2 V-type (Cas12 proteins). Non-limiting examples of Cas 2 class V-type proteins include Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h and Cas12i, homologues thereof or modified forms thereof. As used herein, Cas12 proteins may also be referred to as Cas12 nucleases, Cas12 domains or Cas12 protein domains. In some embodiments, the Cas12 proteins of the present invention include amino acid sequences interrupted by internal fusion protein domains such as deaminase domains.

[0577] In some embodiments, the Cas12 domain is a Cas12 domain or a Cas12 nickase without nuclease activity. In some embodiments, the Cas12 domain is a nuclease activity domain. For example, the Cas12 domain can be a Cas12 domain that forms a nick on a chain of a double-stranded nucleic acid (e.g., a double-stranded DNA molecule). In some embodiments, the Cas12 domain includes any amino acid sequence as described herein. In some embodiments, the Cas12 domain includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to any amino acid sequence described herein. In some embodiments, the Cas12 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any one of the amino acid sequences set forth herein. In some embodiments, the Cas12 domain comprises at least 10, 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 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical consecutive amino acid residues compared to any one of the amino acid sequences set forth herein.

[0578] In some embodiments, a protein comprising a Cas12 fragment is provided. For example, in some embodiments, the protein comprises one of two Cas12 domains: (1) a gRNA binding domain of Cas12; or (2) a DNA cleavage domain of Cas12. In some embodiments, a protein comprising Cas12 or a fragment thereof is referred to as a "Cas12 variant". Cas12 variants have homology with Cas12 or a fragment thereof. For example, a Cas12 variant is at least about 70% identical to wild-type Cas12, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the Cas12 variant can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 4546, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas12. In some embodiments, the Cas12 variant includes a fragment of Cas12 (e.g., a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas12. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% of the amino acid length of the corresponding wild-type Cas12. In some embodiments, the length of the fragment is at least 100 amino acids. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0579] In some embodiments, Cas12 corresponds to or partially or entirely includes a Cas12 amino acid sequence having one or more mutations that change the Cas12 nuclease activity. For example, such mutations include amino acid substitutions within the RuvC nuclease domain of Cas12. In some embodiments, a variant or homolog of Cas12 is provided that is at least about 70% identical to wild-type Cas12, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, a Cas12 having a short or long amino acid sequence of about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or more is provided.

[0580] In some embodiments, the Cas12 fusion protein provided herein includes the full-length amino acid sequence of the Cas12 protein, such as one of the Cas12 sequences provided herein. However, in other embodiments, the fusion protein provided herein does not include the full-length Cas12 sequence, but only includes one or more fragments thereof. Exemplary amino acid sequences of suitable Cas12 domains are provided herein, and other suitable sequences of Cas12 domains and fragments are apparent to those skilled in the art.

[0581] Typically, Class 2 V-type Cas proteins have a single functional RuvC nuclease domain (see, e.g., Chen et al., "CRISPR-Cas12a target binding unleashes indiscriminate-stranded DNase activity," Science 360: 436-439 (2018)). In some cases, the Cas12 protein is a variant of the Cas12b protein. (See Strecker et al., Nature Communications, 2019, 10 (1): Art. No.: 212). In one embodiment, when compared to the wild-type Cas12 protein amino acid sequence, the variant Cas12 polypeptide has an amino acid sequence that differs by 1, 2, 3, 4, 5 or more amino acids (e.g., having a deletion, insertion, substitution, fusion). In some cases, the variant Cas12 polypeptide has an amino acid change (e.g., a deletion, insertion or substitution) that reduces the activity of the Cas12 polypeptide. For example, in some cases, the variant Cas12 is a Cas12b polypeptide having a percentage of nickase activity of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the corresponding wild-type Cas12b protein. In some cases, the variant Cas12b protein has no substantial nickase activity.

[0582] In some cases, the variant Cas12b protein has reduced nickase activity. For example, the variant Cas12b protein exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1% or less than about 0.1% of the nickase activity of the wild-type Cas12b protein.

[0583] In some embodiments, the Cas12 protein includes an RNA-guided endonuclease from the Cas12a / Cpf1 family that exhibits activity in mammalian cells. CRISPR of Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease of the class II CRISPR / Cas system. This acquired immunity mechanism exists in Prevotella and Francisella. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses guide RNA to find and cut viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some limitations of the CRISPR / Cas9 system. Unlike the Cas9 nuclease, the result of Cpf1-mediated DNA cleavage is a double-strand break with a short 3' overhang. The staggered cleavage pattern of Cpf1 can open up the possibility of directional gene transfer, similar to traditional restriction enzyme cloning, which can improve the efficiency of gene editing. Like the above Cas9 variants and orthologs, Cpf1 can also expand the number of CRISPR targetable sites to AT-rich regions or AT-rich genomes that lack the NGG PAM sites favored by SpCas9. The Cpf1 locus includes a mixed α / β domain, a helical region following RuvC-I, RuvC-II, and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. In addition, unlike Cas9, Cpf1 does not have a HNH endonuclease domain, and the N-terminus of Cpf1 does not have the α-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain architecture table shows that Cpf1 is functionally unique and is classified as a Class 2, Type V CRISPR system. The Cas1, Cas2, and Cas4 proteins encoded by the Cpf1 locus are more similar to Class I and Class III systems than to Class II systems. Functional Cpf1 does not require transactivating CRISPR RNA (tracrRNA), therefore, only CRISPR (crRNA) is required. This is advantageous for genome editing because not only is Cpf1 smaller than Cas9, but its sgRNA molecule is also smaller (about half the nucleotides of Cas9). The Cpf1-crRNA complex cleaves the target DNA or RNA by recognizing the adjacent motif 5'-YTN-3' or 5'-TTTN-3' compared to the G-rich PAM targeted by Cas9. Upon recognition of the PAM, Cpf1 introduces sticky-end-like DNA double-strand breaks with overhangs of 4 or 5 nucleotides.

[0584] In some aspects of the invention, a vector encodes a CRISPR enzyme that is mutated relative to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence and can be used. Cas12 can refer to a sequence having at least or about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and / or sequence homology to a wild-type embodiment Cas12 polypeptide (e.g., Cas12 from Bacillus jochii). Cas12 can refer to a polypeptide having up to or about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and / or sequence homology with a wild-type embodiment Cas12 polypeptide (e.g., from Bacillus sutchuenii (BhCas12b), Bacillus V3-13 (BvCas12b), and Alicyclobacillus acidophilus (AaCas12b). Cas12 can refer to a wild-type or modified form of a Cas12 protein, which can include amino acid changes, such as deletions, insertions, substitutions, variations, mutations, fusions, chimeras, or any combination thereof.

[0585] Nucleic acid programmable DNA binding proteins

[0586] Some aspects of the present disclosure provide a fusion protein including a domain that acts as a nucleic acid programmable DNA binding protein, which can be used to guide a protein (such as a base editor) to a specific nucleic acid (e.g., DNA or RNA) sequence. In a particular embodiment, the fusion protein includes a nucleic acid programmable DNA binding protein domain and a deaminase domain. Non-limiting examples of nucleic acid programmable DNA binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. Non-limiting examples of Cas enzymes 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 / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Css1, Css2, Cse5e, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, class II Cas effector protein, type V Cas effector protein, class VI protein, CARF, DinG, homologs thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, even though they may not be specifically listed in the present disclosure.See, e.g., Makarova et al. "Classification 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 type VCRISPR-Cas systems" Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, the entire contents of each of which are hereby incorporated by reference.

[0587] An example of a programmable DNA binding protein with a nucleic acid having a PAM specificity different from that of Cas9 is a clustered regularly interspaced short palindromic repeat sequence from Prevotella and Francisella 1 (Cpf1). Similar to Cas9, Cpf1 is also a Class 2 CRISPR effector. It has been shown that Cpf1 mediates a powerful DNA interference with characteristics different from Cas9. Cpf1 is an endonuclease guided by a single RNA lacking tracrRNA, and it utilizes a T-rich protoporphyrin-adjacent motif (TTN, TTTN or YTN). In addition, Cpf1 cuts DNA via staggered DNA double-strand breaks. Among the 16 Cpf1 family proteins, two enzymes from Acidaminococcus and Lachnospiraceae have been shown to have effective genome editing activity in human cells. Cpf1 proteins are known in the art and have previously been described in, for example, Yamano et al., "Crystal structure of Cpf1 complexed with guide RNA and target DNA". Cell (165) 2016, p. 949-962; the entire contents of which are hereby incorporated by reference.

[0588] Useful in the present compositions and methods are nuclease-inactivated Cpf1 (dCpf1) variants that can be used as guide nucleotide sequence-programmable DNA binding protein domains. The Cpf1 protein has a RuvC-like nuclease domain that is similar to the RuvC domain of Cas9, but does not have a HNH nuclease domain, and the N-terminus of Cpf1 does not have the α-helical recognition lobe of Cas9. Zetsche et al., Cell, 163, 759-771, 2015 (incorporated herein by reference) showed that the RuvC-like domain of Cpf1 is responsible for cutting both DNA strands and inactivating the RuvC-like domain inactivates the Cpf1 nuclease activity. For example, mutations corresponding to D917A, E1006A, or D1255A in Francisella Cpf1 inactivate the Cpf1 nuclease activity. In some embodiments, dCpf1 of the present disclosure includes mutations corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D122. It should be understood that any mutation that inactivates the RuvC domain of Cpf, such as a substitution mutation, deletion, or insertion, can be used in accordance with the present disclosure.

[0589] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein can be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). In some embodiments, the Cpf1 protein is a nuclease-inactivated Cpf1 (dCpf1). In some embodiments, Cpf1, nCpf1 or dCpf1 comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to the Cpf1 sequence disclosed herein. In some embodiments, dCpf1 comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the Cpf1 sequence disclosed herein, and comprises mutations corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It should be understood that Cpf1 from other bacterial species can also be used according to the present disclosure.

[0590] Wild-type Francisella Cpf1 (D917, E1006, and D1255 are in bold and underlined)

[0591]

[0592]

[0593] Francisella Cpf1 D917A (A917, E1006 and D1255 are bold and underlined)

[0594]

[0595]

[0596] Francisella Cpf1 E1006A (D917, A1006 and D1255 are bold and underlined)

[0597]

[0598] Francisella Cpf1 D1255A (D917, E1006 and A1255 are bold and underlined)

[0599]

[0600] Francisella Cpf1 D917A / E1006A (A917, A1006 and D1255 are bold and underlined)

[0601]

[0602]

[0603] Francisella Cpf1 D917A / D1255A (A917, E1006 and A1255 are bold and underlined)

[0604]

[0605]

[0606] Francisella Cpf1 E1006A / D1255A (D917, A1006 and A1255 are bold and underlined)

[0607]

[0608]

[0609] Francisella Cpf1 D917A / E1006A / D1255A (A917, A1006, and A1255 are bold and underlined)

[0610]

[0611] In some embodiments, one of the Cas9 domains present in the fusion protein can be replaced by a guide nucleotide sequence-programmable DNA binding protein domain that has no requirement for a PAM sequence.

[0612] In some embodiments, the Cas9 domain is a 2Cas9 domain from Staphylococcus aureus (SaCas9). In some embodiments, the SaCas9 domain is a nuclease-active SaCas9, a nuclease-inactivated SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, SaCas9 includes an N579A mutation, or a corresponding mutation in any amino acid sequence provided herein.

[0613] In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence with a non-canonical PAM. In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence with a NNGRRT or NNGRRT PAM sequence. In some embodiments, the SaCas9 domain includes one or more of E781X, N967X, and R1014X mutations, or a corresponding mutation in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SaCas9 domain includes one or more of E781K, N967K, and R1014H mutations, or one or more corresponding mutations in any amino acid sequence provided herein. In some embodiments, the SaCas9 domain includes E781K, N967K, or R1014H mutations, or a corresponding mutation in any amino acid sequence provided herein.

[0614] Example SaCas9 sequences

[0615]

[0616] The underlined and bolded residue N579 above can be mutated (e.g., to A579) to generate a SaCas9 nickase.

[0617] Example SaCas9n sequences

[0618]

[0619]

[0620] Residue A579 above can be mutated from N579 to generate the SaCas9 nickase, indicated by underline and bold.

[0621] Example SaKKH Cas9

[0622]

[0623] Residue A579, which can be mutated from N579 to generate the SaCas9 nickase, is underlined and in bold. Residues K781, K967, and H1014, which can be mutated from E781, N967, and R1014 to generate the SaKKH Cas9, are underlined and in italics.

[0624] In some embodiments, the napDNAbp is a circular array. In the following sequences, plain text represents adenosine deaminase sequences, bold sequences represent sequences derived from Cas9, italic sequences represent linker sequences, and underlined sequences represent bipartite nuclear localization sequences.

[0625] CP5 (with MSP "NGC" PID and "D10A" nickase):

[0626]

[0627] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a single effector of a microbial CRISPR-Cas system. Single effectors of a microbial CRISPR-Cas system include, but are not limited to, Cas9, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multi-subunit effector complexes, while Class 2 systems have single protein effectors. For example, Cas9 and Cpf1 are Class 2 effectors. In addition to Cas9 and Cpf1, Shmakov et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems", Mol. Cell, 2015 Nov. 5; 60 (3): 385-397 describes three different Class 2 CRISPR-Cas systems (Cas12b / C2c1 and Cas12c / C2c3), the entire contents of which are hereby incorporated by introduction. The effectors of two of the systems, Cas12b / C2c1 and Cas12c / C2c3, include a RuvC-like nuclease domain associated with Cpf1. The third system includes an effector with two predicted HEPN RNase domains. Unlike the CRISPR RNA produced by Cas12b / C2c1, the production of mature CRISPR RNA is independent of the CRISPR RNA and tracrRNA for DNA cleavage.

[0628] It is reported that the crystal structure of Bacillus acidoterrestris Cas12b / C2c1 (AacC2c1) forms a complex with a chimeric single-molecule guide RNA (sgRNA). See, for example, Liu et al., "C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism", Mol. Cell, 2017 Jan. 19; 65 (2): 310-322, the entire contents of which are hereby incorporated by introduction. Crystal structures have also been reported in Bacillus acidoterrestris C2c1 bound to target DNA in the form of a ternary complex. See, for example, Yang et al., "PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease", Cell, 2016 Dec. 15; 167 (7): 1814-1828, the entire contents of which are hereby incorporated by introduction. The catalytically competent conformations of AacC2c1 (with both target and non-target DNA strands) have been independently localized in a single RuvC catalytic pocket, where Cas12b / C2c1-mediated cleavage results in seven nucleotide staggered breaks of the target DNA. Structural comparisons between the Cas12b / C2c1 ternary complex and previously identified Cas9 and Cpf1 counterparts demonstrate the diversity of mechanisms used by the CRISPR-Cas9 system.

[0629] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein can be Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, napDNAbp is Cas12b / C2c1 protein. In some embodiments, napDNAbp is Cas12c / C2c3 protein. In some embodiments, the amino acid sequence included in napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, napDNAbp is naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the amino acid sequence included in the napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to any one of the napDNAbp sequences provided herein. It should be understood that Cas12b / C2c1 or Cas12c / C2c3 from other bacterial species can also be used according to the present disclosure.

[0630] Cas12b / C2c1((uniprot.org / uniprot / T0D7A2#2)

[0631] sp|T0D7A2|C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acidoterrestris (ATCC 49025 / DSM 3922 / CIP31B 3926 / CIP31B31G / CIP3131B3100 c2c1 PE=1SV=1) amino acid sequence is as follows:

[0632]

[0633] AacCas12b (Alicyclobacter acidophilus) - WP_067623834

[0634]

[0635]

[0636] BhCas12b (Bacillus hisashi) NCBI reference sequence: WP_095142515

[0637]

[0638]

[0639] BvCas12b V4 (changes relative to wild type S893R / K846R / E837G) is expressed as follows: 5'mRNA Cap --- 5'UTR --- bhCas12b --- STOP sequence --- 3'UTR --- 120 polynucleotide tail 5'UTR: GGGAAATAAGAGAGAAAAGAAGAGTAAGAA GAAATATAAGAGCCACC

[0640] 3'UTR (TriLink standard UTR)

[0641]

[0642] Nucleic acid sequence of bhCas12b (V4)

[0643]

[0644]

[0645] In some embodiments, Cas12b is BvCas12B, which is a variant of BhCas12b and includes the following changes relative to BhCas12B: S893R, K846R, and E837G.

[0646] BvCas12b (Bacillus V3-13) NCBI reference sequence: WP_101661451.1

[0647]

[0648]

[0649] In some embodiments, Cas12b is BTCas12b. BTCas12b (Bacillus thermoamylovorans) NCBI reference sequence: WP_041902512

[0650]

[0651] In some embodiments, napDNAbp refers to Cas12c. In some embodiments, the Cas12c protein is a variant of Cas12c1 or Cas12c1. In some embodiments, the Cas12 protein is a variant of Cas12c2 or Cas12c2. In some embodiments, the Cas12 protein is a Cas12c protein from the genus Oleophilus HI0009 (i.e., OspCas12c) or a variant of OspCas12c. These Cas12c molecules have been described in Yan et al., "Functionally Diverse Type V CRISPR-Cas Systems," Science, 2019 Jan. 4; 363: 88-91; the entire contents of which are hereby incorporated by reference. In some embodiments, the amino acid sequence included in napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to naturally occurring Cas12c1, Cas12c2 or OspCas12c proteins. In some embodiments, napDNAbp is a naturally occurring Cas12c1, Cas12c2 or OspCas12c protein. In some embodiments, the amino acid sequence included in napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to any Cas12c1, Cas12c2 or OspCas12c protein described herein. It should be understood that Cas12c1, Cas12c2 or OspCas12c from other bacterial species may also be used according to the present disclosure.

[0652] Cas12c1

[0653]

[0654]

[0655] Cas 12c2

[0656]

[0657]

[0658] OspCas 12c

[0659]

[0660] In some embodiments, napDNAbp refers to Cas12g, Cas12h or Cas12i, which have been described in, for example, Yan et al., "Functionally Diverse Type V CRISPR-Cas Systems," Science, 2019 Jan. 4; 363: 88-91; the entire contents of each of which are hereby incorporated by reference. A new classification of type V Cas proteins was identified by aggregating more than 10TB of sequence data, which showed weak similarity to previously characterized class V proteins (including Cas12g, Cas12h, and Cas12i). In some embodiments, the Cas12 protein is a variant of Cas12g or Cas12g. In some embodiments, the Cas12 protein is a variant of Cas12h or Cas12h. In some embodiments, the Cas12 protein is a variant of Cas12i or Cas12i. It should be understood that other RNA-guided DNA binding proteins can be used as napDNAbp and are within the scope of the present disclosure. In some embodiments, the amino acid sequence included in napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to naturally occurring Cas12g, Cas12h or Cas12i proteins. In some embodiments, napDNAbp is a naturally occurring Cas12g, Cas12h or Cas12i protein. In some embodiments, the amino acid sequence included in napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to any Cas12g, Cas12h or Cas12i protein described herein. It should be understood that Cas12g, Cas12h or Cas12i from other bacterial species can also be used according to the present disclosure. In some embodiments, Cas12i is Cas12i1 or Cas12i2.

[0661] Cas12g1

[0662]

[0663] Cas 12h1

[0664]

[0665] Cas 12i 1

[0666]

[0667] Cas 12i 2

[0668]

[0669] Representative nucleic acid and protein sequences of base editors are as follows: BhCas12b GGSGGS-ABE8-Xten20 at P153

[0670]

[0671]

[0672]

[0673] BhCas12b GGSGGS-ABE8-Xten20 at K255

[0674]

[0675]

[0676]

[0677] BhCas12b GGSGGS-ABE8-Xten20 in D306

[0678]

[0679]

[0680]

[0681] BhCas12b GGSGGS-ABE8-Xten20 in D980

[0682]

[0683]

[0684]

[0685] BhCas12b GGSGGS-ABE8-Xten20 in K1019

[0686]

[0687]

[0688]

[0689] For the above sequences, the Kozak sequence is indicated in bold and underlined; the N-terminal nuclear localization signal (NLS) is marked; lowercase letters indicate the GGGSGGS linker; the sequence encoding ABE8 is marked, and the unmodified sequence encodes BhCas12b; the double underline indicates the Xten20 linker; the single underline indicates the C-terminal NLS; GGATCC indicates the GS linker; italic letters represent the coding sequence of the 3x hemagglutinin (HA) tag.

[0690] Guide polynucleotide

[0691] In one embodiment, the guide polynucleotide is a guide RNA. The RNA / Cas complex can help guide the Cas protein to the target DNA. Cas9 / crRNA / tracrRNA endonucleolytic cleavage is complementary to the linear or circular dsDNA target of the interval. The target chain that is not complementary to the crRNA is first endonucleolytic cleavage, and then trimmed at 3'-5' exonucleolytic cleavage. In nature, DNA binding and cleavage generally require proteins and two RNAs. However, single guide RNA ("sgRNA" or "gNRA" for short) can be engineered to integrate various aspects of crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M.et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes short motifs in CRISPR repeats (PAM or pre-spacer adjacent motifs) to help distinguish self from non-self. Cas9 nuclease sequence and structure are well known to those skilled in the art (see, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti, JJ et al., 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 "Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M. et al, Science 337: 816-821 (2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus. Based on the present disclosure, other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art, and such Cas9 nucleases and sequences include those from "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.In some embodiments, the Cas9 nuclease has an inactive (eg, inactivated) DNA cleavage domain, ie, Cas9 is a nickase.

[0692] In some embodiments, the guide polynucleotide is at least one single guide RNA ("sgRNA" or "gNRA"). In some embodiments, the guide polynucleotide is at least one tracrRNA. In some embodiments, the guide polynucleotide does not require a PAM sequence to guide a polynucleotide-programmable DNA binding domain (e.g., Cas9 or Cpf1) to a target nucleotide sequence.

[0693] The polynucleotide programmable nucleotide binding domains (e.g., domains derived from CRISPR) of the base editors disclosed herein can be identified by associating with guide polynucleotides. Guide polynucleotides (e.g., gRNA) are typically single-stranded and can be programmed to site-specifically bind (i.e., via complementary base pairing) to the target sequence of polynucleotides, thereby guiding the base editor nucleic acid bound to the guide nucleic acid to the target sequence. The guide polynucleotide can be DNA. The guide polynucleotide can be RNA. In some embodiments, the guide polynucleotide includes natural nucleotides (e.g., adenosine). In some embodiments, the guide polynucleotide includes non-natural (or non-natural) nucleotides (e.g., peptide nucleic acids or nucleotide analogs). In some embodiments, the targeting region of the guide nucleic acid sequence can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. The targeting region of the guide nucleic acid can be between 10 and 30 nucleotides in length, or between 15 and 25 nucleotides in length, or between 15 and 20 nucleotides in length.

[0694] In some embodiments, the guide polynucleotide comprises two or more separate polynucleotides that can interact with each other via, for example, complementary base pairing (e.g., dual guide polynucleotides). For example, a guide polynucleotide can include a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). For example, a guide polynucleotide can include one or more trans-activating CRISPR RNAs (tracrRNAs).

[0695] In a class II CRISPR system, CRISPR protein (e.g., Cas9) targeting nucleic acids typically requires complementary base pairing between a first RNA molecule (crRNA) that includes a sequence that recognizes the target sequence and a second RNA molecule (trRNA) that includes a recognition repeat sequence, which forms a scaffold region that stabilizes the guide RNA-CRISPR protein complex. Such dual guide RNA systems can be used as guide polynucleotides to guide the base editors disclosed herein to the target polynucleotide sequence.

[0696] In some embodiments, the base editors provided herein utilize a single guide polynucleotide (e.g., gRNA). In some embodiments, the base editors provided herein utilize dual guide polynucleotides (e.g., dual gRNA). In some embodiments, the base editors provided herein utilize one or more guide polynucleotides (e.g., multiple gRNAs). In some embodiments, a single guide polynucleotide is used for different base editors described herein. For example, a single guide polynucleotide can be used for a cytidine base editor and an adenosine base editor.

[0697] In other embodiments, the guide polynucleotide may include a polynucleotide targeting portion of a nucleic acid and a scaffold portion of a nucleic acid in a single molecule (i.e., a single molecule guide nucleic acid). For example, a single molecule guide polynucleotide may be a single guide RNA (sgRNA or gRNA). As used herein, the term guide polynucleotide sequence encompasses any single, double, or multi-molecule nucleic acid that is capable of interacting with a base editor and guiding the base editor to a target polynucleotide sequence.

[0698] Typically, a guide polynucleotide (e.g., a crRNA / trRNA complex or gRNA) includes a "polynucleotide-targeting segment" comprising a sequence capable of recognizing and binding to a target polynucleotide sequence, and a "protein binding segment" of a guide polynucleotide stabilized within a polynucleotide programmable nucleotide binding domain component of a base editor. In some embodiments, the polynucleotide targeting segment of a guide polynucleotide recognizes and binds to a DNA polynucleotide, thereby facilitating the editing of bases in the DNA. In other embodiments, the polynucleotide targeting segment of a guide polynucleotide recognizes and binds to an RNA polynucleotide, thereby facilitating the editing of bases in the RNA. Herein, a "segment" refers to a segment or region of a molecule, for example, a continuous segment of nucleotides in a guide polynucleotide. A segment may also refer to a region / segment of a complex, such that a segment may include more than, for example, when a guide polynucleotide includes a plurality of individual nucleic acid molecules, a protein binding segment may include, for example, all or a portion of a plurality of individual molecules hybridized along a complementary region. In some embodiments, a protein-binding segment of a DNA-targeting RNA comprising two separate molecules may include (i) 40 to 75 base pairs of a first RNA molecule of 100 base pairs in length; and (ii) 10 to 25 base pairs of a second RNA molecule of 50 base pairs in length. Unless otherwise expressly defined in a particular context, the definition of "segment" is not limited to a specific number of total base pairs, is not limited to any specific number of base pairs of a given RNA molecule, is not limited to a specific number of separate molecules within a complex, and may include regions of RNA molecules of any total length, and may include regions that are complementary to other molecules.

[0699] The guide RNA or guide polynucleotide may include two or more RNAs, such as CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The guide RNA or guide polynucleotide may sometimes include single-stranded RNA or a single guide RNA (sgRNA) formed by fusion of a portion (e.g., a functional portion) of crRNA and tracrRNA. The guide RNA or guide polynucleotide may also be a double RNA including crRNA and tracrRNA. In addition, crRNA may hybridize with a target DNA.

[0700] As described above, the guide RNA or guide polynucleotide can be an expression product. For example, the DNA encoding the guide RNA can be a vector comprising a sequence encoding the guide RNA. The guide RNA or guide polynucleotide can be transferred into the cell by transfecting the cell with an isolated guide RNA or plasmid DNA comprising a sequence encoding the guide RNA and a promoter. The guide RNA or guide polynucleotide can also be transferred into the cell in other ways, such as using virus-mediated gene delivery.

[0701] The guide RNA or guide polynucleotide can be isolated. For example, the guide RNA can be transfected into a cell or organism in the form of an isolated RNA. The guide RNA can be prepared by in vitro transcription using any in vitro transcription system known in the art. The guide RNA can be transferred into a cell in the form of an isolated RNA rather than in the form of a plasmid comprising the coding sequence of the guide RNA.

[0702] The guide RNA or guide polynucleotide may include three regions: a first region at the 5' end that can be complementary to a target site in a chromosomal sequence, a second internal region that can form a stem-loop structure, and a third region that can be single-stranded. The first region of each guide RNA may also be different so that each guide RNA guides the fusion protein to a specific target site. In addition, the second and third regions of each guide RNA may be the same in all guide RNAs.

[0703] The first region of the guide RNA or guide polynucleotide can be complementary to the sequence of the target site in the chromosome sequence, so that the first region of the guide RNA can be base paired with the target site. In some embodiments, the first region of the guide RNA may include or be from about 10 nucleotides to 25 nucleotides (i.e., from 10 nucleotides to about 25 nucleotides; or from about 10 nucleotides to about 25 nucleotides; or from about 10 nucleotides to about 25 nucleotides; or about 10 nucleotides to 25 nucleotides) or more. For example, the base pairing region between the first region of the guide RNA and the target site in the chromosome sequence can be or can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25 or more nucleotides in length. Sometimes, the length of the first region of the guide RNA can be or can be about 19, 20 or 21 nucleotides.

[0704] The guide RNA or guide polynucleotide may also include a second region that forms a secondary structure. For example, the secondary structure formed by the guide RNA may include a stem (or hairpin) and a loop. The length of the loop and the stem may vary. For example, the length of the loop may range from about 3 to 10 nucleotides, and the length of the stem may range from about 6 to 20 base pairs. The stem may include one or more protrusions of 1 to 10 or about 10 nucleotides...

Claims

1. A method of treating a nervous system disorder in a subject, the method comprising: administering to a subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, The adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain. wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 or a corresponding position thereof in SEQ ID NO: 2, And wherein the guide polynucleotide directs the adenosine base editor to effect an A to G nucleobase change in a target gene or a regulatory component thereof associated with the nervous system disorder in the subject, thereby treating the nervous system disorder in the subject. 2 . The method according to claim 1 , wherein the target gene is an α-L-iduronidase (IDUA) gene, and the nervous system disease is Hurler's disease.

3. A method for treating Hurler's disease in a subject, the method comprising administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 or a corresponding position thereof in SEQ ID NO: 2, And wherein the guide polynucleotide directs the adenosine base editor to effect an A to G nucleobase change in the subject's α-L-iduronidase (IDUA) gene or its regulatory components, thereby treating the subject's Hurler's disease.

4. The method of claim 2 or 3, wherein the administering improves at least one symptom associated with Hurler's disease.

5. The method of claim 4, wherein the administration results in faster improvement of at least one symptom associated with Hurler's disease compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase.

6. The method of any one of claims 2 to 5, wherein the IDUA gene or its regulatory element comprises a SNP associated with Hurler's disease.

7. The method of any one of claims 2 to 6, wherein the A to G nucleobase change is at a SNP associated with Hurler's disease.

8. The method of claim 6 or 7, wherein the SNP associated with Hurler's disease results in a W402X or W401X amino acid mutation in the IDUA polypeptide numbered in SEQ ID NO: 4 or a variant thereof encoded by the IDUA gene, wherein X is a stop codon.

9. The method of any one of claims 6 to 8, wherein the A to G nucleobase change changes the SNP associated with Hurler's disease to a wild-type nucleobase.

10. The method of any one of claims 6 to 8, wherein the A to G nucleobase change changes the SNP associated with Hurler's disease to a non-wild-type nucleobase that results in one or more improved symptoms of Hurler's disease.

11. The method of any one of claims 6 to 10, wherein the A to G change at the SNP associated with Hurler's disease changes the stop codon in the IDUA polypeptide encoded by the IDUA gene to tryptophan.

12. The method of any one of claims 6 to 10, wherein the guide polynucleotide comprises a nucleic acid sequence complementary to the IDUA gene or a regulatory element thereof comprising the SNP associated with the Hurler's disease.

13. The method according to any one of claims 6 to 12, wherein the adenosine base editor forms a complex with a single guide RNA (sgRNA), wherein the single guide RNA (sgRNA) comprises a nucleic acid sequence complementary to the IDUA gene or its regulatory component comprising the SNP associated with the Hurler disease.

14. The method of claim 13, wherein the sgRNA comprises a nucleic acid sequence selected from the group consisting of 5'-GACUCUAGGCAGAGGUCUCAA-3', 5'-ACUCUAGGCAGAGGUCUCAA-3', 5'-CUCUAGGCGAAGUGUCGC-3', and 5'-GCUCUAGGCCGAAGUGUCGC-3'.

15. The method of claim 1, wherein the target gene is a leucine-rich repeat kinase 2 (LRRK2) gene, and the nervous system disease is Parkinson's disease.

16. A method of treating Parkinson's disease in a subject, the method comprising: administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 or a corresponding position thereof in SEQ ID NO: 2, And wherein the guide polynucleotide guides the adenosine base editor to affect the leucine-rich repeat kinase-2 (LRRK2) gene or its regulatory components in the subject to achieve an A to G nucleobase change, thereby treating Parkinson's disease in the subject.

17. The method of any one of claims 1, 15, or 16, wherein the administering improves at least one symptom associated with Parkinson's disease.

18. The method of claim 17, wherein the administration results in a faster improvement in at least one symptom associated with Parkinson's disease compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase.

19. The method of any one of claims 15 to 18, wherein the LRRK2 gene or regulatory element thereof comprises a SNP associated with Parkinson's disease.

20. The method of claim 19, wherein the A to G nucleobase change is at a SNP associated with Parkinson's disease.

21. The method of claim 19 or 20, wherein the SNP associated with Parkinson's disease results in an A419V, R1441C, R1441H or G2019S amino acid mutation in the LRRK2 polypeptide numbered in SEQ ID NO: 3 or a variant thereof encoded by the LRRK2 gene.

22. The method of any one of claims 19 to 21, wherein the A to G nucleobase change changes the SNP associated with Parkinson's disease to a wild-type nucleobase.

23. The method of any one of claims 19 to 21, wherein the A to G nucleobase change changes the SNP associated with the Parkinson's disease to a non-wild-type nucleobase that results in one or more improved symptoms of Parkinson's disease.

24. The method of any one of claims 19 to 22, wherein the A to G nucleobase change changes a cysteine ​​or histidine to an arginine in the LRRK2 polypeptide encoded by the LRRK2 gene.

25. The method of any one of claims 19 to 22, wherein the A to G change changes a serine to a glycine in the LRRK2 polypeptide encoded by the LRRK2 gene.

26. The method of any one of claims 19 to 22, wherein the A to G change replaces cysteine ​​(C) or histidine (H) with arginine (R) at position 144 or replaces serine with glycine (G) at position 2019 of the LRRK2 polypeptide or variant thereof numbered in SEQ ID NO: 3 encoded by the LRRK2 gene.

27. A method of treating Parkinson's disease in a subject, the method comprising: A subject is administered (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, and wherein the guide polynucleotide guides the adenosine base editor to effect an A to G nucleobase change at a SNP of the LRRK2 gene associated with Parkinson's disease, wherein the SNP does not encode a G2019S mutation in the LRRK2 polypeptide numbered in SEQ ID NO: 3 or a variant thereof.

28. The method of claim 27, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166, or a position corresponding thereto, in SEQ ID NO:

2.

29. The method of any one of claims 15 to 28, wherein the guide polynucleotide comprises a nucleic acid sequence complementary to a LRRK2 gene or a regulatory element thereof comprising a SNP associated with the Parkinson's disease.

30. The method of any one of claims 15 to 28, wherein the adenosine base editor forms a complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to a LRRK2 gene or a regulatory component thereof comprising a SNP associated with Parkinson's disease.

31. The method of claim 30, wherein the sgRNA comprises the nucleic acid sequence: 5'-AAGCGCAAGCCUGGAGGGAA-3'; or 5'-ACUACAGCAUUGCUCAGUAC-3'.

32. The method of claim 1, wherein the target gene is the methyl CpG binding protein 2 (MECP2) gene and the neurological disease is Rett's disease.

33. A method of treating Rett's disease in a subject, the method comprising administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and the adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 or a corresponding position thereof in SEQ ID NO: 2, And wherein the guide polynucleotide directs the adenosine base editor to achieve an A to G nucleobase change in the methyl CpG binding protein 2 (MECP2) gene or its regulatory components in the subject, thereby treating Rett's disease in the subject.

34. The method of claim 32 or 33, wherein the administering improves at least one symptom associated with Rett's disease.

35. The method of claim 34, wherein the administration results in faster improvement of at least one symptom associated with Rett's disease compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase.

36. The method of any one of claims 32 to 35, wherein the MECP2 gene or its regulatory element comprises a SNP associated with Rett's disease.

37. The method of claim 36, wherein the A to G nucleobase change is at a SNP associated with the Rett syndrome.

38. The method of claim 36 or 37, wherein the SNP associated with Rett's disease results in a R106W or T158M amino acid mutation in the MECP2 polypeptide numbered in SEQ ID NO: 5 or a variant thereof encoded by the MECP2 gene.

39. The method of claim 36 or 37, wherein the SNP associated with Rett's disease results in an R255X or R270X amino acid mutation in the MECP2 polypeptide encoded by the MECP2 gene, wherein X is a stop codon.

40. The method of any one of claims 36 to 39, wherein the A to G nucleobase change changes the SNP associated with Rett's disease to a wild-type nucleobase.

41. The method of any one of claims 36 to 39, wherein the A to G nucleobase change changes the SNP associated with Rett's disease to a non-wild-type nucleobase that results in improved Rett's disease symptoms.

42. The method of any one of claims 36 to 39, wherein the A to G nucleobase change at the SNP associated with Rett's disease changes the stop codon in the MECP2 polypeptide to tryptophan.

43. The method of any one of claims 36 to 42, wherein the guide polynucleotide comprises a nucleic acid sequence complementary to a MECP2 gene or a regulatory component thereof comprising a SNP associated with Rett's disease.

44. The method of any one of claims 36 to 42, wherein the adenosine base editor forms a complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to a MECP2 gene or a regulatory component thereof comprising a SNP associated with Rett syndrome.

45. The method of claim 44, wherein the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of 5'-CUUUUCACUUCCUGCCGGGG-3', 5'-AGCUUCCAUGUCCAGCCUUC-3', 5'-ACCAUGAAGUCAAAAUCAUU-3', and 5'-GCUUUCAGCCCCGUUUCUUG-3'.

46. ​​The method of claim 1, wherein the target gene is the ATP-binding cassette subfamily member 4 (ABCA4) gene and the nervous system disease is Stargardt's disease.

47. A method of treating Stargardt's disease in a subject, the method comprising administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 or a position corresponding thereto in SEQ ID NO: 2, And wherein the guide polynucleotide directs the adenosine base editor to effect an A to G nucleobase change in the ATP-binding cassette subfamily member 4 (ABCA4) gene or a regulatory component thereof in the subject, thereby treating Stargardt's disease in the subject.

48. The method of claim 46 or 47, wherein the administering improves at least one symptom associated with Stargardt's disease.

49. The method of claim 48, wherein the administration results in faster improvement of at least one symptom associated with Stargardt's disease compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase.

50. The method of any one of claims 46 to 49, wherein the ABCA4 gene comprises a SNP associated with Stargardt's disease.

51. The method of claim 50, wherein the A to G nucleobase change is at a SNP associated with Stargardt's disease.

52. The method of claim 50 or 51, wherein the SNP associated with Stargardt's disease results in an A1038V or G1961E amino acid mutation in the ABCA4 polypeptide numbered in SEQ ID NO: 6 or a variant thereof encoded by the ABCA4 gene.

53. The method of claim 52, wherein the SNP associated with Stargardt's disease results in a G1961E amino acid mutation in the ABCA4 polypeptide numbered in SEQ ID NO: 6 or a variant thereof.

54. The method of any one of claims 51 to 53, wherein the A to G nucleobase change changes the SNP associated with Stargardt's disease to a wild-type nucleobase.

55. The method of any one of claims 51 to 53, wherein the A to G nucleobase change changes a SNP associated with the Stargardt's disease to a non-wild-type nucleobase that results in one or more improved symptoms of Stargardt's disease.

56. The method of any one of claims 51 to 55, wherein the guide polynucleotide comprises a nucleic acid sequence complementary to an ABCA4 gene or a regulatory element thereof comprising a SNP associated with the Stargardt's disease.

57. The method of any one of claims 51 to 56, wherein the adenosine base editor forms a complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to the ABCA4 gene or a regulatory component thereof comprising a SNP associated with Stargardt's.

58. The method of claim 57, wherein the sgRNA comprises the sequence 5'-CUCCAGGGCGAACUUCGACACACAGC-3'.

59. The method of any of the preceding claims, wherein the administration results in improved symptoms associated with a nervous system disorder compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase.

60. A method for editing a target gene or a regulatory component thereof associated with a nervous system disorder, the method comprising contacting the target gene or the regulatory component thereof with (i) an adenosine base editor and (ii) a guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 or a corresponding position thereof in SEQ ID NO: 2, The guide polynucleotide directs the adenosine base editor to effect an A to G nucleobase change in the target gene associated with the nervous system disorder or its regulatory component.

61. The method of claim 60, wherein the target gene is the leucine-rich repeat kinase 2 (LRRK2) gene and the nervous system disease is Parkinson's disease.

62. A method for editing a leucine-rich repeat kinase-2 (LRRK2) gene or a regulatory component thereof, the method comprising contacting the LRRK2 gene or a regulatory component thereof with (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 or a corresponding position thereof in SEQ ID NO: 2, And wherein the guide polynucleotide directs the adenosine base editor to effect an A to G nucleobase change in the LRRK2 gene and its regulatory components.

63. The method of claim 62, wherein the A to G nucleobase change is at a SNP associated with the Parkinson's disease.

64. The method of claim 62 or 63, wherein the SNP associated with Parkinson's disease results in an A419V, R1441C, R1441H or G2019S amino acid mutation in the LRRK2 polypeptide numbered in SEQ ID NO: 3 or a variant thereof encoded by the LRRK gene.

65. The method of any one of claims 62 to 64, wherein the A to G nucleobase change changes the SNP associated with Parkinson's disease to a wild-type nucleobase.

66. The method of any one of claims 62 to 64, wherein the A to G nucleobase change changes a SNP associated with Parkinson's disease to a non-wild-type nucleobase that results in one or more improved symptoms of Parkinson's disease.

67. The method of any one of claims 62 to 66, wherein the A to G nucleobase change changes a cysteine ​​or histidine to an arginine in the LRRK2 polypeptide encoded by the LRRK2 gene.

68. The method of any one of claims 62 to 66, wherein the A to G change changes a serine to a glycine in the LRRK2 polypeptide encoded by the LRRK2 gene.

69. The method of any one of claims 62 to 66, wherein the A to G change replaces cysteine ​​(C) or histidine (H) with arginine (R) at position 144 or replaces serine with glycine (G) at position 2019 of the LRRK2 polypeptide numbered in SEQ ID NO: 3 or a variant thereof encoded by the LRRK2 gene.

70. A method for editing a leucine-rich repeat kinase-2 (LRRK2) gene or a regulatory component thereof, the method comprising contacting the LRRK gene or its regulatory component with (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, and wherein the guide polynucleotide directs the adenosine base editor to effect an A to G nucleobase change at a SNP in the LLRK2 gene, wherein the SNP does not encode a G2019S mutation in the LRRK2 polypeptide numbered in SEQ ID NO: 3 or a variant thereof.

71. The method of claim 70, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 numbered in SEQ ID NO:2, or a position corresponding thereto.

72. The method of any one of claims 61 to 71, wherein the guide polynucleotide comprises a nucleic acid sequence complementary to a LRRK2 gene or a regulatory element thereof comprising a SNP associated with the Parkinson's disease.

73. The method of any one of claims 61 to 71, wherein the adenosine base editor forms a complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to a LRRK2 gene or a regulatory component thereof comprising a SNP associated with Parkinson's disease.

74. The method of claim 73, wherein the sgRNA comprises the nucleic acid sequence: 5'-AAGCGCAAGCCUGGAGGGAA-3'; or 5'-ACUACAGCAUUGCUCAGUAC-3'.

75. The method of claim 60, wherein the target gene is an alpha-L-iduronidase (IDUA) gene and the neurological disease is Hurler's disease.

76. A method for editing an α-L-iduronidase (IDUA) gene or a regulatory component thereof, the method comprising contacting the IDUA gene or a regulatory component thereof with (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 or a corresponding position thereof in SEQ ID NO: 2, And wherein the guide polynucleotide directs the adenosine base editor to effect an A to G nucleobase change in the IDUA gene or a regulatory component thereof.

77. The method of any one of claims 75 or 76, wherein the IDUA gene or its regulatory element comprises a SNP associated with Hurler's disease.

78. The method of any one of claims 75 to 77, wherein the A to G nucleobase change is at a SNP associated with Hurler's disease.

79. The method of claim 77 or 78, wherein the SNP associated with Hurler's disease results in a W402X or W401X amino acid mutation in the IDUA polypeptide numbered in SEQ ID NO: 4 or a variant thereof encoded by the IDUA gene, wherein X is a stop codon.

80. The method of any one of claims 77 to 79, wherein the A to G nucleobase change changes the SNP associated with Hurler's disease to a wild-type nucleobase.

81. The method of any one of claims 77 to 79, wherein the A to G nucleobase change changes the SNP associated with Hurler's disease to a non-wild-type nucleobase that results in one or more improved symptoms of Hurler's disease.

82. The method of any one of claims 77 to 81, wherein the A to G change at the SNP associated with Hurler's disease changes the stop codon in the IDUA polypeptide encoded by the IDUA gene to tryptophan.

83. The method of any one of claims 77 to 81, wherein the guide polynucleotide comprises a nucleic acid sequence complementary to an IDUA gene or a regulatory element thereof comprising a SNP associated with Hurler's disease.

84. The method of any one of claims 77 to 83, wherein the adenosine base editor forms a complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to the IDUA gene or a regulatory component thereof comprising a SNP associated with Hurler's disease.

85. The method of claim 84, wherein the sgRNA comprises a nucleic acid sequence selected from the group consisting of 5'-GACUCUAGGCAGAGGUCUCAA-3', 5'-ACUCUAGGCAGAGGUCUCAA-3', 5'-CUCUAGGCCGAAGUGUCGC-3', and 5'-GCUCUAGGCCGAAGUGUCGC-3'.

86. The method of claim 60, wherein the target gene is the methyl CpG binding protein 2 (MECP2) gene and the neurological disease is Rett's disease.

87. A method for editing a methyl CpG binding protein 2 (MECP2) gene or a regulatory component thereof, the method comprising administering to a subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 or a corresponding position thereof in SEQ ID NO: 2, And wherein the guide polynucleotide directs the adenosine base editor to effect an A to G nucleobase change in the MECP2 gene or its regulatory components.

88. The method of claim 86 or 87, wherein the MECP2 gene or its regulatory component comprises a SNP associated with Rett's disease.

89. The method of claim 88, wherein the A to G nucleobase change is at a SNP associated with the Rett syndrome.

90. The method of claim 88 or 89, wherein the SNP associated with Rett's disease results in a R106W or T158M amino acid mutation in the MECP2 polypeptide numbered in SEQ ID NO: 5 or a variant thereof encoded by the MECP2 gene.

91. The method of claim 88 or 89, wherein the SNP associated with Rett's disease results in an R255X or R270X amino acid mutation in the MECP2 polypeptide encoded by the MECP2 gene, wherein X is a stop codon.

92. The method of any one of claims 88 to 81, wherein the A to G nucleobase change changes the SNP associated with Rett's disease to a wild-type nucleobase.

93. The method of any one of claims 88 to 91, wherein the A to G nucleobase change changes the SNP associated with Rett's disease to a non-wild-type nucleobase that results in one or more improved symptoms of Rett's disease.

94. The method of any one of claims 88 to 91, wherein the A to G nucleobase change at the SNP associated with Rett's disease changes the stop codon in the MECP2 polypeptide to tryptophan.

95. The method of any one of claims 88 to 94, wherein the guide polynucleotide comprises a nucleic acid sequence complementary to a MECP2 gene or a regulatory component thereof comprising the SNP associated with Rett's disease.

96. The method of any one of claims 88 to 94, wherein the adenosine base editor forms a complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to a MECP2 gene or a regulatory component thereof comprising a SNP associated with Rett syndrome.

97. The method of claim 96, wherein the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of 5'-CUUUUCACUUCCUGCCGGGG-3', 5'-AGCUUCCAUGUCCAGCCUUC-3', 5'-ACCAUGAAGUCAAAAUCAUU-3', and 5'-GCUUUCAGCCCCGUUUCUUG-3'.

98. The method of claim 60, wherein the target gene is the ATP-binding cassette subfamily member 4 (ABCA4) gene and the nervous system disease is Stargardt's disease.

99. A method for editing an ATP-binding cassette subfamily member 4 (ABCA4) gene or a regulatory component thereof, the method comprising contacting the ABCA4 gene or a regulatory component thereof with (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 or a corresponding position thereof in SEQ ID NO: 2, And wherein the guide polynucleotide directs the adenosine base editor to effect an A to G nucleobase change in the ABCA4 gene or a regulatory component thereof.

100. The method of claim 98 or 99, wherein the administering improves at least one symptom associated with Stargardt's disease.

101. The method of claim 100, wherein the administration results in faster improvement of at least one symptom associated with Stargardt's disease compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase.

102. The method of any one of claims 98 to 101, wherein the ABCA4 gene comprises a SNP associated with Stargardt's disease.

103. The method of claim 102, wherein the A to G nucleobase change is at a SNP associated with the Stargardt's disease.

104. The method of claim 102 or 103, wherein the SNP associated with Stargardt's disease results in an A1038V or G1961E amino acid mutation in the ABCA4 polypeptide numbered in SEQ ID NO:6 or a variant thereof of the gene encoded by ABCA4.

105. The method of claim 104, wherein the SNP associated with Stargardt's disease results in a G1961E amino acid mutation in the ABCA4 polypeptide numbered in SEQ ID NO: 6 or a variant thereof.

106. The method of any one of claims 103 to 105, wherein the A to G nucleobase change changes the SNP associated with Stargardt's disease to a wild-type nucleobase.

107. The method of any one of claims 103 to 105, wherein the A to G nucleobase change changes the SNP associated with Stargardt's disease to a non-wild-type nucleobase that results in one or more improved symptoms of Stargardt's disease.

108. The method of any one of claims 103 to 107, wherein the guide polynucleotide comprises a nucleic acid sequence complementary to an ABCA4 gene or a regulatory element thereof comprising a SNP associated with Stargardt's disease.

109. The method of any one of claims 103 to 108, wherein the adenosine base editor forms a complex with a single guide RNA (sgRNA), wherein the single guide RNA (sgRNA) comprises a nucleic acid sequence complementary to the ABCA4 gene or its regulatory components comprising a SNP associated with Stargardt's disease.

110. The method of claim 109, wherein the sgRNA comprises the sequence 5'-CUCCAGGGCGAACUUCGACACACAGC-3'.

111. The method of any one of claims 60 to 110, wherein the contacting is performed in a cell.

112. The method of claim 111, wherein the contacting results in less than 10% indels in the genome of the cell, wherein the indel rate is measured by the mismatch frequency between the sequence flanking the single nucleotide modification and the unmodified sequence.

113. The method of claim 111, wherein the contacting results in less than 5% indels in the genome of the cell, wherein the indel rate is measured by the mismatch frequency between the sequence flanking the single nucleotide modification and the unmodified sequence.

114. The method of claim 111, wherein the contacting results in less than 1% indels in the genome of the cell, wherein the indel rate is measured by the mismatch frequency between the sequence flanking the single nucleotide modification and the unmodified sequence.

115. The method of any one of claims 111 to 114, wherein the cell is a neuron.

116. The method of any one of claims 60 to 110, wherein the contacting is in a population of cells.

117. The method of claim 116, wherein after the contacting step, the contacting results in an A to G nucleobase change in at least 40% of the cell population.

118. The method of claim 116, wherein after the contacting step, the contacting results in an A to G nucleobase change in at least 50% of the cell population.

119. The method of claim 116, wherein after the contacting step, the contacting results in an A to G nucleobase change in at least 70% of the cell population.

120. The method of any one of claims 116 to 119, wherein at least 90% of the cells are viable after the contacting step.

121. The method of any one of claims 116 to 120, wherein the cell population is not enriched after the contacting step.

122. The method of any one of claims 116 to 121, wherein the cell population is neurons.

123. The method of any one of claims 111 to 122, wherein the contacting is in vivo or ex vivo.

124. The method of any one of claims 1 to 123, wherein the polynucleotide programmable DNA binding domain is Cas9.

125. The method of claim 124, wherein the Cas9 is SpCas9, SaCas9, or a variant thereof.

126. The method of claim 124 or 125, wherein the polynucleotide-programmable DNA-binding domain comprises a modified SpCas9 with an altered protospacer adjacent motif (PAM) specificity.

127. The method of claim 126, wherein the Cas9 is specific for a PAM sequence selected from the group consisting of NGG, NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, and NGC; wherein N is A, G, C, or T; and wherein R is A or G.

128. The method of any one of claims 124 to 127, wherein the polynucleotide programmable DNA binding domain is a nuclease inactive variant.

129. A method according to any one of claims 124 to 127, wherein the polynucleotide programmable DNA binding domain is a nickase variant.

130. The method of claim 129, wherein the nickase variant comprises the amino acid substitution D10A or its corresponding amino acid replacement.

131. The method of any one of claims 1-130, wherein the adenosine deaminase domain comprises a TadA domain.

132. The method of claim 131, wherein the adenosine deaminase comprises a TadA deaminase comprising a V82S change and / or a T166R change.

133. The method of any one of claims 1 to 132, wherein the adenosine deaminase further comprises one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, Q154R, or a combination thereof.

134. The method of any one of claims 1 to 133, wherein the adenosine deaminase comprises a combination of alterations selected from the group consisting of: Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y147T+Q154R; Y147T+Q154S; and Y123H+Y147R+Q154R+I76Y.

135. The method of any one of claims 1 to 134, wherein the adenosine base editor domain comprises an adenosine deaminase monomer.

136. The method of any one of claims 1 to 135, wherein the adenosine base editor comprises an adenosine deaminase dimer.

137. The method of any one of claims 131 to 136, wherein the TadA deaminase is a TadA*8 variant.

138. The method of claim 137, wherein the TadA*8 variant is selected from the group consisting of: TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12 and TadA*8.

13.

139. The method of claim 138, wherein the adenosine base editor is an ABE8 base editor selected from the group consisting of ABE8.1, ABE8.2, ABE8.3, ABE8.4, ABE8.5, ABE8.6, ABE8.7, ABE8.8, ABE8.9, ABE8.10, ABE8.11, ABE8.12, and ABE8.

13.

140. A cell produced according to the method of any one of claims 111 to 115.

141. A cell population produced according to the method of any one of claims 116 to 122.

142. A base editor system comprising (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 or a corresponding position thereof in SEQ ID NO: 2, And wherein the guide polynucleotide directs the adenosine base editor to effect an A to G nucleobase change in a target gene or a regulatory component thereof associated with a nervous system disorder.

143. The base editor system of claim 142, wherein the target gene is a leucine-rich repeat kinase-2 (LRRK2) gene and the neurological disease is Parkinson's disease.

144. A base editor system comprising (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 or a position corresponding thereto in SEQ ID NO: 2, And wherein the guide polynucleotide directs the adenosine base editor to effect an A to G nucleobase change in the LRRK2 gene and its regulatory components.

145. The base editor system of claim 144, wherein the A to G nucleobase change is at a SNP associated with Parkinson's disease in the LRRK2 gene or a regulatory component thereof.

146. The base editor system of claim 144 or 145, wherein the SNP associated with Parkinson's disease results in an A419V, R1441C, R1441H or G2019S amino acid mutation in the LRRK2 polypeptide numbered in SEQ ID NO: 3 or a variant thereof encoded by the LRRK2 gene.

147. The base editor system of any one of claims 145 or 146, wherein the A to G nucleobase change changes the SNP associated with Parkinson's disease to a wild-type nucleobase.

148. A base editor system according to any one of claims 145 or 146, wherein the A to G nucleobase change changes the SNP associated with Parkinson's disease to a non-wild-type nucleobase, which results in one or more improved symptoms of Parkinson's disease.

149. The base editor system of any one of claims 145 to 148, wherein the A to G nucleobase change changes a cysteine ​​or histidine to an arginine in the LRRK2 polypeptide encoded by the LRRK2 gene.

150. The base editor system of any one of claims 145 to 148, wherein the A to G change changes a serine to a glycine in the LRRK2 polypeptide encoded by the LRRK2 gene.

151. The base editor system of any one of claims 145 to 148, wherein the A to G change replaces cysteine ​​(C) or histidine (H) with arginine (R) at position 144 or replaces serine with glycine (G) at position 2019 of the LRRK2 polypeptide or variant thereof numbered in SEQ ID NO:3 encoded by the LRRK2 gene.

152. The base editor system of claim 151, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 numbered in SEQ ID NO: 2 or a position corresponding thereto.

153. The base editor system of any one of claims 143 to 152, wherein the guide polynucleotide comprises a nucleic acid sequence complementary to a LRRK2 gene or a regulatory component thereof comprising a SNP associated with Parkinson's disease.

154. A base editor system according to any one of claims 143 to 152, wherein the adenosine base editor forms a complex with a single guide RNA (sgRNA), wherein the single guide RNA (sgRNA) comprises a nucleic acid sequence complementary to the LRRK2 gene or its regulatory component comprising a SNP associated with Parkinson's disease.

155. The base editor system of claim 154, wherein the sgRNA comprises the nucleic acid sequence: 5'-AAGCGCAAGCCUGGAGGGAA-3'; or 5'-ACUACAGCAUUGCUCAGUAC-3'.

156. The base editor system of claim 142, wherein the target gene is an α-L-iduronidase (IDUA) gene and the neurological disease is Hurler's disease.

157. A base editor system comprising (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 or a position corresponding thereto in SEQ ID NO: 2, And wherein the guide polynucleotide directs the adenosine base editor to effect an A to G nucleobase change in an α-L-iduronidase (IDUA) gene or a regulatory component thereof.

158. The base editor system of claim 156 or 157, wherein the IDUA gene or its regulatory component comprises a SNP associated with Hurler's disease.

159. The base editor system of any one of claims 156 to 158, wherein the A to G nucleobase change is at a SNP associated with Hurler's disease.

160. The base editor system of claim 158 or 159, wherein the SNP associated with Hurler's disease results in a W402X or W401X amino acid mutation in the IDUA polypeptide numbered in SEQ ID NO:4 or a variant thereof encoded by the IDUA gene, wherein X is a stop codon.

161. The base editor system of any one of claims 158 to 160, wherein the A to G nucleobase change changes a SNP associated with Hurler's disease to a wild-type nucleobase.

162. The base editor system of any one of claims 158 to 160, wherein the A to G nucleobase change changes the SNP associated with Hurler's disease to a non-wild-type nucleobase that results in one or more improved symptoms of Hurler's disease.

163. The base editor system of any one of claims 158 to 162, wherein the A to G change at the SNP associated with Hurler's disease changes the stop codon in the IDUA polypeptide encoded by the IDUA gene to tryptophan.

164. The base editor system of any one of claims 158 to 162, wherein the guide polynucleotide comprises a nucleic acid sequence complementary to an IDUA gene or a regulatory component thereof comprising a SNP associated with Hurler's disease.

165. The base editor system of any one of claims 158 to 164, wherein the adenosine base editor forms a complex with a single guide RNA (sgRNA), wherein the single guide RNA (sgRNA) comprises a nucleic acid sequence complementary to the IDUA gene or its regulatory component comprising a SNP associated with Hurler's disease.

166. The base editor system of claim 165, wherein the sgRNA comprises a nucleic acid sequence selected from the group consisting of 5'-GACUCUAGGCAGAGGUCUCAA-3', 5'-ACUCUAGGCAGAGGUCUCAA-3', 5'-CUCUAGGCCGAAGUGUCGC-3', and 5'-GCUCUAGGCCGAAGUGUCGC-3'.

167. The base editor system of claim 142, wherein the target gene is the methyl CpG binding protein 2 (MECP2) gene and the neurological disease is Rett's disease.

168. A base editor system comprising (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 or a corresponding position thereof in SEQ ID NO: 2, And wherein the guide polynucleotide directs the adenosine base editor to effect an A to G nucleobase change in the methyl CpG binding protein 2 (MECP2) gene or a regulatory component thereof.

169. A base editor system according to claim 167 or 168, wherein the MECP2 gene or its regulatory component includes a SNP associated with Rett syndrome.

170. The base editor system of claim 169, wherein the A to G nucleobase change is at the SNP associated with Rett syndrome.

171. The base editor system of claim 169 or 170, wherein the SNP associated with Rett syndrome causes an R106W or T158M amino acid mutation in the MECP2 polypeptide numbered in SEQ ID NO:5 encoded by the MECP2 gene or a variant thereof.

172. The base editor system of claim 169 or 170, wherein the SNP associated with Rett's disease results in an R255X or R270X amino acid mutation in the MECP2 polypeptide encoded by the MECP2 gene, wherein X is a stop codon.

173. The base editor system of any one of claims 169 to 172, wherein the A to G nucleobase change changes the SNP associated with Rett's disease to a wild-type nucleobase.

174. The base editor system of any one of claims 169 to 172, wherein the A to G nucleobase change changes the SNP associated with Rett's disease to a non-wild-type nucleobase, which results in one or more improved symptoms of Rett's disease.

175. The base editor system of any one of claims 169 to 172, wherein the A to G nucleobase change at the SNP associated with Rett's disease changes the stop codon in the MECP2 polypeptide to tryptophan.

176. The base editor system of any one of claims 169 to 175, wherein the guide polynucleotide comprises a nucleic acid sequence complementary to a MECP2 gene or a regulatory component thereof comprising a SNP associated with the Rett syndrome.

177. A base editor system according to any one of claims 169 to 175, wherein the adenosine base editor forms a complex with a single guide RNA (sgRNA), and the single guide RNA (sgRNA) comprises a nucleic acid sequence complementary to the MECP2 gene or its regulatory component including the SNP associated with the Rett disease.

178. The base editor system of claim 177, wherein the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of 5'-CUUUUCACUUCCUGCCGGGG-3', 5'-AGCUUCCAUGUCCAGCCUUC-3', 5'-ACCAUGAAGUCAAAAUCAUU-3', and 5'-GCUUUCAGCCCCGUUUCUUG-3'.

179. The base editor system of claim 142, wherein the target gene is the ATP-binding cassette subfamily member 4 (ABCA4) gene, and the neurological disease is Stargardt's disease.

180. A base editor system comprising contacting (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 or a corresponding position thereof in SEQ ID NO: 2, And wherein the guide polynucleotide directs the adenosine base editor to effect an A to G nucleobase change in the ATP-binding cassette subfamily member 4 (ABCA4) gene or a regulatory component thereof.

181. A base editor system according to claim 179 or 180, wherein the administration improves at least one symptom associated with Stargardt's disease.

182. The base editor system of claim 181, wherein said administration results in faster improvement of at least one symptom associated with Stargardt's disease compared to treatment with a base editor without said amino acid substitution in said adenosine deaminase.

183. A base editor system according to any one of claims 179 to 182, wherein the ABCA4 gene includes a SNP associated with Stargardt's disease.

184. The base editor system of claim 183, wherein the A to G nucleobase change is at a SNP associated with the Stargardt's disease.

185. A base editor system according to claim 183 or 184, wherein the SNP associated with Stargardt's disease causes an A1038V or G1961E amino acid mutation in the ABCA4 polypeptide numbered in SEQ ID NO: 6 or a variant thereof encoded by the ABCA4 gene.

186. A base editor system according to claim 185, wherein the SNP associated with Stargardt's disease causes a G1961E amino acid mutation in the ABCA4 polypeptide numbered in SEQ ID NO: 6 or a variant thereof.

187. A base editor system according to any one of claims 184 to 186, wherein the A to G nucleobase change changes the SNP associated with Stargardt's disease to a wild-type nucleobase.

188. A base editor system according to any one of claims 184 to 186, wherein the A to G nucleobase change changes the SNP associated with Stargardt's disease to a non-wild-type nucleobase, which results in improved Stargardt's disease symptoms.

189. A base editor system according to any one of claims 184 to 188, wherein the guide polynucleotide comprises a nucleic acid sequence complementary to the ABCA4 gene or its regulatory components comprising a SNP associated with Stargardt's disease.

190. A base editor system according to any one of claims 184 to 189, wherein the adenosine base editor forms a complex with a single guide RNA (sgRNA), wherein the single guide RNA (sgRNA) comprises a nucleic acid sequence complementary to the ABCA4 gene or its regulatory components including a SNP associated with Stargardt's disease.

191. The base editor system of claim 190, wherein the sgRNA comprises the sequence 5'-CUCCAGGGCGAACUUCGACACACAGC-3'.

192. The base editor system of any one of claims 142 to 191, wherein the polynucleotide programmable DNA binding domain is Cas9.

193. The base editor system of claim 192, wherein the Cas9 is SpCas9, SaCas9 or a variant thereof.

194. A base editor system according to claim 192 or 193, wherein the polynucleotide programmable DNA binding domain comprises a modified SpCas9 with an altered pre-spacer adjacent motif (PAM) specificity.

195. A base editor system according to claim 194, wherein the Cas9 is specific for a PAM sequence selected from the group consisting of NGG, NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN and NGC, wherein N is A, G, C or T, and wherein R is A or G.

196. A base editor system according to any one of claims 192 to 195, wherein the polynucleotide programmable DNA binding domain is a nuclease inactive variant.

197. A base editor system according to any one of claims 192 to 195, wherein the polynucleotide programmable DNA binding domain is a nickase variant.

198. The base editor system of claim 197, wherein the nickase variant comprises the amino acid substitution D10A or its corresponding amino acid substitution.

199. The base editor system of any one of claims 142 to 198, wherein the adenosine deaminase domain comprises a TadA domain.

200. The base editor system of claim 199, wherein the adenosine deaminase comprises a TadA deaminase comprising a V82S change and / or a T166R change.

201. The base editor system of any one of claims 142 to 200, wherein the adenosine deaminase further comprises one or more of the following changes: Y147T, Y147R, Q154S, Y123H, Q154R, or a combination thereof.

202. The base editor system of any one of claims 142 to 201, wherein the adenosine deaminase comprises a combination of alterations selected from the group consisting of: Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y147T+Q154R; Y147T+Q154S; and Y123H+Y147R+Q154R+I76Y.

203. The base editor system of any one of claims 142 to 202, wherein the adenosine base editor domain comprises an adenosine deaminase monomer.

204. The base editor system of any one of claims 142 to 203, wherein the adenosine base editor comprises an adenosine deaminase dimer.

205. The base editor system of any one of claims 199 to 204, wherein the TadA deaminase is a TadA*8 variant.

206. The base editor system of claim 205, wherein the TadA*8 variant is selected from the group consisting of TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, and TadA*8.

13.

207. The base editor system of claim 206, wherein the adenosine base editor is an ABE8 base editor selected from the group consisting of ABE8.1, ABE8.2, ABE8.3, ABE8.4, ABE8.5, ABE8.6, ABE8.7, ABE8.8, ABE8.9, ABE8.10, ABE8.11, ABE8.12, and ABE8.

13.

208. A vector comprising a nucleic acid sequence encoding the adenosine base editor of any one of claims 142 to 207.

209. A vector comprising a nucleic acid sequence encoding an adenosine base editor and a guide polynucleotide according to any one of claims 142 to 207.

210. The vector of claim 208 or 209, wherein the vector is a viral vector, a lentiviral vector or an AAV vector.

211. A cell comprising a base editor system according to any one of claims 142 to 207 or a vector according to any one of claims 208 to 210.

212. The cell of claim 211, wherein the cell is a central nervous system cell.

213. The cell of claim 211, wherein the cell is a neuron.

214. The cell of claim 211, wherein the cell is a photoreceptor.

215. The cell of any one of claims 211 to 214, wherein the cell is in vitro, in vivo or ex vivo.

216. A pharmaceutical composition comprising a base editor according to any one of claims 142 to 207, a vector according to any one of claims 208 to 210, or a cell according to any one of claims 211 to 215, and a pharmaceutically acceptable carrier.

217. A pharmaceutical composition according to claim 216, further comprising a lipid.

218. A pharmaceutical composition according to claim 216, further comprising a virus.

219. A kit comprising a base editor according to any one of claims 142 to 207 or a vector according to any one of claims 208 to 210.

220. The method of any one of claims 1 to 139, wherein at least one nucleotide of the guide polynucleotide comprises a non-naturally occurring modification.

221. The method of any one of claims 14, 31, 45, 58, 74, 85, 97 or 110, wherein at least one nucleotide of the nucleic acid sequence comprises a non-naturally occurring modification.

222. The base editor system of any one of claims 155, 166, 178 or 191, wherein at least one nucleotide of the nucleic acid sequence comprises a non-naturally occurring modification.

223. A base editor system according to claim 222, wherein the non-naturally occurring modification is a chemical modification.

224. A base editor system according to claim 223, wherein the chemical modification is 2'-O-methylation.

225. The base editor system of claim 223, wherein the nucleic acid sequence comprises a phosphorothioate.

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