Deaminase for base editing and variants thereof

Abstract

The present application provides deaminases for base editing and variants thereof, nucleobase editors or fusion proteins comprising the deaminases, their encoding polynucleotides or expression vectors, and methods of producing modifications in target nucleobase sequences using such editors. The invention also provides a mutant Cas9.

Description

Technical Field

[0001] The present application relates to deaminases for use with, for example, nucleic acid-guided nucleases to modify target nucleic acid sequences. More specifically, the present application relates to adenosine deaminases and cytidine deaminases with high base editing efficiency. Background Art

[0002] Targeted editing of nucleic acid sequences, such as targeted cutting or targeted modification of genomic DNA, is an effective 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 (e.g., CBE4) that convert target C•G base pairs to T•A base pairs, and adenosine base editors (e.g., ABE7.10) that convert A•T base pairs to G•C base pairs. The art needs improved base editors that can induce modifications within target sequences with greater specificity and efficiency. Summary of the invention

[0003] In a first aspect, the present application provides an adenosine deaminase comprising:

[0004] (i) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 1-47 (e.g., SEQ ID NOs: 2-47); or

[0005] (ii) one or more alterations selected from the group consisting of position D12X, H21X, A61X, V95X, S155X, S156X, C159X, E164X, E167X, K169X relative to SEQ ID NO: 1, wherein X is any amino acid, preferably, one or more substitutions selected from the group consisting of D12H, H21D or H21Q, A61C, V95T, S155R, S156N or S156K, C159R, E164V or E164W, E167R or E167W, K169N relative to SEQ ID NO: 1, wherein the amino acid positions are numbered relative to SEQ ID NO: 1.

[0006] In some embodiments, the adenosine deaminase comprises:

[0007] (i) an amino acid having a sequence identity of 80% to 99.5% (e.g., 85% to 99.5%, 90% to 99.5%, or 95% to 99.5%) to SEQ ID NO: 1, and

[0008] (ii) one or more alterations selected from the group consisting of position D12X, H21X, A61X, V95X, S155X, S156X, C159X, E164X, E167X, K169X relative to SEQ ID NO: 1, wherein X is any amino acid, preferably, one or more substitutions selected from the group consisting of D12H, H21D or H21Q, A61C, V95T, S155R, S156N or S156K, C159R, E164V or E164W, E167R or E167W, K169N relative to SEQ ID NO: 1, wherein the amino acid positions are numbered relative to SEQ ID NO: 1.

[0009] In some embodiments, the adenosine deaminase is capable of deaminating adenosine in DNA. In some embodiments, the adenosine deaminase is capable of deaminating cytosine in DNA, but with low efficiency.

[0010] In some embodiments, the adenosine deaminase comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1-47 (eg, SEQ ID NOs: 2-47).

[0011] In some embodiments, the adenosine deaminase comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1-19 (eg, SEQ ID NOs: 2-19).

[0012] In some embodiments, the adenosine deaminase comprises, or consists of, the amino acid sequence of any one of SEQ ID NOs: 20-47.

[0013] In a second aspect, the present application provides a cytidine deaminase comprising:

[0014] (i) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 48-63 or 64-99 (e.g., SEQ ID NOs: 48-63 or 65-99); or

[0015] (ii) one or more changes selected from the group consisting of positions A17X, G64X, Y70X, L73X, V79X, F81X, M91X, H93X, G102X, D112X, A119X, N124X, R130X, S139X, A140X, C143X, R151X, K153X relative to SEQ ID NO: 64, wherein X is any amino acid, preferably selected from the group consisting of positions A17X, G64X, Y70X, L73X, V79X, F81X, M91X, H93X, G102X, D112X, A119X, N124X, R130X, S139X, A140X, C143X, R151X, K153X relative to SEQ ID NO: one or more substitutions in the group consisting of A17N, G64C, Y70G or Y70H or Y70K, L73F, V79T, F81L, M91L, H93N, G102S, D112N, A119G, N124V or N124P, R130V, S139K, A140R, C143A, R151M, K153G of SEQ ID NO: 64, wherein the amino acid positions are numbered relative to SEQ ID NO: 64.

[0016] In some embodiments, the cytidine deaminase comprises:

[0017] (i) an amino acid having a sequence identity of 80% to 99.5% (e.g., 85% to 99.5%, 90% to 99.5%, or 95% to 99.5%) to SEQ ID NO: 64, and

[0018] (ii) one or more changes selected from the group consisting of positions A17X, G64X, Y70X, L73X, V79X, F81X, M91X, H93X, G102X, D112X, A119X, N124X, R130X, S139X, A140X, C143X, R151X, K153X relative to SEQ ID NO: 64, wherein X is any amino acid, preferably selected from the group consisting of positions A17X, G64X, Y70X, L73X, V79X, F81X, M91X, H93X, G102X, D112X, A119X, N124X, R130X, S139X, A140X, C143X, R151X, K153X relative to SEQ ID NO: one or more substitutions in the group consisting of A17N, G64C, Y70G or Y70H or Y70K, L73F, V79T, F81L, M91L, H93N, G102S, D112N, A119G, N124V or N124P, R130V, S139K, A140R, C143A, R151M, K153G of SEQ ID NO: 64, wherein the amino acid positions are numbered relative to SEQ ID NO: 64.

[0019] In some embodiments, the cytidine deaminase is capable of deaminating cytidine in DNA. In some embodiments, the cytidine deaminase is capable of deaminating adenosine in DNA, but with lower efficiency.

[0020] In some embodiments, the cytidine deaminase comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 48-63 or 64-99 (e.g., SEQ ID NOs: 48-63 or 65-99).

[0021] In a third aspect, the present application provides a nucleobase editor, comprising:

[0022] (i) a polynucleotide programmable DNA binding domain; and

[0023] (ii) a deaminase, wherein the deaminase is the adenosine deaminase described in the first aspect or the cytidine deaminase described in the second aspect.

[0024] In some embodiments, the nucleobase editor further comprises one or more uracil glycosylase inhibitor (UGI) domains.

[0025] In some embodiments, the nucleobase editor is an adenosine base editor (ABE), wherein the deaminase is an adenosine deaminase. In a preferred embodiment, the adenosine deaminase comprises:

[0026] (i) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 1-47 (e.g., SEQ ID NOs: 2-47); or

[0027] (ii) one or more alterations selected from the group consisting of position D12X, H21X, A61X, V95X, S155X, S156X, C159X, E164X, E167X, K169X relative to SEQ ID NO: 1, wherein X is any amino acid, preferably, one or more substitutions selected from the group consisting of D12H, H21D or H21Q, A61C, V95T, S155R, S156N or S156K, C159R, E164V or E164W, E167R or E167W, K169N relative to SEQ ID NO: 1, wherein the amino acid positions are numbered relative to SEQ ID NO: 1.

[0028] In some embodiments, the nucleobase editor is a cytidine base editor (CBE). In a preferred embodiment, the cytidine deaminase comprises: The cytidine deaminase comprises:

[0029] (i) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 48-63 or 64-99 (e.g., SEQ ID NOs: 48-63 or 65-99); or

[0030] (ii) one or more changes selected from the group consisting of positions A17X, G64X, Y70X, L73X, V79X, F81X, M91X, H93X, G102X, D112X, A119X, N124X, R130X, S139X, A140X, C143X, R151X, K153X relative to SEQ ID NO: 64, wherein X is any amino acid, preferably selected from the group consisting of positions A17X, G64X, Y70X, L73X, V79X, F81X, M91X, H93X, G102X, D112X, A119X, N124X, R130X, S139X, A140X, C143X, R151X, K153X relative to SEQ ID NO: one or more substitutions in the group consisting of A17N, G64C, Y70G or Y70H or Y70K, L73F, V79T, F81L, M91L, H93N, G102S, D112N, A119G, N124V or N124P, R130V, S139K, A140R, C143A, R151M, K153G of SEQ ID NO: 64, wherein the amino acid positions are numbered relative to SEQ ID NO: 64.

[0031] In some embodiments, the polynucleotide programmable DNA binding domain comprises a domain selected from the group consisting of a Cas9 domain, a Cas12 domain, a TnpB domain, an IscB domain, a homing nuclease (meganuclease) domain, a zinc finger DNA binding domain, and a transcription activator-like effector (TALE) DNA binding domain. In some embodiments, the polynucleotide programmable DNA binding domain comprises a Cas9 domain. In some embodiments, the Cas9 domain is Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), Streptococcus pyogenes Cas9 (SpCas9) or a variant thereof. In some embodiments, the polynucleotide programmable DNA binding domain is an inactive nuclease or nickase variant.

[0032] In some embodiments, the polynucleotide programmable DNA binding domain is a Cas9 domain, preferably a Cas9 comprising a mutation of one or two amino acid substitutions selected from D700R or E1243R, more preferably a Cas9 comprising D700R or a Cas9 comprising E1243R. In a preferred embodiment, the mutant Cas9 comprises an amino acid sequence of SEQ ID NO: 115 or 116, or consists of an amino acid sequence of SEQ ID NO: 115 or 116.

[0033] In some embodiments, the base editor comprises a nucleic acid-guided nuclease protein and an embedded nucleotide base editor (NBE) domain. In some embodiments, the embedded NBE domain is an adenosine base editor (ABE) domain. In some embodiments, the embedded ABE domain is an embedded adenosine deaminase protein domain. In some embodiments, the polynucleotide programmable DNA binding domain comprises a linker flanking at least one embedded domain protein. In some embodiments, the embedded NBE domain is a cytidine base editor (CBE) domain. In some embodiments, the embedded CBE domain is an embedded cytidine deaminase protein domain.

[0034] In some embodiments, the polynucleotide programmable DNA binding domain is associated with the deaminase, for example, directly connected together or connected together through a linker. Generally, a person skilled in the art will be able to select a suitable linker. In one embodiment, the linker comprises an amino acid sequence of any one of SEQ ID NOs: 104-107, or consists of an amino acid sequence of any one of SEQ ID NOs: 104-107. Preferably, the linker is SEQ ID NO: 106.

[0035] In some embodiments, the nucleobase editor can be in the form of a kit.

[0036] In a fourth aspect, the present application provides a fusion protein comprising a polynucleotide programmable DNA binding domain and at least one nucleobase editor domain comprising a deaminase linked together. The polynucleotide programmable DNA binding domain and at least one nucleobase editor domain comprising a deaminase can be directly linked or linked via a linker.

[0037] Generally, those skilled in the art can select suitable joints. In one embodiment, the joint comprises an amino acid sequence of any one of SEQ ID NO: 104-107, or is composed of an amino acid sequence of any one of SEQ ID NO: 104-107. Preferably, the joint is SEQ ID NO: 106.

[0038] In some embodiments, the polynucleotide programmable DNA binding domain is a Cas9 domain, preferably a mutant Cas9 comprising one or more amino acid mutations (e.g., amino acid substitutions), for example, a mutant Cas9 comprising one or two amino acid substitutions selected from D700R or E1243R, more preferably a Cas9 comprising D700R or a Cas9 comprising E1243R. In a preferred embodiment, the mutant Cas9 comprises an amino acid sequence of SEQ ID NO: 115 or 116, or consists of an amino acid sequence of SEQ ID NO: 115 or 116.

[0039] In some embodiments, the deaminase is an adenosine deaminase or a cytidine deaminase described herein.

[0040] In a fifth aspect, the present application provides a polynucleotide encoding an adenosine deaminase, the adenosine deaminase comprising:

[0041] (i) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 1-47 (e.g., SEQ ID NOs: 2-47); or

[0042] (ii) one or more alterations selected from the group consisting of position D12X, H21X, A61X, V95X, S155X, S156X, C159X, E164X, E167X, K169X relative to SEQ ID NO: 1, wherein X is any amino acid, preferably, one or more substitutions selected from the group consisting of D12H, H21D or H21Q, A61C, V95T, S155R, S156N or S156K, C159R, E164V or E164W, E167R or E167W, K169N relative to SEQ ID NO: 1, wherein the amino acid positions are numbered relative to SEQ ID NO: 1.

[0043] In some embodiments, the adenosine deaminase comprises:

[0044] (i) an amino acid having a sequence identity of 80% to 99.5% (e.g., 85% to 99.5%, 90% to 99.5%, or 95% to 99.5%) to SEQ ID NO: 1, and

[0045] (ii) one or more alterations selected from the group consisting of position D12X, H21X, A61X, V95X, S155X, S156X, C159X, E164X, E167X, K169X relative to SEQ ID NO: 1, wherein X is any amino acid, preferably, one or more substitutions selected from the group consisting of D12H, H21D or H21Q, A61C, V95T, S155R, S156N or S156K, C159R, E164V or E164W, E167R or E167W, K169N relative to SEQ ID NO: 1, wherein the amino acid positions are numbered relative to SEQ ID NO: 1.

[0046] In some embodiments, the polynucleotide may be codon optimized for better expression in a host cell.

[0047] In a sixth aspect, the present application provides a polynucleotide encoding a cytidine deaminase, the cytidine deaminase comprising:

[0048] (i) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 48-63 or 64-99 (e.g., SEQ ID NOs: 48-63 or 65-99); or

[0049] (ii) one or more changes selected from the group consisting of positions A17X, G64X, Y70X, L73X, V79X, F81X, M91X, H93X, G102X, D112X, A119X, N124X, R130X, S139X, A140X, C143X, R151X, K153X relative to SEQ ID NO: 64, wherein X is any amino acid, preferably selected from the group consisting of positions A17X, G64X, Y70X, L73X, V79X, F81X, M91X, H93X, G102X, D112X, A119X, N124X, R130X, S139X, A140X, C143X, R151X, K153X relative to SEQ ID NO: one or more substitutions in the group consisting of A17N, G64C, Y70G or Y70H or Y70K, L73F, V79T, F81L, M91L, H93N, G102S, D112N, A119G, N124V or N124P, R130V, S139K, A140R, C143A, R151M, K153G of SEQ ID NO: 64, wherein the amino acid positions are numbered relative to SEQ ID NO: 64.

[0050] In some embodiments, the cytidine deaminase comprises:

[0051] (i) an amino acid having a sequence identity of 80% to 99.5% (e.g., 85% to 99.5%, 90% to 99.5%, or 95% to 99.5%) to SEQ ID NO: 64, and

[0052] (ii) one or more changes selected from the group consisting of positions A17X, G64X, Y70X, L73X, V79X, F81X, M91X, H93X, G102X, D112X, A119X, N124X, R130X, S139X, A140X, C143X, R151X, K153X relative to SEQ ID NO: 64, wherein X is any amino acid, preferably selected from the group consisting of positions A17X, G64X, Y70X, L73X, V79X, F81X, M91X, H93X, G102X, D112X, A119X, N124X, R130X, S139X, A140X, C143X, R151X, K153X relative to SEQ ID NO: one or more substitutions in the group consisting of A17N, G64C, Y70G or Y70H or Y70K, L73F, V79T, F81L, M91L, H93N, G102S, D112N, A119G, N124V or N124P, R130V, S139K, A140R, C143A, R151M, K153G of SEQ ID NO: 64, wherein the amino acid positions are numbered relative to SEQ ID NO: 64.

[0053] In some embodiments, the polynucleotide may be codon optimized for better expression in a host cell.

[0054] In a seventh aspect, the present application provides an expression vector comprising a polynucleotide encoding an adenosine deaminase of the present invention. In some embodiments, the expression vector is a viral vector selected from the group consisting of adeno-associated virus (AAV), retroviral vector, adenoviral vector, lentiviral vector, Sendai virus vector and herpes virus vector.

[0055] In an eighth aspect, the present application provides an expression vector comprising a polynucleotide encoding a cytidine deaminase of the present invention. In some embodiments, the expression vector is a viral vector selected from the group consisting of adeno-associated virus (AAV), retroviral vector, adenoviral vector, lentiviral vector, Sendai virus vector and herpes virus vector.

[0056] In the ninth aspect, the present application provides a polynucleotide encoding a nucleobase editor, the nucleobase editor comprising: (i) a polynucleotide programmable DNA binding domain; and (ii) a deaminase, wherein the deaminase is the adenosine deaminase described in the first aspect or the cytidine deaminase described in the second aspect.

[0057] In some embodiments, the deaminase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 1-47, SEQ ID NOs: 48-63 or 64-99 (e.g., SEQ ID NOs: 2-47, SEQ ID NOs: 48-63 or 65-99).

[0058] In some embodiments, the deaminase is an adenosine deaminase, and the adenosine deaminase comprises:

[0059] (i) an amino acid having a sequence identity of 80% to 99.5% (e.g., 85% to 99.5%, 90% to 99.5%, or 95% to 99.5%) to SEQ ID NO: 1, and

[0060] (ii) one or more alterations selected from the group consisting of position D12X, H21X, A61X, V95X, S155X, S156X, C159X, E164X, E167X, K169X relative to SEQ ID NO: 1, wherein X is any amino acid, preferably, one or more substitutions selected from the group consisting of D12H, H21D or H21Q, A61C, V95T, S155R, S156N or S156K, C159R, E164V or E164W, E167R or E167W, K169N relative to SEQ ID NO: 1, wherein the amino acid positions are numbered relative to SEQ ID NO: 1.

[0061] In some embodiments, the deaminase is a cytidine deaminase, and the cytidine deaminase comprises:

[0062] (i) an amino acid having a sequence identity of 80% to 99.5% (e.g., 85% to 99.5%, 90% to 99.5%, or 95% to 99.5%) to SEQ ID NO: 64, and

[0063] (ii) one or more changes selected from the group consisting of positions A17X, G64X, Y70X, L73X, V79X, F81X, M91X, H93X, G102X, D112X, A119X, N124X, R130X, S139X, A140X, C143X, R151X, K153X relative to SEQ ID NO: 64, wherein X is any amino acid, preferably selected from the group consisting of positions A17X, G64X, Y70X, L73X, V79X, F81X, M91X, H93X, G102X, D112X, A119X, N124X, R130X, S139X, A140X, C143X, R151X, K153X relative to SEQ ID NO: one or more substitutions in the group consisting of A17N, G64C, Y70G or Y70H or Y70K, L73F, V79T, F81L, M91L, H93N, G102S, D112N, A119G, N124V or N124P, R130V, S139K, A140R, C143A, R151M, K153G of SEQ ID NO: 64, wherein the amino acid positions are numbered relative to SEQ ID NO: 64.

[0064] In some embodiments, the polynucleotide may be codon-optimized for better expression in a host cell. In the tenth aspect, the present application provides an expression vector comprising a polynucleotide encoding a nucleobase editor according to the present invention. In some embodiments, the vector is a viral vector selected from the group consisting of adeno-associated virus (AAV), retroviral vector, adenoviral vector, lentiviral vector, Sendai virus vector, and herpes virus vector.

[0065] In the eleventh aspect, the present application provides a polynucleotide encoding the fusion protein of the present invention and an expression vector comprising the polynucleotide.

[0066] In some embodiments, the polynucleotide may be codon optimized for better expression in a host cell.

[0067] Those skilled in the art will readily understand that the expression vector of the present invention may contain transcriptional regulatory elements such as promoters and enhancers.

[0068] In the twelfth aspect, the present application provides a cell comprising the expression vector of the present invention.

[0069] In some embodiments, the cell can be any suitable type of cell, for example, a prokaryotic cell or a eukaryotic cell, including, but not limited to, a bacterial cell, a fungal cell, a plant cell, an insect cell, or a mammalian cell.

[0070] In the thirteenth aspect, the present application provides a base editing method, comprising contacting a polynucleotide sequence with a nuclear base editor, wherein the nuclear base editor comprises: (i) a polynucleotide programmable DNA binding domain; and (ii) a deaminase, wherein the deaminase is the adenosine deaminase described in the first aspect or the cytidine deaminase described in the second aspect, wherein the deaminase deaminates the nuclear bases in the polynucleotide, thereby editing the polynucleotide sequence.

[0071] In some embodiments, the deaminase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-47, SEQ ID NOs: 48-63, or 64-99 (e.g., SEQ ID NOs: 2-47, SEQ ID NOs: 48-63, or 65-99).

[0072] In a fourteenth aspect, the present application provides a method for correcting a genetic defect in a subject, the method comprising administering a nucleobase editor or a polynucleotide encoding a nucleobase editor to the subject, the nucleobase editor comprising: (i) a polynucleotide programmable DNA binding domain; and (ii) a deaminase, wherein the deaminase is the adenosine deaminase described in the first aspect or the cytidine deaminase described in the second aspect, to deaminate a target nucleobase in the target nucleotide sequence of the subject, thereby correcting the genetic defect.

[0073] In some embodiments, the deaminase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-47, SEQ ID NOs: 48-63, or 64-99 (e.g., SEQ ID NOs: 2-47, SEQ ID NOs: 48-63, or 65-99).

[0074] In some embodiments, the method comprises delivering the nucleobase editor or a polynucleotide encoding the nucleobase editor, and one or more guide polynucleotides to a cell of the subject. In some embodiments, the subject is a mammal, including a non-human mammal or a human. In some embodiments, deamination of the target nucleobase causes the target nucleobase to be replaced by a wild-type nucleobase.

[0075] In a fifteenth aspect, the present invention provides medical uses of the adenosine deaminase or cytidine deaminase, nucleobase editor or fusion protein of the present invention or their encoding polynucleotides.

[0076] In some embodiments, the present application provides the use of the adenosine deaminase or cytidine deaminase described in the present invention or their encoding polynucleotides in the preparation of a drug or a kit for base editing or correcting a genetic defect in a subject.

[0077] In some embodiments, the present application provides use of the fusion protein or its encoding polynucleotide described in the present invention in the preparation of a drug or a kit, wherein the drug or kit is used for base editing or correcting a genetic defect in a subject.

[0078] In some embodiments, the present application provides the use of the nucleobase editor described in the present invention or the polynucleotide encoding the nucleobase editor in the preparation of a drug or a kit for base editing or correcting a genetic defect in a subject.

[0079] In the sixteenth aspect, the present application provides a molecular complex comprising the base editor described in the present invention and one or more guide RNA sequences, tracrRNA sequences, or target DNA sequences.

[0080] In the seventeenth aspect, the present application provides a molecular complex comprising the fusion protein described in the present invention and one or more guide RNA sequences, tracrRNA sequences, or target DNA sequences.

[0081] In the eighteenth aspect, the present application provides a pharmaceutical composition comprising the base editor or fusion protein described in the present invention, or an expression vector comprising a polynucleotide encoding the base editor or fusion protein, or a molecular complex, and a pharmaceutically acceptable excipient.

[0082] In the nineteenth aspect, the present application provides a kit comprising: the base editor or fusion protein described in the present invention, or an expression vector comprising a polynucleotide encoding the base editor or fusion protein, or a molecular complex, and instructions for use.

[0083] The pharmaceutical composition or kit of the present invention can be used for base editing or correcting genetic defects in a subject.

[0084] In the twentieth aspect, the present application provides a mutated Cas9 comprising one or two amino acid substitutions selected from D700R or E1243R.

[0085] In some embodiments, the mutant Cas comprises D700R, in other embodiments, the mutant Cas comprises E1243R. In a preferred embodiment, the mutant Cas9 comprises the amino acid sequence of SEQ ID NO: 115 or 116, or consists of the amino acid sequence of SEQ ID NO: 115 or 116.

[0086] In the present invention, the terms "mutated Cas" and "Cas variant" are used interchangeably.

[0087] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification, including definitions, shall prevail.

[0088] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 The A to G editing efficiency of the different TadA variants validated in Example 1 in the sgRNA library cell lines is shown.

[0090] Figure 2 Shown is the fold change in the percentage of mutations in surviving colonies before and after selection (16 or 64 µg / ml chloramphenicol) of E. coli cells transformed with the cytidine deaminase variant library in Example 7.

[0091] Figure 3 The percentage of mutations identified in Example 7 by next generation sequencing (NGS) in surviving colonies on selective plates for the engineered CBE variants of the present invention are shown.

[0092] Figure 4 The C to T editing efficiencies of the variants T88.74-V10, T88.74-V14, T88.74-V16, and T88.74-V17 tested in Example 8 are shown relative to T88.74-V0.

[0093] Figure 5 Shown is the fold change in the percentage of mutations in surviving colonies before and after selection (64 or 128 µg / ml chloramphenicol) of E. coli cells transformed with the cytidine deaminase variant library in Example 8.

[0094] Figure 6 The C to T editing efficiencies of variants T88.74-V21 and T88.74-V22 tested in Example 8 relative to variant T88.74-V14 are shown.

[0095] Figure 7The C to T editing efficiencies of variants T88.74-V27, T88.74-V29, T88.74-V32, and T88.74-V33 tested in Example 8 are shown relative to variant T88.74-V17. DETAILED DESCRIPTION

[0096] Base editors

[0097] Disclosed herein are nuclear base editors, such as adenosine base editors (ABEs) or cytidine base editors (CBEs), for editing, modifying, or changing a target nucleotide sequence of a polynucleotide. Nuclear base editors are described herein, comprising a programmable nucleotide binding domain (e.g., a polynucleotide programmable nucleotide binding domain (e.g., Cas9) or a zinc finger protein DNA binding domain or a TALE DNA binding domain) and at least one nuclear base editing domain (e.g., adenosine deaminase or cytidine deaminase). When a polynucleotide programmable nucleotide binding domain (e.g., Cas9 or Cas12) is present in a cell and is bound to a bound guide polynucleotide (e.g., gRNA), it can specifically bind to a target polynucleotide sequence (i.e., by complementary base pairing between the bases of the bound guide nucleic acid and the bases of the target polynucleotide sequence) and thereby position the base editor to the target nucleic acid sequence desired to be edited.

[0098] In some embodiments, base editing activity is assessed by editing efficiency. Base editing can be determined by any suitable method, such as by Sanger sequencing or next generation sequencing. In some embodiments, base editing efficiency is measured by the total sequencing read percentage of the nuclear base conversion achieved by the base editor, for example, for adenosine deaminase, the total sequencing read percentage of the target AT base pair converted to the GC base pair is measured; for cytidine deaminase, the total sequencing read percentage of the target CG base pair converted to the TA base pair is measured. In some embodiments, when base editing is performed in a cell population, the base editing efficiency is measured by the total cell percentage of the nuclear base conversion achieved by the base editor.

[0099] The term "base editor system" refers to the components required to edit the nucleobases of a target nucleotide sequence. In various embodiments, the base editor system comprises: (1) a polynucleotide programmable nucleotide binding domain (e.g., Cas9); (2) a deaminase domain for deaminating the nucleobase (e.g., adenosine deaminase and / or cytidine deaminase; see PCT / US2019 / 044935, PCT / US2020 / 016288, the entire contents of each of which are incorporated herein by reference); and (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 adenosine base editor (ABE). In other embodiments, the base editor is a cytidine base editor (CBE).

[0100] 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 or one or more cytidine 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 pair of guide polynucleotides may be used to target different deaminases to a target nucleic acid sequence.

[0101] The deaminase domain and the polynucleotide programmable nucleotide binding component of the base editor system may be covalently or non-covalently associated with each other, or with any combination of their association and interaction. 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 association with the deaminase domain. For example, in some embodiments, the deaminase domain may include additional heterologous portions or domains that are capable of interacting, associating, or forming a complex with additional heterologous portions or domains that are part of the polynucleotide programmable nucleotide binding domain.

[0102] In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may 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.

[0103] The base editor system may also include a guide polynucleotide component. It should be understood that the components of the base editor system may be associated with each other by covalent bonds, non-covalent interactions, or any combination of their associations and interactions. In some embodiments, the deaminase domain may target the target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the deaminase domain may include an additional heterologous portion or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) that can interact, associate, or form a complex with a portion or segment of the guide polynucleotide (e.g., a polynucleotide motif). In some embodiments, the additional heterologous portion or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) may be fused or connected to the deaminase domain. In some embodiments, the additional heterologous portion may be able to bind, interact, associate, or form a complex with a polypeptide. In some embodiments, the additional heterologous portion may be able to bind, interact, associate, 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 may be capable of binding to a polynucleotide linker. The additional heterologous moiety may be a protein domain. In some embodiments, the additional heterologous moiety may 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.

[0104] Guide polynucleotide

[0105] In some embodiments, the guide polynucleotide is a guide RNA. The RNA / Cas complex can help "guide" the Cas protein to the target DNA. Cas9 / crRNA / tracrRNA cleaves linear or circular dsDNA targets complementary to the spacer in an endonuclease manner. The target strand that is not complementary to the crRNA is first cut in an endonuclease manner, and then trimmed 3'-5' in an exonuclease manner. In nature, DNA binding and cleavage generally require proteins and two RNAs. However, a single guide RNA ("sgRNA" or simply "gRNA") can be engineered to integrate various aspects of both 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 (PAM or protospacer adjacent motifs) in CRISPR repeat sequences to help distinguish self from non-self.

[0106] 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 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 clear to those skilled in the art based on this application, 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 this reference are incorporated herein by reference). In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase. 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 protospacer adjacent motif (PAM) sequence to guide a polynucleotide programmable DNA binding domain (e.g., Cas9 or Cpf1) to a target nucleotide sequence. The polynucleotide programmable nucleotide binding domains (e.g., CRISPR-derived domains) of the base editors disclosed herein can recognize a target polynucleotide sequence by associating with a guide polynucleotide.A guide polynucleotide (e.g., a gRNA) is typically single-stranded and can be programmed to site-specifically bind (i.e., by complementary base pairing) to a target sequence of a polynucleotide, thereby directing a base editor 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 comprises natural nucleotides (e.g., adenosine). In some embodiments, the guide polynucleotide comprises non-natural (or unnatural) nucleotides (e.g., peptide nucleic acids or nucleotide analogs). In some embodiments, the length of 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. The length of the targeting region of the guide nucleic acid can be 10-30 nucleotides, or 15-25 nucleotides, or 15-20 nucleotides.

[0107] Programmable nucleotide binding domain

[0108] The programmable nucleotide binding domain of the base editor may itself comprise one or more domains. For example, a polynucleotide programmable nucleotide binding domain may comprise one or more nuclease domains. In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain may comprise an endonuclease or an exonuclease. The term "exonuclease" herein refers to a protein or polypeptide capable of digesting a nucleic acid (e.g., RNA or DNA) from a free end, and the term "endonuclease" refers to a protein or polypeptide capable of catalyzing (e.g., cleaving) an internal region in a nucleic acid (e.g., DNA or RNA). In some embodiments, an endonuclease can cleave a single strand in a double-stranded nucleic acid.

[0109] Any DNA destabilizing molecule can be used in any combination in the compositions described herein, including but not limited to Cas9 or Cas12 nickases, Cas9 or Cas12 proteins operably linked to a single guide RNA (sgRNA) (e.g., dCas), any RNA programmable system, zinc finger nuclease nickases (ZFN nickases), TALEN nickases, and / or one or more nucleotides (e.g., one or more peptide nucleic acids (PNAs), locked nucleic acids (LNAs), and / or bridge nucleic acids (BNAs)). In certain embodiments, the base editing composition comprises more than one DNA destabilizing molecule, such as one or more proteins (e.g., nickases, etc.) and / or one or more nucleotides. In certain embodiments, the composition comprises a ZFN nickase and one or more additional proteins and / or nucleotide DNA destabilizing molecules (e.g., one or more nucleotides as described herein). In certain aspects, the base editing composition does not comprise a Cas9 protein, but may comprise other Cas proteins (e.g., non-Cas9 RNA programmable systems). In certain embodiments, the DNA destabilizing molecule comprises a zinc finger nuclease (ZFN) nickase.

[0110] In some embodiments, the nuclease is a zinc finger nuclease (ZFN) or a TALE DNA binding domain-nuclease fusion (TALEN). ZFNs and TALENs contain a DNA binding domain (zinc finger protein or TALE DNA binding domain) and a cleavage domain or cleavage half-domain that has been engineered to bind to a target site in a selected gene.

[0111] At least one zinc finger protein (ZFP) DNA binding domain of the base editing composition can be operably connected to one or more other components of the base editing composition, for example, connected to one or more DNA destabilizing molecules (e.g., connected to Cas9 nickase, dCas9, etc.) and / or connected to at least one adenine or cytosine deaminase. In certain embodiments, at least one ZFP DNA binding domain is operably connected to an adenine or cytosine deaminase. In other embodiments, the base editing composition comprises a first and a second ZFP DNA binding domain, wherein the first ZFP DNA binding domain is operably connected to a Cas9 nickase. The ZFP DNA binding domain may comprise 3, 4, 5, 6 or more fingers and may bind to a target site on either side (5' or 3') of the targeted base to be edited. In certain embodiments, the target site bound by the ZFP is located at 1 to 100 (or any number between) nucleotides on either side of the targeted base. In other embodiments, the target site to which the ZFP binds is located 1 to 50 (or any number in between) nucleotides on either side of the targeted base.

[0112] In some embodiments, the endonuclease can cleave both strands of a double-stranded nucleic acid molecule. In some embodiments, the polynucleotide programmable nucleotide binding domain can be a deoxyribonuclease. In some embodiments, the polynucleotide programmable nucleotide binding domain can be a ribonuclease.

[0113] 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. The term "nickase" herein refers to a polynucleotide programmable nucleotide binding domain, which includes a nuclease domain that can cleave only one of the two chains in a double-stranded nucleic acid molecule (e.g., DNA). In some embodiments, the nickase can be derived from the fully catalytically active (e.g., natural) form of the polynucleotide programmable nucleotide binding domain by introducing one or more mutations into the active polynucleotide programmable nucleotide binding domain. For example, when the polynucleotide programmable nucleotide binding domain includes a nickase domain derived from Cas9, the Cas9-derived nickase domain may include a D10A mutation and a histidine at position 840. In such an embodiment, residue H840 remains catalytically active, thereby cleaving a single strand of a nucleic acid duplex. In another embodiment, the Cas9-derived nickase domain 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 that is not required for nickase activity. For example, when the polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can comprise a deletion of all or part of a RuvC domain or a HNH domain.

[0114] Thus, a base editor comprising a polynucleotide programmable nucleotide binding domain (comprising a nickase domain) is capable of producing 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 double-stranded target polynucleotide sequence of the nucleic acid cleaved by the base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain) is a chain that is not edited by the base editor (i.e., the chain cleaved by the base editor is opposite to the chain containing the base to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain) can cleave the chain of a DNA molecule that is targeted for editing. In such an embodiment, the chain that is not targeted is not cleaved.

[0115] Also provided herein is a base editor comprising a catalytically inactive (i.e., incapable of cleaving a target polynucleotide sequence) polynucleotide programmable nucleotide binding domain. Herein, the terms "catalytically inactive" and "nuclease inactive" are used interchangeably to refer to a polynucleotide programmable nucleotide binding domain having one or more mutations and / or deletions, which results in its inability to cleave a nucleic acid chain while retaining its ability and specificity to bind to a target polynucleotide. In some embodiments, a catalytically inactive 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 of a base editor comprising a Cas9 domain, Cas9 may simultaneously comprise a D10A mutation and an H840A mutation. Such mutations inactivate two nuclease domains, resulting in a loss of nuclease activity. In other embodiments, a catalytically inactive polynucleotide programmable nucleotide binding domain may comprise one or more deletions of all or part of a catalytic domain (e.g., a RuvCl and / or HNH domain). In further embodiments, the catalytically inactive polynucleotide programmable nucleotide binding domain comprises a point mutation (eg, D10A or H840A) and a deletion of all or part of the nuclease domain.

[0116] Some aspects of the present application provide fusion proteins comprising a domain acting as a polynucleotide 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 comprises a nucleic acid programmable DNA binding protein domain and one or more deaminase domains. Non-limiting examples of polynucleotide programmable DNA binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpf1, 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, Cas sl2h, Casl2i, Csyl, Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm 4. Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csxl0, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csxl 1, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, type II Cas effector protein, type V Cas effector protein, type VI Cas effector protein, CARF, DinG, their homologs, or their modified or engineered forms. Other nucleic acid programmable DNA binding proteins are also within the scope of the present application, although they may not be specifically listed in the present application.For example, see Makarova et al. "Classification and Nomenclature of CRISPR-Cas Systems: Wherefrom 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. l 126 / science.aav7271, the entire contents of each reference are incorporated herein by reference.

[0117] In some embodiments, the polynucleotide programmable DNA binding domain is a Cas9 domain, preferably a Cas9 comprising a mutation of one or two amino acid substitutions selected from D700R or E1243R, more preferably a Cas9 comprising D700R or a Cas9 comprising E1243R. In a preferred embodiment, the mutant Cas9 comprises an amino acid sequence of SEQ ID NO: 115 or 116, or consists of an amino acid sequence of SEQ ID NO: 115 or 116.

[0118] In some embodiments, the present application provides a fusion protein comprising a V-type CRISPR / Cas effector protein. The V-type CRISPR / Cas effector protein is a subtype of a 2-type CRISPR / Cas effector protein. For examples of V-type CRISPR / Cas systems and effector proteins thereof (e.g., Cas12 family proteins, such as Cas12a), see, for example, Shmakov et al., Nat Rev Microbial. 2017 March; 15(3):169-182: "Diversity and evolution of class 2 CRISPR-Cas systems.". Examples include, but are not limited to: Cas12 family (Cas12a, Cas12b, Cas12c), C2c4, C2c8, C2c5, C2c10, and C2c9; and CasX (Cas12e) and CasY (Cas12d). See also, e.g., Koonin et al., Curr Opin Microbial. 2017 June; 37:67-78: "Diversity, classification and evolution of CRISPR-Cas systems." In some embodiments, a CBE disclosed herein comprises a type V CRISPR / Cas effector protein.

[0119] In some embodiments, the present application provides a TALE base editor, which may comprise a TALE domain, a deaminase domain and / or a cofactor protein (e.g., a FokI endonuclease) domain, wherein the TALE base editor comprises a fusion protein having the general structure NH2-[TALE]-[deaminase domain]-COOH, NH2-[deaminase domain]-[TALE]-COOH, NH2-[TALE]-[deaminase domain]-[cofactor protein]-COOH, NH2-[cofactor protein]-[deaminase domain]-[TALE]-COOH, NH2-[cofactor protein]-[TALE]-[deaminase]-COOH, or NH2-[deaminase domain]-[TALE]-[cofactor protein]-COOH; wherein each instance of "]-[" includes an optional linker, such as a peptide linker.

[0120] In some embodiments, the disclosed methods involve transducing (e.g., by transfection) cells with multiple complexes, each complex comprising a fusion protein and a cofactor protein, the fusion protein comprising a TAL effector domain and a deaminase domain, wherein each cofactor protein localizes the fusion protein to a different target sequence. Reference Yang L. et al., Engineering and optimizing deaminase fusions for genome editing, Nature Comms., 2016. In specific embodiments, the methods disclosed herein involve TAL effector domains that bind to target sites not by Watson-Crick hybridization but by binding to the major groove of the DNA double helix. In certain embodiments, the method involves transfecting a nucleic acid construct (e.g., a plasmid), each nucleic acid construct (or together) encoding a component of a plurality of complexes of a TALE base editor and a cofactor protein, the TALE base editor comprising a TALE domain and a deaminase domain. In certain embodiments, the disclosed fusion protein comprises a cofactor protein domain, i.e., the domain is integrated into the fusion protein construct. In other embodiments, the TALE base editor comprises a TALE domain and a deaminase domain, and the cofactor protein is introduced into the cell separately from the base editor.

[0121] In certain embodiments of the disclosed methods, constructs encoding TALE base editors are transfected into cells separately from constructs encoding cofactor proteins. In certain embodiments, these components are encoded on a single construct and transfected together. In specific embodiments, these single constructs encoding TALE base editors and cofactor proteins can be iteratively transfected into cells, with each iteration associated with a subset of target sequences. In specific embodiments, these single constructs can be transfected into cells within a few days. In other embodiments, they can be transfected into cells within weeks.

[0122] A to GEdit

[0123] In some embodiments, the base editor described herein may include a deaminase domain, which includes an adenosine deaminase. Such an adenosine deaminase domain of a base editor can facilitate the editing of an adenine (A) nucleobase to a guanine (G) nucleobase by deaminating adenosine or adenine (A) to form inosine (I), which exhibits base pairing properties similar to G, i.e., inosine can pair with cytosine, thereby introducing guanine at the adenine site deaminated by adenosine deaminase during subsequent transcription. Adenosine deaminase is capable of deaminating (i.e., removing an amine group) adenine of a deoxyadenosine residue in deoxyribonucleic acid (DNA).

[0124] In some embodiments, the nucleobase editors provided herein can be prepared by fusing one or more protein domains together to produce a fusion protein. In certain embodiments, the fusion protein provided herein comprises one or more features that improve the base editing activity (e.g., efficiency, selectivity, and specificity) of the fusion protein. For example, the fusion protein provided herein may include a Cas9 domain with reduced nuclease activity. In some embodiments, the fusion protein provided herein may have a Cas9 domain (dCas9) that does not have nuclease activity, or a Cas9 domain (referred to as Cas9 nickase (nCas9)) that cuts one strand of a double-stranded DNA molecule. Without wishing to be bound by any particular theory, the presence of a catalytic residue (e.g., H840) maintains the activity of Cas9 to cut an unedited (e.g., undeaminated) strand containing a T opposite to the targeted A. Mutations of the catalytic residues of Cas9 (e.g., D10 to A10) prevent cleavage of the editing strand containing the targeted A residue. Such Cas9 variants are capable of generating single-stranded DNA breaks (nicks) at specific locations based on the target sequence defined by the gRNA, resulting in repair of the unedited strand, ultimately resulting in a T to C change on the unedited strand. In some embodiments, the A to G base editor also comprises an inhibitor of inosine base excision repair, such as a uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine-specific nuclease. Without wishing to be bound by any particular theory, the UGI domain or the catalytically inactive inosine-specific nuclease can inhibit or prevent base excision repair of deaminated adenosine residues (e.g., inosine), which can improve the activity or efficiency of the base editor.

[0125] Base editors comprising adenosine deaminase can act on any polynucleotide, including DNA, RNA, and DNA-RNA hybrids. In certain embodiments, base editors comprising adenosine deaminase can deaminate target A of polynucleotides (including RNA). For example, a base editor may include an adenosine deaminase domain capable of deaminating target A of RNA polynucleotides and / or DNA-RNA hybrid polynucleotides. In one embodiment, the adenosine deaminase integrated into the base editor includes all or part of an adenosine deaminase (ADAR, e.g., ADAR1 or ADAR2) acting on RNA. In another embodiment, the adenosine deaminase integrated into the base editor includes all or part of an adenosine deaminase (ADAT) acting on tRNA. Base editors comprising adenosine deaminase domains can also deaminate the A core base of DNA polynucleotides. In one embodiment, the adenosine deaminase domain of the base editor includes all or part of ADAT, which includes one or more mutations that allow ADAT to deaminate target A in DNA.

[0126] Adenosine deaminase can be derived from any suitable organism (e.g., E. coli). In some embodiments, adenosine deaminase is a naturally occurring adenosine deaminase that includes one or more mutations corresponding to the amino acid site mutations provided herein (e.g., mutations occurring in any one of SEQ ID NO: 1-47 (e.g., SEQ ID NO: 2-47)). The corresponding residues in any homologous protein can be identified by, for example, sequence alignment and determination of homologous residues. Mutations in any naturally occurring adenosine deaminase corresponding to any one of the mutations described herein can be generated accordingly.

[0127] Adenosine deaminase

[0128] Some aspects of the present application provide adenosine deaminase. In some embodiments, the adenosine deaminase provided herein can deaminate adenine. In some embodiments, the adenosine deaminase provided herein can deaminate adenine in the deoxyadenosine residues of DNA. As mentioned herein, the term "adenosine deaminase" refers to a deaminase that can deaminate adenine in the deoxyadenosine residues of DNA, and in some cases, the adenosine deaminase can also deaminate cytosine in the deoxycytidine residues of DNA. In some embodiments, the adenosine deaminase provided herein can deaminate cytosine. In some embodiments, the adenosine deaminase provided herein can deaminate cytosine in the deoxycytidine residues of DNA, but with lower efficiency. Adenosine deaminase can be derived from any suitable organism (e.g., Escherichia coli). In some embodiments, adenine deaminase is a naturally occurring adenosine deaminase comprising one or more mutations corresponding to the amino acid site mutations provided herein. Those skilled in the art will be able to identify any homologous proteins and corresponding residues in the respective encoding nucleic acids by methods well known in the art (e.g., by sequence alignment and determination of homologous residues). Thus, those skilled in the art will be able to generate mutations corresponding to any of the mutations described herein in any naturally occurring adenosine deaminase. In some embodiments, the adenosine deaminase is from a prokaryotic organism. In some embodiments, the adenosine deaminase is from bacteria. In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is from Escherichia coli.

[0129] In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is a TadA variant.

[0130] In some embodiments, the adenosine deaminase 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 100% identical to any of the amino acid sequences shown in any of SEQ ID NOs: 1-47 (e.g., SEQ ID NOs: 2-47), or to any of the adenosine deaminase enzymes provided herein. It should be understood that the adenosine deaminase enzymes provided herein may comprise one or more mutations (e.g., any of the mutations provided herein). The present application provides any deaminase domain having a certain percentage of identity plus any mutation described herein or a combination thereof. In some embodiments, the adenosine deaminase comprises an amino acid sequence having 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 shown in SEQ ID NOs: 1-47 or any one of the adenosine deaminase provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence having at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical consecutive amino acid residues compared to any one of the amino acid sequences shown in SEQ ID NOs: 1-47, or any adenosine deaminase provided herein.

[0131] In some embodiments, the deaminases provided herein are capable of deaminating adenine.In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine in a deoxyadenosine residue of DNA.

[0132] In some embodiments, the deaminases provided herein are capable of deaminating cytosine or 5-methylcytosine to uracil or thymine. In some embodiments, the deaminases provided herein are capable of deaminating cytosine in DNA.

[0133] In some embodiments, the adenosine deaminase comprises one or more amino acid substitutions selected from the group consisting of D12H, H21D or H21Q, A61C, V95T, S156K, C159R, E164V or E164W, E167W numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having an amino acid sequence of any one of SEQ ID NOs: 1-47 (e.g., SEQ ID NOs: 2-47). In one embodiment, the adenosine deaminase consists of an amino acid sequence of any one of SEQ ID NOs: 1-47 (e.g., SEQ ID NOs: 2-47).

[0134] In some embodiments, the adenosine deaminase comprises a V95T amino acid substitution numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having the amino acid sequence of SEQ ID NO: 2.

[0135] In some embodiments, the adenosine deaminase comprises V95T and A61C amino acid substitutions numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having the amino acid sequence of SEQ ID NO: 3.

[0136] In some embodiments, the adenosine deaminase comprises V95T and C159R amino acid substitutions numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having the amino acid sequence of SEQ ID NO: 4.

[0137] In some embodiments, the adenosine deaminase comprises V95T and D12H amino acid substitutions numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having the amino acid sequence of SEQ ID NO: 5.

[0138] In some embodiments, the adenosine deaminase comprises V95T and E164V amino acid substitutions numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having the amino acid sequence of SEQ ID NO: 6.

[0139] In some embodiments, the adenosine deaminase comprises V95T and E164W amino acid substitutions numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having the amino acid sequence of SEQ ID NO: 7.

[0140] In some embodiments, the adenosine deaminase comprises V95T and E167R amino acid substitutions numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having the amino acid sequence of SEQ ID NO: 8.

[0141] In some embodiments, the adenosine deaminase comprises V95T and E167W amino acid substitutions numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having the amino acid sequence of SEQ ID NO: 9.

[0142] In some embodiments, the adenosine deaminase comprises V95T, E164V, and E167W amino acid substitutions numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having the amino acid sequence of SEQ ID NO: 10.

[0143] In some embodiments, the adenosine deaminase comprises V95T and H21D amino acid substitutions numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having the amino acid sequence of SEQ ID NO: 11.

[0144] In some embodiments, the adenosine deaminase comprises an amino acid substitution selected from V95T, C159R, E164V, and E167W numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having an amino acid sequence of SEQ ID NO: 12.

[0145] In some embodiments, the adenosine deaminase comprises A61C, E164V, and E167W amino acid substitutions numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having the amino acid sequence of SEQ ID NO: 13.

[0146] In some embodiments, the adenosine deaminase comprises C159R, E164V, and E167W amino acid substitutions numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having the amino acid sequence of SEQ ID NO: 14.

[0147] In some embodiments, the adenosine deaminase comprises C159R and E167W amino acid substitutions numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having the amino acid sequence of SEQ ID NO: 15.

[0148] In some embodiments, the adenosine deaminase comprises H21Q and A61C amino acid substitutions numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having an amino acid sequence of SEQ ID NO: 16.

[0149] In some embodiments, the adenosine deaminase comprises S155R and K169N amino acid substitutions numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having the amino acid sequence of SEQ ID NO: 17.

[0150] In some embodiments, the adenosine deaminase comprises one or more amino acid substitutions from the group consisting of S156N and E164V numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having an amino acid sequence of SEQ ID NO: 18.

[0151] In some embodiments, the adenosine deaminase comprises one or more amino acid substitutions from the group consisting of V95T, S156K, C159R, E164V, and E167W numbered relative to SEQ ID NO: 1. In one embodiment, the adenosine deaminase is an adenosine deaminase having the amino acid sequence of SEQ ID NO: 19.

[0152] It should be recognized that any mutation provided herein can be introduced into other adenosine deaminases, such as Staphylococcus aureus TadA (saTadA), or other adenosine deaminases (e.g., bacterial adenosine deaminases). It will be clear to one skilled in the art how to identify amino acid residues homologous to the mutated residues in SEQ ID NOs: 1-47 from other adenosine deaminases. Thus, any mutation in SEQ ID NOs: 1-47 can be made in other adenosine deaminases. It should also be understood that any mutation provided herein can be made in another adenosine deaminase alone or in any combination.

[0153] Table A. Amino acid sequence of adenosine deaminase

[0154] Name SEQ ID NO Amino acid sequence T8.4-V0 1 MYNAPRFSTGVDALSETELNHEYWMRHALNLAQRAREEGEVPVGAVLVYQDKVIGEGWNRAIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSSLLCDFYREPREQKNALKRASQDPRSSNA T8.4-V1 2 MYNAPRFSTGVDALSETELNHEYWMRHALNLAQRAREEGEVPVGAVLVYQDKVIGEGWNRAIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSSLLCDFYREPREQKNALKRASQDPRSSNA T8.4-V2 3 MYNAPRFSTGVDALSETELNHEYWMRHALNLAQRAREEGEVPVGAVLVYQDKVIGEGWNRCIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSSLLCDFYREPREQKNALKRASQDPRSSNA T8.4-V3 4 MYNAPRFSTGVDALSETELNHEYWMRHALNLAQRAREEGEVPVGAVLVYQDKVIGEGWNRAIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSSLLRDFYREPREQKNALKRASQDPRSSNA T8.4-V4 5 MYNAPRFSTGVHALSETELNHEYWMRHALNLAQRAREEGEVPVGAVLVYQDKVIGEGWNRAIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSSLLCDFYREPREQKNALKRASQDPRSSNA T8.4-V5 6 MYNAPRFSTGVDALSETELNHEYWMRHANLLAQRAREEGEVPVGAVLVYQDKVIGEGWNRAIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSSLCDFYRVPREQKNALKRASQDPRSNA T8.4-V6 7 MYNAPRFSTGVDALSETELNHEYWMRHANLLAQRAREEGEVPVGAVLVYQDKVIGEGWNRAIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSSLCDFYRWPREQKNALKRASQDPRSNA T8.4-V7 8 MYNAPRFSTGVDALSETELNHEYWMRHANLLAQRAREEGEVPVGAVLVYQDKVIGEGWNRAIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSSLCDFYREPRRQKNALKRASQDPRSSNA T8.4-V8 9 MYNAPRFSTGVDALSETELNHEYWMRHANLLAQRAREEGEVPVGAVLVYQDKVIGEGWNRAIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSSLCDFYREPRWQKNALKRASQDPRSNA T8.4-V9 10 MYNAPRFSTGVDALSETELNHEYWMRHANLLAQRAREEGEVPVGAVLVYQDKVIGEGWNRAIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSSLCDFYRVPRWQKNALKRASQDPRSNA T8.4-V10 11 MYNAPRFSTGVDALSETELNDEYWMRHALNLAQRAREEGEVPVGAVLVYQDKVIGEGWNRAIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSSLLCDFYREPREQKNALKRASQDPRSSNA T8.4-V11 12 MYNAPRFSTGVDALSETELNHEYWMRHALNLAQRAREEGEVPVGAVLVYQDKVIGEGWNRAIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSSLLRDFYRVPRWQKNALKRASQDPRSSNA T8.4-V12 13 MYNAPRFSTGVDALSETELNHEYWMRHANLLAQRAREEGEVPVGAVLVYQDKVIGEGWNRCIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSSLCDFYRVPRWQKNALKRASQDPRSNA T8.4-V13 14 MYNAPRFSTGVDALSETELNHEYWMRHALNLAQRAREEGEVPVGAVLVYQDKVIGEGWNRAIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSSLLRDFYRVPRWQKNALKRASQDPRSSNA T8.4-V14 15 MYNAPRFSTGVDALSETELNHEYWMRHALNLAQRAREEGEVPVGAVLVYQDKVIGEGWNRAIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSSLLRDFYREPRWQKNALKRASQDPRSSNA T8.4-V15 16 MYNAPRFSTGVDALSETELNQEYWMRHANLLAQRAREEGEVPVGAVLVYQDKVIGEGWNRCIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSSLCDFYREPREQKNALKRASQDPRSNA T8.4-V16 17 MYNAPRFSTGVDALSETELNHEYWMRHALNLAQRAREEGEVPVGAVLVYQDKVIGEGWNRAIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCRSLCDFYREPREQNNALKRASQDPRSSNA T8.4-V17 18 MYNAPRFSTGVDALSETELNHEYWMRHANLLAQRAREEGEVPVGAVLVYQDKVIGEGWNRAIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSNLCDFYRVPREQKNALKRASQDPRSSNA T8.4-V18 19 MYNAPRFSTGVDALSETELNHEYWMRHALNLAQRAREEGEVPVGAVLVYQDKVIGEGWNRAIGLHDPTAHAEIMALRQGGMVLQNYRLIDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLINVLNYPGMNHRVEVTEGVLAESCSKLLRDFYRVPRWQKNALKRASQDPRSSNA A-DN-1 20 SGTDEDWMARVLELAEEARAAREVPVGAILVLNNELVGEGWNRAIILNDPTAHAEIIALNEGKKKLKSYRLIGATLYVNFEPCVMCANAMIHARIGKVVYGVRNSKRGAAGSTQNILNYPGMNHKVKVVSGVLAEEVGKLLCDFYRMPRQVFNAQKKAQSSIN A-DN-2 21 SGTREDWMERVIELARKAREAREPVPVGAILVLNNELVGEGWNRAIVLNDPTAHAEIIALEEGKKLKSYRLIGATLYVNFEPCVMCALAMIHARIGEVVYGVRNSKRGAAGSVRNILNYPGMNHKVKVVSGVLAEEVGRLLCDFYRMPRQVFNAQKKAQSSIN A-DN-3 22 PGTLEDWMRRALELARKAREREPVPVGAILVLDGKLIGEGWNRAIVLNDPTAHAEIIALEKGKEVTKSYRLIGATLYVNFEPCVMCALAMIHARIGRVVYGVRNSKRGAAGSLMNVLNYPGMNHRVEIVEGVLADEVGKLLCDFYRMPRQVFNAQKKAQSSIN A-DN-4 23 AGTDEDWMARALALAEQARAAREVPVGAILVLNDELVGEGWNRAIILNDPTAHAEIIALEEGKKKTNSYRLIGATLYVNFEPCVMCALAMIHARIGKVVYGVRNSKRGAAGSLMNVLNYPGMNHRVEVVSGVLAEEVGKLLCDFYRMPRQVFNAQKKAQSSIN A-DN-5 24 AGTEEDWMRRALALAEQARAAREVPVGAILVLDGELVGEGWNRAIVLHDPTAHAEIIALREAGKKLQNYRLIGATLYVNFEPCVMCAGAMIHSRIGKVVYGVRNSKRGAAGSLMNVLNYPGMNHRVEVVSGVLAEEVGRLLCDFYRMPRQVFNAQKKAQSSIN A-DN-6 25 AGTEEDWMRRALALAEQARQAREVPVGAILVLDGELVGEGWNRAIVLHDPTAHAEIIALREAGRRTQNYRLIGATLYVNFEPCVMCAGAMIHSRIGKVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEVVEGVLADEVGKLLCDFYRMPRQVFNAQKKAQSSIN A-DN-7 26 SGTDEDWMRRALELAEQAREAREVPVGAVLVLDGELVGEGWNRAIVLHDPTAHAEIIALRQGGERLQNYRLIGATLYVTFEPCVMCAGAMIHSRIGRVVYGVRNSKRGAAGSLMNVLNYPGMNHRVEVVSGVLAKEVGKLLCDFYRMPRQVFNAQKKAQSSIN A-DN-8 27 MGTDEDWMERALELAKKAREAREVPVGAVLVLNDELIGEGWNRAIVLHDPTAHAEIIALREGGRKTQNYRLIGATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEVVSGVLAEEVGKLLCDFYRMPRQVFNAQKKAQSSIN A-DN-9 28 AGTDEDWMREAALAEKAREAREVPVGAVLVLDNEVIGEGWNRAIVLHDPTAHAEIIALRKGGERMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEIVEGILAEECGKLLCDFYRMPRQVFNAQKKAQSSIN A-DN-10 29 PGTDEYWMRRALELARRAREAREVPVGAVLVLNNKVIGEGWNRAIILHDPTAHAEIIALRKGGERMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEIVSGILAEECGRLLCDPYRMPRQVFNAQKKAQSSIN A-DN-11 30 SGTDEDWMRRALRLAEKAREAREVPVGAVLVLDNEVIGEGWNRAIVLHDPTAHAEIIALRKGGEGQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEIVSGILAEECGKLLCDFYRMPRQVFNAQKKAQSSIN A-DN-12 31 SGSDEYWMRLALELAEKAREAREVPVGAVLVLNNEVIGEGWNRAIVLHDPTAHAEIIALRQGGEKMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHKVEIVEGILAEECGKLLCDFYRMPRQVFNAQKKAQSSIN A-DN-13 32 MTDADWMARALELARKALEAGEVPVGAVLVLDGEVVGEGFNNAIRLNDPTAHAEIIALRKGGKKKKDYRLIDATLYVTFEPCVMCAGAMIHARIGKVVFGVRNAKTGAAGSLMDVLKHPGMNHQVEVVSGVLADECAELLCEFFRMPRRVFNAQKKAQSSTD A-DN-14 33 MSDADWMALALEEARKALAAGEVPVGAVLVLDGEVVGRGYNNAIRLNDPTAHAEIIAIRKGGKKKKDYRLIDATLYVTFEPCVMCAGAMIHARIKRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEVVSGVLADECAALLCEFFRMPRRVFNAQKKAQSSTD A-DN-15 34 MTHEDWMRMALEEARKAKEAREVPVGAVLVLNNEVIGRGYNSAIRLNDPTAHAEIIALRKGGKKMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLNHPGMNHKVEIVSGILADECAELLCDFFRMPRRVFNAQKKAQSSTD A-DN-16 35 MSHEDWMRLALEEARKALEAREVPVGAVLVLNNEVIGRGYNNAIKLNDPTAHAEIIALRKGGKTMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLRHPGMNHKVEIVSGILAEECAELLCKFFRMPRRVFNAQKKAQSSTD A-DN-17 36 MTDADWMRRALAEARKALAAGEVPVGAVLVLDGEVVGEGYNAAIRLHDPTAHAEIIALRKGGLTKQNYRLIDATLYVTFEPCVMCAGAMIHSRIARVVFGVRNAKTGAAGSLMDVLNHPGMNHKVEVVSGVLADEAAALLCDFFRMPRRVFNAQKKAQSSTD A-DN-18 37 MSDEDWMRLALEEARKALEAGEVPVGAVLVLDGEVVGRGYNSAIRLHDPTAHAEIIALRKGGLRKQNYRLIDAVLYVTFEPCVMCAGAMIHSRIKKVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEVVSGVLAEECAELLCEFFRMPRRVFNAQKKAQSSTD A-DN-19 38 MTHEDWMRLALAEAEKALADREVPVGAVLVLNNEVIGSGYNNAIILHDPTAHAEIIALRKGGLKMQNYRLIDATLYVTFEPCVMCAGAMIHSRIKRVVFGVRNAKTGAAGSLMDVLNHPGMNHKVEIVSGILADECAELLCRFFRMPRRVFNAQKKAQSSTD A-DN-20 39 MSHEDWMRLALEEARKALEAREVPVGAVLVLNNEVIGRGYNNAITLHDPTAHAEIIALRKGGLKMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLNHPGMNHKVEIVSGILADECAELLCEFFRMPRRVFNAQKKAQSSTD A-DN-21 40 MTDEDWMAEALKLAEKAASLREVPVGAVLVLNDEIIGKGYNAAITLSDTTAHAEINALREGGKKTKNYRLYDATLYSTFEPCVMCAGAMLHARIARYVYGVRNAKTGAAGSLMDVLNHPGMNHRVEVVSGVRAEECAELLCRFFRMPRSVFNAQKKAQSSTD A-DN-22 41 MTDEDWMAIALEEAKKAAELREVPVGAVLVLNDEIIGTGYNAAIVLNDTTAHAEINALRDGGKKLKDYRLYDATLYSTFEPCVMCAGAMLHARIKRVVYGVRNAKTGAAGSLMDVLNHPGMNHRVEVVRGVRADEAAELLCRFFRMPRSVFNAQKKAQSSTD A-DN-23 42 MTHEDWMALALELAKKAAANREVPVGAVLVLDNRVIGKGYNAAITLNDPTAHAEINALRDGGKVMQNYRLYDATLYSTFEPCVMCAGAMLHSRIKRVVFGVRNAKTGAAGSLMDVLNHPGMNHRVEIVEGILADECAELLCRFFRMPRTVFNAQKKAQSSTD A-DN-24 43 MTHEDWMRIALELAKIAEEENREVPVGAVLVLNNEVIGKGYNAAITLNDPTAHAEINALRDGGKKMQNYRLYDATLYSTFEPCVMCAGAMLHSRIKRVVFGVRNAKTGAAGSLMDVLNHPGMNHRVEIVEGILAEECEAELLCRFFRMPRSFNAQKKAQSSTD A-DN-25 44 MTDEDWMALALELAEKAAALREVPVGAVLVLDDEVIGRGYNAAIVLHDPTAHAEINALRDGGLRLQNYRLYDATLYSTFEPCVMCAGAMIHSRIKRYVYGVRNAKTGAAGSLMDVLNHPGMNHRVEVVRGVRAEECAELLCRFFRMPRSVFNAQKKAQSSTD A-DN-26 45 MTDEDWMARALELAEKAAAAREVPVGAVLVLDDEIIGEGYNAAITLHDPTAHAEILALRQGGLKLQNYRLYDATLYSTFEPCVMCAGAMIHSRIKRYVYGVRNAKTGAAGSLMDVLNHPGMNHRVEVVRGVRAEECEAELLCRFFRMPRSFFNAQKKAQSSTD A-DN-27 46 MTHEDWMRIALELAEKALAAREVPVGAVLVLNNEVIGKGWNAAITLHDPTAHAEINALRHGGLVMQNYRLYDATLYSTFEPCVMCAGAMIHSRIKRVVFGVRNAKTGAAGSLMDVLNHPGMNHRVEIVEGILAEECAELLCRFFRMPRSVFNAQKKAQSSTD A-DN-28 47 MTHEDWMRIALELAEKAAAAREVPVGAVLVLNNEVIGKGWNRAITLHDPTAHAEINALRDGGLKMQNYRLYDATLYSTFEPCVMCAGAMIHSRIKRVVFGVRNAKTGAAGSLMDVLNHPGMNHRVEIVEGILAEECAELLCRFFRMPRSVFNAQKKAQSSTD

[0155] Cytidine deaminase and C to T (U) editing

[0156] "Cytidine deaminase" means a polypeptide or fragment thereof that can catalyze a deamination reaction that converts an amino group into a carbonyl group. In one embodiment, the cytidine deaminase converts cytosine into uracil, or converts 5-methylcytosine into thymine. The cytidine deaminase provided herein (e.g., an engineered cytidine deaminase) can be from any organism, such as bacteria.

[0157] In some embodiments, the cytidine deaminase of the base editor may include all or part of the apolipoprotein B mRNA editing complex (APOBEC) family deaminase or their variants. APOBEC is a family of evolutionarily conserved cytidine deaminases. Members of this family are C-to-U editing enzymes. In some embodiments, the cytidine deaminase includes but is not limited to: an APOBEC family member, including but not limited to: APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (now called "APOBEC3E"), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, activation-induced (cytidine) deaminase (AID), hAPOBEC1 (derived from Homo sapiens), rAPOBEC1 (derived from Rattus norvegicus), ppAPOBEC1 (derived from Bornean orangutan (Pongo pygmaeus)), AmAPOBEC1 (BEM3.31) (derived from American alligator (Alligator mississippiensis)), ocAPOBEC1 (derived from Oryctolagus cuniculus), SsAPOBEC2 (BEM3.39) (derived from Eurasian wild boar (Sus scrofa)), hAPOBEC3A (derived from Homo sapiens), maAPOBEC1 (derived from golden hamster (Mesocricetus auratus)), mdAPOBEC1 (derived from Monodelphis domestica); cytidine deaminase 1 (CDA1), hA3A (i.e., APOBEC3A derived from Homo sapiens), RrA3F (BEM3.14) (which is APOBEC3F derived from Rhinopithecus roxellana); PmCDA1 (derived from sea lamprey (Petromyzon marinus)); AID (activation-induced cytidine deaminase; AICDA), which is derived from mammals (e.g., humans, pigs, cattle, horses, monkeys, etc.); hAID (derived from Homo sapiens); and FENRY.

[0158] The term "deaminase" or "deaminase domain", as used herein, refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase, which catalyzes the hydrolytic deamination of cytidine to uridine or deoxycytidine to deoxyuridine. In some embodiments, the deaminase or deaminase domain is a cytosine deaminase, which catalyzes the hydrolytic deamination of cytosine to uracil, thereby achieving editing from C:G to T:A (i.e., editing of C to T).

[0159] In some embodiments, the cytidine deaminase is a de novo designed TadA variant having cytidine deaminase activity. In some embodiments, the cytidine deaminase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 48-63.

[0160] In some embodiments, the cytidine deaminase is generated by directed evolution based on SEQ ID NO: 64. In some embodiments, the cytidine deaminase comprises one or more substitutions selected from the group consisting of A17N, G64C, Y70G or Y70H or Y70K, L73F, V79T, F81L, M91L, H93N, G102S, D112N, A119G, N124V or N124P, R130V, S139K, A140R, C143A, R151M, K153G relative to SEQ ID NO: 64, wherein amino acid positions are numbered relative to SEQ ID NO: 64. In some embodiments, the cytidine deaminase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 64-99.

[0161] In some embodiments, the cytidine deaminase comprises Y70H and M91L amino acid substitutions numbered relative to SEQ ID NO: 64. In one embodiment, the cytidine deaminase is a cytidine deaminase having the amino acid sequence of SEQ ID NO: 74.

[0162] In some embodiments, the cytidine deaminase comprises Y70H, M91L, and F81L amino acid substitutions numbered relative to SEQ ID NO: 64. In one embodiment, the cytidine deaminase is a cytidine deaminase having the amino acid sequence of SEQ ID NO: 78.

[0163] In some embodiments, the cytidine deaminase comprises Y70H, M91L, F81L, C143A, G64C, and L73F amino acid substitutions numbered relative to SEQ ID NO: 64. In one embodiment, the cytidine deaminase is a cytidine deaminase having the amino acid sequence of SEQ ID NO: 80.

[0164] In some embodiments, the cytidine deaminase comprises Y70H, M91L, F81L, G64C, and L73F amino acid substitutions numbered relative to SEQ ID NO: 64. In one embodiment, the cytidine deaminase is a cytidine deaminase having an amino acid sequence of SEQ ID NO: 81.

[0165] In some embodiments, the cytidine deaminase comprises Y70H, M91L, F81L, and N124V amino acid substitutions numbered relative to SEQ ID NO: 64. In one embodiment, the cytidine deaminase is a cytidine deaminase having an amino acid sequence of SEQ ID NO: 85.

[0166] In some embodiments, the cytidine deaminase comprises Y70H, M91L, F81L, G64C, L73F, and N124V amino acid substitutions numbered relative to SEQ ID NO: 64. In one embodiment, the cytidine deaminase is a cytidine deaminase having the amino acid sequence of SEQ ID NO:91.

[0167] In some embodiments, the cytidine deaminase comprises Y70H, M91L, F81L, G64C, L73F, R130V, A140R, D112N, and N124V amino acid substitutions numbered relative to SEQ ID NO: 64. In one embodiment, the cytidine deaminase is a cytidine deaminase having the amino acid sequence of SEQ ID NO: 97.

[0168] In some embodiments, the cytidine deaminase comprises the amino acid sequence of any one of SEQ ID NOs: 48-63 and SEQ ID NOs: 64-99, preferably, the cytidine deaminase consists of the amino acid sequence of any one of SEQ ID NOs: 48-63 and SEQ ID NOs: 64-99.

[0169] Table B-1. Amino acid sequences of de novo designed TadA variants with cytidine deaminase activity

[0170] Name SEQ ID NO Amino acid sequence C-DN-1 48 GMHSDEEWMAIALEEARKARDAGKAPVGAVLVLDDEIIGKGYNAAIDLNDPTAHAEIIAIREGGKKLKDYRLIDATLYVTFEPCVMCAGALLNARIARVVYGVRNSKRGAAGSLMNVLNYPGLNHRVEVRSGVLADEALALLCDFYRMPRQVFNAQKKAQSSIN C-DN-2 49 GMHSDEYWMDLALEEARKAREAGKAPVGAVLVLDDEVIGRGYNSAIDLNDPTAHAEIIALREGGKRLKDYRLIDATLYVTFEPCVMCAGALLNARIKRVVYGVRNSKRGAAGSLMNVLNYPGLNHRVEVVSGVKAEEALQLLCDFYRMPRQVFNAQKKAQSSIN C-DN-3 50 GMHSHEEWMKIALAEAEKARKERKAPVGAVLVLNNEVIGKGYNSAIELNDPTAHAEIIALREGGKKMQDYRLIDATLYVTFEPCVMCAGAMLNSRIKRVVFGVRNSKRGAAGSLMNVLNYPGLNHKVEIVEGILADECLELLCDFYRMPRQVFNAQKKAQSSIN C-DN-4 51 SSHSHEYWMEKALALARKAREERKAPVGAVLVLDNEVIGEGYNSAIDLNDPTAAHAEIIALREGGKKMQDYRLIDATLYVTFEPCVMCAGAMLNSRIKRVVFGVRNSKRGAAGSLMNVLNYPGLNHRVEIVSGILADECLGLCDFYRMPRQVFNAQKKAQSSIN C-DN-5 52 MTHSDEEWMRVALAEAEKAREAGKAPVGAVLVLNDEIIGRGYNSAIDHLHDPTAHAEIIALRQGGLYLQNYRLIDATLYVTFEPCVMCAGALINSRIRRRVVYGVRNSKRGAAGDLMNVLNYPGMNHRVEVRSGVLAEEALALLCEFYRMPRQVFNAQKKAQSSIN C-DN-6 53 HMHSDEHWMRLALEEARKAREEGKAPVGAVLVLDDEVIGRGYNNAISLHDPTAHAEIIALRQGGLRRQNYRLIDATLYVTFEPCVMCAGALINSRIKRVVYGVRNSKRGAAGGLMNVLNYPGMNHKVEVVSGVLAEEALALLCEFYRMPRQVFNAQKKAQSSIN C-DN-7 54 GSHSHEHWMRLALAEARKARDARKAPVGAVLVLNNEVIGRGYNSAIALHDPTAHAEIIALRQGGLKMQNYRLIDATLYVTFEPCVMCAGAMINSRIKRVVFGVRNSKRGAAGSLMNVLNYPGMNHKVEIVEGILADECLELLCEFYRMPRQVFNAQKKAQSSIN C-DN-8 55 DMHSHEYWMRLALEEARKAREERKAPVGAVLVLDNEVIGKGYNSAIKLHDPTAHAEIIALRQGGLKMQNYRLIDATLYVTFEPCVMCAGAMINSRIKRVVFGVRNSKRGAAGSLMNVLNYPGMNHKVEIVSGILADECLLDLCEFYRMPRQVFNAQKKAQSSIN C-DN-9 56 SSHSDEHWMAKALELAEKARAAGHVPVGAVLVLNDEIVGTGFNAAKTLNDPTAHAEIIAIRKGGKTLKDYRLWGATLYTTFEPCVMCAGAMIHARIKRVVFGVCNAKTHACMGMMDVLGLPGLNHRVEVVSGVLADEALDLLCRFFRMPRRVFNAQKKAQSSTD C-DN-10 57 ASHSDAHWMARALELAEKAREDGHVPVGAVLVLDDEVIGEGYNAAKTLNDPTAHAEILALRAGGKRLRDYRLWGATLYTTFEPCVMCAGAMIHARIKRVVFGVCNAKTHACMERMDVLGTPGLNHRVEVVRGVLADECLELLCRFFRMPRRVFNAQKKAQSSTD C-DN-11 58 GLHSDEEWMAIALREAEKARENRHVPVGAVLVLDNEVIGTGYNAAKTLNDPTAHAEILALRAGGKKMQDYRLWGATLYTTFEPCVMCAGAMIHSRIERVVFGVCNAKTHACMSRMDVLGLPGLNHKVEIVSGILADECLDLLCRFFRMPRRVFNAQKKAQSSTD C-DN-12 59 GAHSDEEWMAKALELAEKAREARHVPVGAVLVLDNEVIGTGFNAAKTLNDPTAHAEIIALRRGGQTMQDYRLWGATLYTTFEPCVMCAGAMIHSRIKRVVFGVCNAKTHACMSMMDVLGLPGLNHRVEIVAGILADECLDLLCRFFRMPRRVFNAQKKAQSSTD C-DN-13 60 SSHSDAHWMARALELAEKAREAGHVPVGAVLVLDDEVIGEGYNAAKTLHDPTAHAEIIALRQGGLRLQNYRLWGATLYTTFEPCVMCAGAMIHSRIKRVVFGVCNAKTHACMGLMDVLGHPGMNHKVEVVGGVLADECLDLLCRFFRMPRRVFNAQKKAQSSTD C-DN-14 61 ASHSDEHWMRRALELAERAREAGHVPVGAVLVLDDEVVGEGYNAAKTLHDPTAHAEIIALRQGGLRRQNYRLWGATLYTTFEPCVMCAGAMIHSRIARVVYGVCNAKTHACMGLMDVLGHPGMNHRVEVVGGVLADEALELLCRFFRMPRRVFNAQKKAQSSTD C-DN-15 62 DMHSDEYWMRRALELAEKARERRHVPVGAVLVLNNEVIGEGFNAAKSLHDPTAHAEIIALRKGGLKMQNYRLWGATLYTTFEPCVMCAGAMIHSRIKRVVFGVCNAKTHACMSLMDVLGHPGMNHRVEIVRGILADECLDLLCRFFRMPRRVFNAQKKAQSSTD C-DN-16 63 GSHSDEYWMRRALELAEKAREDRHVPVGAVLVLNNEVIGEGYNAAKTLHDPTAHAEIIALRKGGLKMQNYRLWGATLYTTFEPCVMCAGAMIHSRIKRVVFGVCNAKTHACMSLMDVLGHPGMNHKVEIVRGILREECEELLCRFFRMPRRVFNAQKKAQSSTD

[0171] Table B-2. Amino acid sequence of cytidine deaminase derived by directed evolution based on SEQ ID NO: 64

[0172] Name SEQ ID NO Amino acid sequence T88.74-V0 64 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNYRLLECTLYVTFEPCMMCAGAMVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V1 65 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNGRLLECTLYVTFEPCMMCAGAMVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V2 66 MNEQDEYWMRRAMALAARAEQEGEVVPGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNIRLLECTLYVTFEPCMMCAGAMVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACKAQLCRFFRRPRRVKNAARKQAAGG T88.74-V3 67 MNEQDEYWMRRAMALAARAEQEGEVVPGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNYRLLECTLYVTFEPCMMCAGAMVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLARFFRRPRRVKNAARKQAAGG T88.74-V4 68 MNEQDEYWMRRAMALAARAEQEGEVVPGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQACAHLGNIRLLECTLYVTFEPCMMCAGAMVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFRFRPRRVKNAARKQAAGG T88.74-V5 69 MNEQDEYWMRRAMALAARAEQEGEVVPGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNYRLLECTLYVTLEPCMMCGAMVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFRRPRRVKNAARKQAAGG T88.74-V6 70 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNYRLFECTLYVTFEPCMMCAGAMVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFRFRPRRVKNAARKQAAGG T88.74-V7 71 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNYRLLECTLYTTFEPCMMCAGAMVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V8 72 MNEQDEYWMRRAMALANRAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNYRLLECTLYVTFEPCMMCAGAMVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V9 73 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNKRLLECTLYVTFEPCMMCAGAMVHSRIQRLVYSASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V10 74 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNHRLLECTLYVTFEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V11 75 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNKRLLECTLYVTFEPCMMCAGAMVNSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V12 76 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNHRLLECTLYVTFEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLARFFRRPRRVKNAARKQAAGG T88.74-V13 77 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQACAHLGNHRLLECTLYVTFEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V14 78 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNHRLLECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V15 79 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNHRLFECTLYVTFEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V16 80 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQACAHLGNHRLFECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLARFFRRPRRVKNAARKQAAGG T88.74-V17 81 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQACAHLGNHRLFECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V18 82 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNHRLFECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V19 83 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQACAHLGNHRLLECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V20 84 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNHRLLECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLARFFRRPRRVKNAARKQAAGG T88.74-V21 85 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNHRLLECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLVHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V22 86 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNHRLLECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLPHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V23 87 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNHRLLECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRMVGNAARKQAAGG T88.74-V24 88 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNHRLLECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVNSVLQLLGHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V25 89 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNHRLLECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVNSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V26 90 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQAGAHLGNHRLLECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWVAGVLADACSRQLCRFFRRPRRVKNAARKQAAGG T88.74-V27 91 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQACAHLGNHRLFECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLVHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V28 92 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQACAHLGNHRLFECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLPHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V29 93 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQACAHLGNHRLFECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWVAGVLADACSRQLCRFFRRPRRVKNAARKQAAGG T88.74-V30 94 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQACAHLGNHRLFECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRMVGNAARKQAAGG T88.74-V31 95 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQACAHLGNHRLFECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVNSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V32 96 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQACAHLGNHRLFECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLVHRVDWVAGVLADACSRQLCRFFRRPRRVKNAARKQAAGG T88.74-V33 97 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQACAHLGNHRLFECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVNSVLQLLAHPGLVHRVDWVAGVLADACSRQLCRFFRRPRRVKNAARKQAAGG T88.74-V34 98 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQACAHLGNHRLFECTLYVTLEPCMMCAGALVHSRIQRLVYSASNAKTGAVDSVLQLLAHPGLNHRVDWRAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG T88.74-V35 99 MNEQDEYWMRRAMALAARAEQEGEVPVGALVVYHGDCVGEGWNRSIGHHDATAHAEIMALRQACAHLGNHRLFECTLYVTLEPCMMCAGALVHSRIQRLVYGASNAKTGAVDSVLQLLAHPGLNHRVDWVAGVLADACSAQLCRFFRRPRRVKNAARKQAAGG

[0173] Table C. Nucleotide sequences encoding adenosine deaminase and cytidine deaminase

[0174] Name SEQ IDNO Amino acid sequence ABE8e 100 ATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTCTCTGAGGTGGAGTTTTCCCACGAGTACTGGATGAGACATGCCCTGACCCTGGCCAAGAGGGCACGGGATGAGAGGGAGGTGCCTGTGGGAGCCGTGCTGGTGCTGAACAATAGAGTGATCGGCGAGGGCTGGAACAGAGCCATCGGCCTGCACGACCCAACAGCCCATGCCGAAATTATGGCCCTGAGACAGGGCGGCCTGGTCATGCAGAACTACAGACTGATTGACGCCACCCTGTACGTGACATTCGAGCCTTGCGTGATGTGCGCCGGCGCCATGATCCACTCTAGGATCGGCCGCGTGGTGTTTGGCGTGAGGAACTCAAAAAGAGGCGCCGCAGGCTCCCTGATGAACGTGCTGAACTACCCCGGCATGAATCACCGCGTCGAAATTACCGAGGGAATCCTGGCAGATGAATGTGCCGCCCTGCTGTGCGATTTCTATCGGATGCCTAGACAGGTGTTCAATGCTCAGAAGAAGGCCCAGAGCTCCATCAACTCCGGAGGATCTAGCGGAGGCTCCTCTGGCTCTGAGACACCTGGCACAAGCGAGAGCGCAACACCTGAAAGCAGCGGGGGCAGCAGCGGGGGGTCAGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGTGACTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAGGAAAGTC ecTadA8e 101 TCTGAGGTGGAGTTTTCCCACGAGTACTGGATGAGACATGCCCTGACCCTGGCCAAGAGGGCACGGGATGAGAGGGAGGTGCCTGTGGGAGCCGTGCTGGTGCTGAACAATAGAGTGATCGGCGAGGGCTGGAACAGAGCCATCGGCCTGCACGACCCAACAGCCCATGCCGAAATTATGGCCCTGAGACAGGGCGGCCTGGTCATGCAGAACTACAGACTGATTGACGCCACCCTGTACGTGACATTCGAGCCTTGCGTGATGTGCGCCGGCGCCATGATCCACTCTAGGATCGGCCGCGTGGTGTTTGGCGTGAGGAACTCAAAAAGAGGCGCCGCAGGCTCCCTGATGAACGTGCTGAACTACCCCGGCATGAATCACCGCGTCGAAATTACCGAGGGAATCCTGGCAGATGAATGTGCCGCCCTGCTGTGCGATTTCTATCGGATGCCTAGACAGGTGTTCAATGCTCAGAAGAAGGCCCAGAGCTCCATCAAC BE4max 102 ATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTCTCCTCAGAGACTGGGCCTGTCGCCGTCGATCCAACCCTGCGCCGCCGGATTGAACCTCACGAGTTTGAAGTGTTCTTTGACCCCCGGGAGCTGAGAAAGGAGACATGCCTGCTGTACGAGATCAACTGGGGAGGCAGGCACTCCATCTGGAGGCACACCTCTCAGAACACAAATAAGCACGTGGAGGTGAACTTCATCGAGAAGTTTACCACAGAGCGGTACTTCTGCCCCAATACCAGATGTAGCATCACATGGTTTCTGAGCTGGTCCCCTTGCGGAGAGTGTAGCAGGGCCATCACCGAGTTCCTGTCCAGATATCCACACGTGACACTGTTTATCTACATCGCCAGGCTGTATCACCACGCAGACCCAAGGAATAGGCAGGGCCTGCGCGATCTGATCAGCTCCGGCGTGACCATCCAGATCATGACAGAGCAGGAGTCCGGCTACTGCTGGCGGAACTTCGTGAATTATTCTCCTAGCAACGAGGCCCACTGGCCTAGGTACCCACACCTGTGGGTGCGCCTGTACGTGCTGGAGCTGTATTGCATCATCCTGGGCCTGCCCCCTTGTCTGAATATCCTGCGGAGAAAGCAGCCCCAGCTGACCTTCTTTACAATCGCCCTGCAGTCTTGTCACTATCAGAGGCTGCCACCCCACATCCTGTGGGCCACAGGCCTGAAGTCTGGAGGATCTAGCGGAGGATCCTCTGGCAGCGAGACACCAGGAACAAGCGAGTCAGCAACACCAGAGAGCAGTGGCGGCAGCAGCGGCGGCAGCGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGTGACAGCGGCGGGAGCGGCGGGAGCGGGGGGAGCACTAATCTGAGCGACATCATTGAGAAGGAGACTGGGAAACAGCTGGTCATTCAGGAGTCCATCCTGATGCTGCCTGAGGAGGTGGAGGAAGTGATCGGCAACAAGCCAGAGTCTGACATCCTGGTGCACACCGCCTACGACGAGTCCACAGATGAGAATGTGATGCTGCTGACCTCTGACGCCCCCGAGTATAAGCCTTGGGCCCTGGTCATCCAGGATTCTAACGGCGAGAATAAGATCAAGATGCTGAGCGGAGGATCCGGAGGATCTGGAGGCAGCACCAACCTGTCTGACATCATCGAGAAGGAGACAGGCAAGCAGCTGGTCATCCAGGAGAGCATCCTGATGCTGCCCGAAGAAGTCGAAGAAGTGATCGGAAACAAGCCTGAGAGCGATATCCTGGTCCATACCGCCTACGACGAGAGTACCGACGAAAATGTGATGCTGCTGACATCCGACGCCCCAGAGTATAAGCCCTGGGCTCTGGTCATCCAGGATTCCAACGGAGAGAACAAAATCAAAATGCTGTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAGGAAAGTC rAPOBEC1 103 TCCTCAGAGACTGGGCCTGTCGCCGTCGATCCAACCCTGCGCCGCCGGATTGAACCTCACGAGTTTGAAGTGTTCTTTGACCCCCGGGAGCTGAGAAAGGAGACATGCCTGCTGTACGAGATCAACTGGGGAGGCAGGCACTCCATCTGGAGGCACACCTCTCAGAACACAAATAAGCACGTGGAGGTGAACTTCATCGAGAAGTTTACCACAGAGCGGTACTTCTGCCCCAATACCAGATGTAGCATCACATGGTTTCTGAGCTGGTCCCCTTGCGGAGAGTGTAGCAGGGCCATCACCGAGTTCCTGTCCAGATATCCACACGTGACACTGTTTATCTACATCGCCAGGCTGTATCACCACGCAGACCCAAGGAATAGGCAGGGCCTGCGCGATCTGATCAGCTCCGGCGTGACCATCCAGATCATGACAGAGCAGGAGTCCGGCTACTGCTGGCGGAACTTCGTGAATTATTCTCCTAGCAACGAGGCCCACTGGCCTAGGTACCCACACCTGTGGGTGCGCCTGTACGTGCTGGAGCTGTATTGCATCATCCTGGGCCTGCCCCCTTGTCTGAATATCCTGCGGAGAAAGCAGCCCCAGCTGACCTTCTTTACAATCGCCCTGCAGTCTTGTCACTATCAGAGGCTGCCACCCCACATCCTGTGGGCCACAGGCCTGAAG

[0175] Other sequences used in this application, for example, linker sequences or sgRNA sequences, are listed in Tables D and E.

[0176] Table D. Adapter sequences

[0177] SEQ ID NO Amino acid sequence 104 SGGSSGGSSGSETPGTSESATPESSGGSSGGS 105 SGGS 106 PAPAPAP 107 SGSETPGTSESATPES

[0178] Table E. sgRNA sequences

[0179] Name SEQ ID NO Nucleotide sequence gRNA_1 108 CCCATACCTTGGAGCAACGG gRNA_2 109 CCCGCACCTTGGCGCAGCGG sgRNA_3 110 GGAATCCCTTCTGCAGCACC CAM-gRNA1 111 TGATCCGAACGTGGCCAATA CAM-gRNA2 112 TACGGCGCGGTGCACCTGGA

[0180] Table F. Other sequences

[0181] Name SEQ IDNO Amino acid sequence ABE8.8 113 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD nCas9 (nickase D10A) 114 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Mutated nCas9 (D700R) 115 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDRSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Mutated nCas9 (E1243R) 116 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYRKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Mutated nCas9 (A987R) 117 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDRYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0182] Split System

[0183] In some embodiments, the nuclear base editor disclosed herein comprises a "split" system, in which the Cas protein, deaminase, or both are split into two components and can be reconstructed to form a functional protein to perform base editing. "Split Cas9 protein", "split Cas9" or "split deaminase" refers to a Cas9 or deaminase protein provided with an N-terminal fragment and a C-terminal fragment encoded by two separate nucleotide sequences. The polypeptide corresponding to the N-terminal portion and the C-terminal portion of the Cas9 protein or deaminase can be spliced ​​to form a "reconstructed" Cas9 protein or deaminase. In some embodiments, the Cas9 protein or deaminase 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, both of which are each incorporated herein by reference.

[0184] Embedded Systems

[0185] In some embodiments, the nucleobase editor system disclosed herein comprises an embedded system in which a deaminase (e.g., an adenosine deaminase or a cytidine deaminase) is inserted into Cas9. In some embodiments, the present application provides a fusion protein comprising a nucleic acid-guided nuclease protein (e.g., Cas9 or any nuclease disclosed herein) and an embedded nucleotide deaminase protein domain. In some embodiments, the embedded nucleotide deaminase protein domain is an embedded adenosine deaminase protein domain. In some embodiments, the embedded adenosine deaminase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 1-47 (e.g., SEQ ID NOs: 2-47). In some embodiments, the embedded nucleotide deaminase protein domain is an embedded cytidine deaminase protein domain. In some embodiments, the embedded cytidine deaminase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 48-63 or 64-99 (e.g., SEQ ID NOs: 48-63 or 65-99).

[0186] Example

[0187] The compositions and methods disclosed herein are further described in the following examples, which are exemplary only and are not intended to limit the scope of the invention. The scope of the invention is defined by the claims.

[0188] Example 1. Evaluation of engineered TadA variants in sgRNA library cells

[0189] TadA deaminase comes from nature. Through rational design, mutations were introduced into it to obtain a highly active DNA adenosine deaminase active mutant T8.4-V0 (SEQ ID NO: 1). Subsequently, directed evolution was used to further improve its activity, and a series of TadA mutants were obtained by introducing single or multiple mutations (Table 1).

[0190] Table 1. Mutations contained in different adenosine deaminase variants relative to T8.4-V0 (SEQ ID NO: 1)

[0191] Variant Name Compared with the mutations introduced by T8.4-V0 T8.4-V1 V95T T8.4-V2 V95T-A61C T8.4-V3 V95T-C159R T8.4-V4 V95T-D12H T8.4-V5 V95T-E164V T8.4-V6 V95T-E164W T8.4-V7 V95T-E167R T8.4-V8 V95T-E167W T8.4-V9 V95T-E164V-E167W T8.4-V10 V95T-H21D T8.4-V11 V95T-C159R-E164V-E167W T8.4-V12 A61C-E164V-E167W T8.4-V13 C159R-E164V-E167W T8.4-V14 C159R-E167W T8.4-V16 S155R-K169N T8.4-V17 S156N-E164V T8.4-V18 V95T-S156K-C159R-E164V-E167W

[0192] To systematically evaluate the ability of each mutant to perform base editing in mammalian cells, we used a sgRNA library cell line (Max W. Shen et al., Nature, 2018 Nov;563(7733):646-651. doi:10.1038 / s41586-018-0686-x, cloned and constructed by GeneWei Biotech). This library cell line is derived from HEK293T and integrates more than 10,000 target sites and their paired homologous sgRNAs.

[0193] The nucleotide sequence encoding ecTadA8e in the ABE8e expression vector (Michelle F. Richter et al., Nat Biotechnol, 2020 Jul;38(7):883-891. doi: 10.1038 / s41587-020-0453-z, gene cloning was completed by GenScript Biotech) was replaced with the nucleotide sequence encoding the TadA variant designed by the inventors. In the experiment, a series of mRNAs encoding ABE were generated by in vitro transcription and tested for activity in the expression library cell line. HEK293T library cells (1x10 5 ) were seeded in 24-well plates (Corning). Following the manufacturer's protocol, 400 ng of mRNA encoding the ABE variant was transfected into the cells using Lipofectamine MessengerMAX (ThermoFisher, catalog number LMRNA003). After 72 hours, the genomic DNA of the cells was extracted and the editing efficiency was analyzed by next-generation sequencing (NGS). sgRNAs detected in all samples were defined as effective sgRNAs, and the A to G editing efficiency of these paired target sites was analyzed. To quantify the efficacy of each variant, its average editing efficiency value was calculated and normalized to the average editing efficiency value of T8.4-V0. As shown in Tables 2 and Figure 1 As shown, the editing efficiency of T8.4-V0 is slightly higher than that of ABE8.8 (a known ABE variant, used as a control; Gaudelli NM et al., Nat Biotechnol. 2020 Jul;38(7):892-900. doi: 10.1038 / s41587-020-0491-6.), and the average editing efficiency of many engineered variants of the present invention is higher than that of T8.4-V0, demonstrating that the engineered variants of the present invention are suitable as adenine base editors with improved activity.

[0194] Table 2. A to G editing efficiency of different TadA variants in library cells

[0195] Editor Average A to G editing efficiency ratio normalized by T8.4-V0 activity in the same experiment T8.4-V0 1.00 T8.4-V1 1.13 T8.4-V2 1.20 T8.4-V3 1.19 T8.4-V4 1.16 T8.4-V5 1.24 T8.4-V6 1.18 T8.4-V7 1.17 T8.4-V8 1.28 T8.4-V9 1.24 T8.4-V10 1.15 T8.4-V11 1.21 T8.4-V12 1.25 T8.4-V13 1.30 T8.4-V14 1.26 T8.4-V16 1.10 T8.4-V17 1.17 T8.4-V18 1.23 ABE8.8 (control) 0.97

[0196] Figure 1 The overall A to G editing efficiency (i.e., the conversion rate of A:T to G:C) of some adenosine deaminase variants of the present invention (T8.4-V1, T8.4-V2, T8.4-V4, T8.4-V7, T8.4-V10, T8.4-V12) and the known ABE variant ABE8.8 (SEQ ID NO: 113) compared with T8.4-V0 is shown. Compared with T8.4-V0, some variants of the present invention (T8.4-V1, T8.4-V2, T8.4-V4, T8.4-V10) show similar editing windows but higher editing efficiency, and other variants (T8.4-V7, T8.4-V12) show wider editing windows and higher editing efficiency. Here, "editing window" refers to the position of A that can be edited in the original spacer. "Wider editing window" means that there are more positions of A that can be edited in the original spacer.

[0197] Example 2. Evaluation of the activity of adenine base editor (ABE) variants in mice

[0198] Use wild-type mice (mouse strain , purchased from Biocytogen, with an age of about 7-9 weeks and a body weight of 20-25g when used for the experiment) to further evaluate the editing effect of the base editor constructed according to the adenosine deaminase variant selected in Example 1 in vivo. The inventors designed a specific guide RNA (gRNA_1: CCCATACCTTGGAGCAACGG, SEQID NO: 108) for disrupting the mouse Pcsk9 gene. The mRNA encoding the ABE variant obtained by in vitro transcription and the chemically synthesized gRNA_1 were combined and encapsulated into lipid nanoparticles (LNPs) and encapsulated according to the method described in patent application WO / 2023 / 185697A2. These formulated LNPs were then injected into mice through the tail vein at 0.15 mg per kilogram of body weight (0.15 mg / kg body weight (mpk)).

[0199] One week after the injection, the mouse liver samples were collected, and the regions near the editing sites were amplified by PCR and analyzed by NGS sequencing after DNA extraction. As shown in Table 3, compared with T8.4-V0, mice treated with the engineered mutants of the present invention showed higher A to G editing efficiency.

[0200] Table 3. DNA editing efficiency (%) achieved by the adenosine deaminase variants of the present invention in wild-type mice

[0201] Experimental batch Editor Number of animals Average A to G editing efficiency (%) B10 T8.4-V0 3 24.37 B10 T8.4-V12 3 45.61 B10 T8.4-V1 3 47.75 B10 T8.4-V8 3 43.59 B10 T8.4-V14 3 52.13 B10 T8.4-V13 3 45.12 B12 T8.4-V0 3 26.17 B12 T8.4-V3 3 35.04 B13 T8.4-V0 3 21.44 B13 T8.4-V2 3 25.18 B13 T8.4-V10 3 31.25

[0202] Example 3. Effect of different linker sequences on the activity of adenine base editor (ABE) variants

[0203] The adenosine deaminase variants tested in Examples 1 and 2 were connected to nCas9 (D10A) via a 32-amino acid linker (SGGSSGGSSGSETPGTSESATPESSGGSSGGS, SEQ ID NO: 104). To test the effect of different linker sequences on the editing activity of ABE variants, we tested the effect of changing the length and type of the linker or not using a linker on the activity of T8.4-V0. The same experimental system as in Example 1 was used for detection, and the results are shown in Table 4. The editor (T8.4-L0) obtained by connecting T8.4-V0 and nCas9 (D10A) via the linker shown in SEQ ID NO: 104 was used as a control. The replacement of the linker did not reduce the base editing activity of T8.4-V0, and T8.4-L3 obtained by connecting T8.4-V0 to nCas9 using the linker sequence PAPAPAP (SEQ ID NO: 106) had significantly improved editing activity.

[0204] Table 4. A to G editing efficiency of different linker sequences in library cells

[0205] Editor Adapter sequence Average A to G editing efficiency ratio normalized for activity using T8.4-L0 T8.4-L0 SGGSSGGSSGSETPGTSESATPESSGGSSGGS 1.00 T8.4-L1 No connector 0.99 T8.4-L2 SGGS 0.99 T8.4-L3 PAPAPAP 1.04 T8.4-L4 SGSETPGTSESATPES 1.01

[0206] Example 4. Modification of Cas9 to enhance base editing activity

[0207] The editing activity of adenine base editor (ABE) variants was enhanced by modifying nCas9 (nickase D10A, SEQ ID NO: 114). Mutations were introduced into Cas9 based on rational structural design, and activity was tested using the same experimental system as in Example 1. T8.4-V0 and the editor T8.4-C0 composed of nCas9 without mutations were used as controls. The results are shown in Table 5. The introduction of D700R or E1243R into nCas9 can improve the editing activity and editing efficiency of adenine base editors.

[0208] Table 5. A to G editing efficiency of T8.4-V0 and mutant Cas9 in library cells

[0209] Editor Mutations introduced in nCas9 Average A to G editing efficiency ratio normalized for activity using T8.4-C0 T8.4-C0 none 1.00 T8.4-C1 nCas9-D700R (SEQ ID NO: 115) 1.26 T8.4-C2 nCas9-E1243R (SEQ ID NO: 116) 1.25 T8.4-C3 nCas9-A987R (SEQ ID NO: 117) 0.45

[0210] Example 5. De novo design of adenosine deaminase with high activity

[0211] Based on the structure and sequence information, a new TadA with adenosine deaminase activity was designed, and the polynucleotide sequence encoding ecTadA8e was replaced with the polynucleotide sequence encoding the newly designed TadA variant in the ABE8e expression vector to generate a series of ABE variants. The editing activity of these variants was measured in HEK293T cells as described below.

[0212] 20-24 hours before transfection, HEK293T cells (1.5x10 4 ) were inoculated in 96-well plates (Corning). For targeted editing experiments, cells were transfected with 80 ng of expression vector encoding ABE variants, 40 ng of spCas9 gRNA_2 (CCCGCACCTTGGCGCAGCGG, SEQ ID NO: 109) expression plasmid and 3.6 μL FuGENE HD transfection reagent (E2312, purchased from Promega) according to the manufacturer's protocol. After 72 hours, the genomic DNA of the cells was extracted and the editing efficiency of the ABE variants was analyzed by NGS. As shown in Table 6, many newly designed ABE variant sequences have A to G editing activity, among which A-DN-8, A-DN-9, A-DN-10, A-DN-11 and A-DN-12 show more than 60% A to G editing efficiency.

[0213] Table 6. A to G editing efficiency of different ABEs

[0214] Editor A to G editing efficiency (%) C to T editing efficiency (%) A-DN-1 4.64 0.09 A-DN-2 6.45 0.08 A-DN-3 5.67 0.09 A-DN-4 3.94 0.09 A-DN-5 4.59 0.10 A-DN-6 3.43 0.07 A-DN-7 36.12 0.20 A-DN-8 70.07 0.34 A-DN-9 69.55 0.45 A-DN-10 65.40 0.82 A-DN-11 66.30 0.81 A-DN-12 62.01 0.28 A-DN-13 8.27 0.12 A-DN-14 22.62 0.12 A-DN-15 21.27 0.11 A-DN-16 25.79 0.08 A-DN-17 25.98 0.09 A-DN-18 16.84 0.12 A-DN-19 21.58 0.12 A-DN-20 19.45 0.14 A-DN-21 9.18 0.06 A-DN-22 12.91 0.02 A-DN-23 40.40 0.29 A-DN-24 49.87 0.24 A-DN-25 2.17 0.06 A-DN-26 33.58 0.27 A-DN-27 40.30 0.16 A-DN-28 40.49 0.33

[0215] Example 6. De novo design of TadA variants with cytidine deaminase activity

[0216] TadA was modified to mediate C to T editing, and has the advantages of smaller size and less off-target compared to natural cytidine deaminases such as rAPOBEC used in BE4max. To this end, based on the structure and sequence information, a new TadA with cytidine deaminase activity was designed, and the polynucleotide sequence encoding rAOPBEC (SEQ ID NO: 103) was replaced with a polynucleotide sequence encoding a newly designed TadA variant in the BE4max (encoding nucleotide sequence is SEQ ID NO: 102) expression vector to generate a series of CBE variants. The editing activity was measured in the HEK293T cell line using the experimental method described in Example 5, and the sgRNA sequence used was sgRNA_3 (GGAATCCCTTCTGCAGCACC, SEQ ID NO: 110), and its A to G and C to T editing activities were detected by NGS sequencing. As shown in Table 7, many newly designed CBE variants have C to T editing activity, among which the editing activities of variants C-DN-7 and C-DN-8 are comparable to those of the control BE4max. Furthermore, none of these CBE variants had A to G editing activity, demonstrating that they were specific cytosine deaminases.

[0217] Table 7. A to G and C to T editing efficiencies of different CBE variants

[0218] Editor A to G editing efficiency (%) C to T editing efficiency (%) C-DN-1 0.02 14.07 C-DN-2 0.03 17.10 C-DN-3 0.20 36.64 C-DN-4 0.08 20.73 C-DN-5 0.02 2.98 C-DN-6 0.04 22.96 C-DN-7 0.33 41.63 C-DN-8 0.31 42.63 C-DN-9 0.03 5.20 C-DN-10 0.05 6.37 C-DN-11 0.03 5.75 C-DN-12 0.02 2.03 C-DN-13 0.13 23.38 C-DN-14 0.06 16.00 C-DN-15 0.06 30.29 C-DN-16 0.37 32.73 BE4max 0.03 42.36

[0219] Example 7. Directed evolution of T88.74 to improve its C to T editing activity

[0220] To enhance the activity of the cytidine deaminase T88.74-V0 (SEQ ID NO: 64) constructed by the inventors, we constructed a site-saturated mutation library of T88.74 and performed directed evolution on it. The experimental system used three plasmids: a selection plasmid containing a chloramphenicol resistance gene (CamR) with two inactivating mutations (L158P and H193R); an sgRNA expression plasmid that produces two sgRNAs targeting the mutation sites in the CamR gene; and an editor (i.e., CBE variant) plasmid library that expresses T88.74-dCas9-2xUGI variants from the library. Library members that are able to introduce C:G to T:A edits at both mutation sites will restore chloramphenicol resistance and allow the bacteria to survive in the presence of chloramphenicol.

[0221] We co-transformed all three plasmids into the E. coli DH10B strain. After overnight induction of expression of the editor library, bacteria were plated on culture plates containing chloramphenicol (selective; 16 or 64 µg / ml) or without chloramphenicol (non-selective). After overnight incubation at 37°C, plasmids from colonies growing on selective and non-selective plates were extracted and analyzed using next-generation sequencing (NGS). We calculated the percentage of mutations and the fold enrichment on the selective plates. The top 50 variants were identified on Figure 3 and Figure 2 Displayed in.

[0222] Example 8. Validation of beneficial mutations in bacterial and mammalian sgRNA library cells

[0223] We first evaluated the CBE activity of the enriched T88.74 variants in E. coli using the system described in Example 7, and measured and compared the editing efficiency of each variant at two target sites on the selected plasmid by NGS (CAM-gRNA1: TGATCCGAACGTGGCCAATA, SEQ ID NO: 111; CAM-gRNA2: TACGGCGCGGTGCACCTGGA, SEQ ID NO: 112). The 11 highly active variants and their corresponding editing efficiencies are listed in Table 8.

[0224] Table 8. C to T editing efficiency of different variants of T88.74 in bacteria

[0225] Editor Based on the mutation site of T88.74 CAM-gRNA1 editing efficiency CAM-gRNA2 editing efficiency T88.74-V0 Original sequence 0.91 7.11 T88.74-V8 A17N 1.02 4.18 T88.74-V3 C143A 2.18 15.78 T88.74-V5 F81L 1.59 7.96 T88.74-V4 G64C 0.87 8.25 T88.74-V6 L73F 2.23 16.31 T88.74-V2 S139K 1.37 6.51 T88.74-V7 V79T 0.94 3.51 T88.74-V1 Y70G 1.42 12.18 T88.74-V10 Y70H; M91L 16.49 39.80 T88.74-V9 Y70K; G102S 5.29 23.95 T88.74-V11 Y70K; H93N 2.36 2.83

[0226] We then selected mutations in the top seven T88.74 variants validated in E. coli for combination and further tested their CBE activity in HEK293T sgRNA library cells. In this HEK293T sgRNA library, more than 10,000 different sgRNAs and their corresponding target sequences were inserted into hotspot regions of the HEK293T genome, allowing analysis of CBE activity on multiple sgRNAs in one simple experiment.

[0227] To evaluate the CBE activity of the selected T88.74 variants, 400 ng of T88.74 variant-based CBE mRNA was transfected into the sgRNA library cells using Lipofectamine MessengerMAX (ThermoFisher, Cat. No. LMRNA003) according to the manufacturer's instructions. After 72 hours, genomic DNA of the cells was extracted and the editing efficiency was analyzed by NGS. The sgRNAs detected in all samples were defined as effective sgRNAs. The C to T editing efficiency of these paired target sites was analyzed. The average editing efficiency value of each variant in the sgRNA library was calculated. As shown in Tables 9 and Figure 4 As shown, the average editing efficiency of many engineered CBE variants was significantly higher than that of the original T88.74-V0.

[0228] Table 9. C to T editing efficiency of different variant versions of T88.74 in library cells

[0229] Editor Based on the mutation site of T88.74 Average C to T editing efficiency ratio normalized for activity using T88.74-V0 T88.74-V0 Original sequence 1.00 T88.74-V10 Y70H; M91L 2.03 T88.74-V12 Y70H; M91L; C143A 2.24 T88.74-V13 Y70H; M91L; G64C 2.24 T88.74-V14 Y70H; M91L; F81L 2.67 T88.74-V15 Y70H; M91L; L73F 2.28 T88.74-V16 Y70H; M91L; F81L; C143A; G64C; L73F 3.28 T88.74-V17 Y70H; M91L; F81L; G64C; L73F 3.27 T88.74-V18 Y70H; M91L; F81L; L73F 3.05 T88.74-V19 Y70H; M91L; F81L; G64C 3.02 T88.74-V20 Y70H; M91L; F81L; C143A 2.80

[0230] We then introduced three enhanced mutations (T88.74-V14) validated in bacteria and mammalian cells into the original directed evolution library and performed selection again using higher concentrations of chloramphenicol (64 or 128 µg / ml) as described in Example 7. The top 50 variants in the second round of directed evolution were Figure 5 Displayed in.

[0231] Next, we selected some enhancing mutations and evaluated their activity in HEK293T sgRNA library cells as described above. Variants T88.74-V21 and T88.74-V22 showed significantly further enhanced C to T editing efficiency compared to the T88.74-V14 variant, as shown in Tables 10 and Figure 6 shown.

[0232] Table 10. C to T editing efficiency of different variant versions of T88.74 in library cells

[0233] Editor Based on the mutation site of T88.74 Average C to T editing efficiency ratio normalized for activity using T88.74-V14 T88.74-V14 Y70H; M91L; F81L 1.00 T88.74-V21 Y70H; M91L; F81L; N124V 1.25 T88.74-V22 Y70H; M91L; F81L; N124P 1.20 T88.74-V23 Y70H; M91L; F81L; R151M; K153G 1.05 T88.74-V24 Y70H; M91L; F81L; D112N; A119G 1.07 T88.74-V25 Y70H; M91L; F81L; D112N 1.06 T88.74-V26 Y70H; M91L; F81L; R130V; A140R 1.08

[0234] We then added a portion of the newly enhanced mutations confirmed based on T88.74-V14 (Table 10) to T88.74-V17, one of the strongest variants screened previously, and evaluated its activity in HEK293T sgRNA library cells according to the above method. Variants T88.74-V27 and T88.74-V33 showed significantly further enhanced C to T editing efficiency compared to the T88.74-V17 variant, as shown in Tables 11 and Figure 7 shown.

[0235] Table 11. C to T editing efficiency of different variant versions of T88.74 in library cells

[0236] Editor Based on the mutation site of T88.74 Average C to T editing efficiency ratio normalized for activity using T88.74-V17 T88.74-V17 Y70H; M91L; F81L; G64C; L73F 1.00 T88.74-V27 Y70H; M91L; F81L; G64C; L73F; N124V 1.17 T88.74-V28 Y70H; M91L; F81L; G64C; L73F; N124P 1.04 T88.74-V29 Y70H; M91L; F81L; G64C; L73F; R130V; A140R 1.09 T88.74-V30 Y70H; M91L; F81L; G64C; L73F; R151M; K153G 1.00 T88.74-V31 Y70H; M91L; F81L; G64C; L73F; D112N 1.05 T88.74-V32 Y70H; M91L; F81L; G64C; L73F; N124V; R130V; A140R 1.06 T88.74-V33 Y70H; M91L; F81L; G64C; L73F; N124V; R130V; A140R; D112N 1.10 T88.74-V35 Y70H; M91L; F81L; G64C; L73F; R130V 1.02

[0237] Other Implementations

[0238] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and improvements are within the scope of the following claims.

Claims

1. An adenosine deaminase, comprising: (i) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 2-47; or (ii) one or more substitutions selected from the group consisting of D12H, H21D or H21Q, A61C, V95T, S155R, S156N or S156K, C159R, E164V or E164W, E167R or E167W, K169N relative to SEQ ID NO: 1, wherein the amino acid positions are numbered relative to SEQ ID NO:

1.

2. A cytidine deaminase, comprising: (i) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 48-63 or 65-99; or (ii) one or more substitutions selected from the group consisting of A17N, G64C, Y70G or Y70H or Y70K, L73F, V79T, F81L, M91L, H93N, G102S, D112N, A119G, N124V or N124P, R130V, S139K, A140R, C143A, R151M, K153G relative to SEQ ID NO: 64, wherein amino acid positions are numbered relative to SEQ ID NO:

64.

3. A nucleobase editor comprising: (i) a polynucleotide programmable DNA binding domain; and (ii) a deaminase, wherein the deaminase is the adenosine deaminase according to claim 1 or the cytidine deaminase according to claim 2.

4. The nucleobase editor of claim 3, wherein the nucleobase editor further comprises one or more uracil glycosylase inhibitor (UGI) domains.

5. The nucleobase editor of claim 3, wherein the nucleobase editor is an adenosine base editor (ABE).

6. The nucleobase editor of claim 3, wherein the nucleobase editor is a cytidine base editor (CBE).

7. The nucleobase editor of any one of claims 3-6, wherein the polynucleotide programmable DNA binding domain comprises a domain selected from the group consisting of a Cas9 domain, a Cas12 domain, a TnpB domain, an IscB domain, a homing endonuclease domain, a zinc finger DNA binding domain, and a transcription activator-like effector (TALE) DNA binding domain.

8. The nucleobase editor of claim 7, wherein the polynucleotide programmable DNA binding domain comprises a Cas9 domain.

9. The nucleobase editor of claim 8, wherein the Cas9 domain is Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), Streptococcus pyogenes Cas9 (SpCas9) or a variant thereof.

10. A nucleobase editor according to claim 8, wherein the Cas9 domain is a mutated Cas9 comprising one or two amino acid substitutions selected from D700R or E1243R, and wherein the mutated Cas9 comprises an amino acid sequence of SEQ ID NO: 115 or 116, or consists of an amino acid sequence of SEQ ID NO: 115 or 116.

11. The nucleobase editor of any one of claims 3-6, wherein the polynucleotide programmable DNA binding domain is an inactive nuclease or nickase variant.

12. The nucleobase editor of any one of claims 3-6, wherein the nucleobase editor comprises a nucleic acid-guided nuclease protein and an embedded nucleotide base editor domain.

13. The nuclear base editor of claim 12, wherein the embedded nucleotide base editor domain is an embedded adenosine base editor (ABE) domain or an embedded cytidine base editor (CBE) domain.

14. The nucleobase editor of claim 13, wherein the embedded adenosine base editor (ABE) domain is an embedded adenosine deaminase protein domain, or the embedded cytidine base editor (CBE) domain is an embedded cytidine deaminase protein domain.

15. The nucleobase editor of claim 14, wherein the polynucleotide programmable DNA binding domain comprises a linker flanked by at least one embedded domain protein.

16. A fusion protein comprising a polynucleotide programmable DNA binding domain and at least one nucleobase editor domain comprising a deaminase linked together, wherein the deaminase is the adenosine deaminase of claim 1 or the cytidine deaminase of claim 2. 17 . A polynucleotide encoding the adenosine deaminase according to claim 1 or the cytidine deaminase according to claim 2 .

18. A polynucleotide encoding a nucleobase editor according to any one of claims 3-15. A polynucleotide encoding the fusion protein according to claim 16 .

20. An expression vector comprising the polynucleotide according to any one of claims 17-19.

21. The expression vector according to claim 20, wherein the vector is a viral vector selected from the group consisting of adeno-associated virus (AAV), retroviral vector, adenoviral vector, lentiviral vector, Sendai virus vector and herpes virus vector.

22. A cell comprising the expression vector according to claim 20 or 21.

23. Use of a nuclear base editor according to any one of claims 3-15 in the preparation of a drug or kit for base editing, wherein the base editing comprises contacting a polynucleotide sequence with a nuclear base editor according to any one of claims 3-15, wherein a deaminase deaminates the nuclear base in the polynucleotide, thereby editing the polynucleotide sequence.

24. Use of a nucleobase editor according to any one of claims 3-15 in the preparation of a drug or kit for correcting a genetic defect in a subject, wherein the correction of the genetic defect in the subject comprises administering to the subject: a nucleobase editor according to any one of claims 3-15 or a polynucleotide encoding a nucleobase editor according to any one of claims 3-15, and one or more guide polynucleotides that guide the nucleobase editor to deaminize a target nucleobase in the target nucleotide sequence of the subject, thereby correcting the genetic defect.

25. The use according to claim 24, comprising delivering the nucleobase editor or a polynucleotide encoding the nucleobase editor, and one or more guide polynucleotides to cells of the subject.

26. The use according to claim 24, wherein the subject is a non-human mammal or a human.

27. The use according to any one of claims 24-26, wherein deamination of the target nucleobase results in replacement of the target nucleobase with a wild-type nucleobase.

28. A molecular complex comprising: a nucleobase editor according to any one of claims 3-15 or a fusion protein according to claim 16, and one or more guide RNA sequences, tracrRNA sequences, or target DNA sequences.

29. A kit comprising: a nucleobase editor according to any one of claims 3-15 or a fusion protein according to claim 16, or an expression vector according to claim 20, or a molecular complex according to claim 28, and instructions for use.

30. A pharmaceutical composition comprising: a nucleobase editor according to any one of claims 3-15 or a fusion protein according to claim 16, or an expression vector according to claim 20, or a molecular complex according to claim 28, and a pharmaceutically acceptable excipient.

31. A mutant Cas9 comprising one or two amino acid substitutions selected from D700R or E1243R, and wherein the mutant Cas9 comprises, or consists of, the amino acid sequence of SEQ ID NO: 115 or 116.

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