Base editing tool and application thereof

Abstract

The invention belongs to the field of nucleic acid editing, and particularly relates to the technical field of regularly clustered interval short palindromic repeat (CRISPR). Specifically, the invention provides adenosine deaminase which is fused with DNA binding protein, can be used for single base editing of target nucleic acid, and has a wide application prospect.

Description

[0001] This application claims the priority of Chinese patent application CN202311769067.7 filed on December 21, 2023 and Chinese patent application CN202410713487.1 filed on June 4, 2024. This application cites the full text of the above Chinese patent application. Technical Field

[0002] The present invention relates to the field of gene editing, in particular to the field of clustered regularly interspaced short palindromic repeats (CRISPR). Specifically, the present invention relates to a base editing tool, in particular to base editing based on adenosine deaminase. Background Art

[0003] CRISPR / Cas technology is a widely used gene editing technology that uses RNA to guide specific binding to target sequences on the genome and cut DNA to produce double-strand breaks, and uses biological non-homologous end joining or homologous recombination for site-specific gene editing.

[0004] The development of single-base gene editing tools can achieve precise modification of specific gene sites without causing double-strand breaks in DNA. Adenine base editor (ABE) based on adenosine deaminase achieves the conversion of A>G (from A to G). At present, there are few types of adenosine deaminases. Therefore, the present invention screened a variety of adenosine deaminases and constructed a tool that can perform single-base editing, expanding the application scope of single-base editing technology. Summary of the invention

[0005] On the one hand, the present invention provides an adenosine deaminase, which is referred to as GZ-71, GZ-68, GZ-65, GZ-64, GZ-46, GZ-74, GZ-67, GZ-13 and GZ-76 in the present invention, and the amino acid sequences of the above adenosine deaminase are shown in SEQ ID No.3-11 respectively.

[0006] In one embodiment, the amino acid sequence of the adenosine deaminase has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity compared to any of SEQ ID No.3-11, and substantially retains the biological function of the sequence from which it is derived. Preferably, the adenosine deaminase is derived from the same species as GZ-71, GZ-68, GZ-65, GZ-64, GZ-46, GZ-74, GZ-67, GZ-13 or GZ-76. More preferably, the adenosine deaminase is derived from Enterobacteriaceae, Escherichia, Citrobacter, Rahnella aceris, Shigella flexneri or Salmonella enterica subsp.

[0007] In one embodiment, the amino acid sequence of the adenosine deaminase has a sequence of one or more amino acid substitutions, deletions or additions compared to any of the sequences of SEQ ID No.3-11; and the biological function of the sequence from which it is derived is substantially retained; the one or more amino acid substitutions, deletions or additions include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid substitutions, deletions or additions. Preferably, the adenosine deaminase is derived from the same species as GZ-71, GZ-68, GZ-65, GZ-64, GZ-46, GZ-74, GZ-67, GZ-13 or GZ-76. More preferably, the adenosine deaminase is derived from Enterobacteriaceae, Escherichia, Citrobacter, Rahnella aceris, Shigella flexneri or Salmonella enterica subsp.

[0008] In one embodiment, the adenosine deaminase comprises an amino acid sequence shown in any one of SEQ ID No.3-11.

[0009] In one embodiment, the amino acid sequence of the adenosine deaminase is shown in any one of SEQ ID No.3-11.

[0010] In one embodiment, the amino acid sequence of the adenosine deaminase has a mutation (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids) at any one or more of the following amino acid positions corresponding to the amino acid sequence shown in SEQ ID No.7 compared to SEQ ID No.7: position 55, position 110, position 113, position 123, position 126, position 127, position 153, position 156, position 159, position 160, position 161, position 167.

[0011] In one embodiment, the amino acid sequence of the adenosine deaminase has mutations at the 110th, 153rd, 161st and 167th amino acid positions corresponding to the amino acid sequence shown in SEQ ID No.7 compared with SEQ ID No.7.

[0012] In one embodiment, the amino acid sequence of the adenosine deaminase has mutations at amino acid positions 110, 153, 161, 167 and 113 corresponding to the amino acid sequence shown in SEQ ID No.7 compared with SEQ ID No.7.

[0013] In one embodiment, the amino acid sequence of the adenosine deaminase has mutations at amino acid positions 110, 153, 161, 167 and 126 corresponding to the amino acid sequence shown in SEQ ID No.7 compared with SEQ ID No.7.

[0014] In one embodiment, the amino acid sequence of the adenosine deaminase has mutations at amino acid positions 110, 153, 161, 167 and 127 corresponding to the amino acid sequence shown in SEQ ID No.7 compared with SEQ ID No.7.

[0015] In one embodiment, the amino acid sequence of the adenosine deaminase has mutations at amino acid positions 110, 153, 161, 167 and 159 corresponding to the amino acid sequence shown in SEQ ID No.7 compared with SEQ ID No.7.

[0016] In one embodiment, the amino acid sequence of the adenosine deaminase has mutations compared to SEQ ID No.7 at amino acid positions 110, 153, 161, 167, 55, 113, 123, 126, 127, 156, 159 and 160 corresponding to the amino acid sequence shown in SEQ ID No.7.

[0017] In one embodiment, the amino acid sequence of the adenosine deaminase has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity compared to the sequence of SEQ ID No.7; and the amino acid sequence of the adenosine deaminase has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity compared to the sequence of SEQ ID No.7; There is a mutation at any one or several of the following amino acid positions of the amino acid sequence shown in ID No. 7 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids): position 55, position 110, position 113, position 123, position 126, position 127, position 153, position 156, position 159, position 160, position 161, and position 167.

[0018] In one embodiment, the amino acid at position 55 is mutated to a non-L amino acid, e.g., G, A, V, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, R, H; preferably, T.

[0019] In one embodiment, the amino acid at position 110 is mutated to a non-V amino acid, for example, G, A, L, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, R, H; preferably, W, K, R or H; more preferably, K.

[0020] In one embodiment, the amino acid at position 113 is mutated to a non-S amino acid, for example, G, A, L, I, P, F, Y, W, V, T, C, M, N, Q, D, E, K, R, H; preferably, N or R; more preferably, N.

[0021] In one embodiment, the amino acid at position 123 is mutated to a non-N amino acid, for example, G, A, L, I, P, F, Y, W, V, T, C, M, S, Q, D, E, K, R, H; preferably, D.

[0022] In one embodiment, the amino acid at position 126 is mutated to a non-N amino acid, for example, G, A, L, I, P, F, Y, W, V, T, C, M, S, Q, D, E, K, R, H; preferably, Q.

[0023] In one embodiment, the amino acid at position 127 is mutated to a non-Y amino acid, for example, G, A, L, I, P, F, N, W, V, T, C, M, S, Q, D, E, K, R, H; preferably, H, L or M; more preferably, H.

[0024] In one embodiment, the amino acid at position 153 is mutated to an amino acid other than Y, e.g., L, A, V, I, P, F, N, W, S, T, C, M, G, Q, D, E, K, R, H; preferably, F.

[0025] In one embodiment, the amino acid at position 156 is mutated to an amino acid other than P, e.g., L, A, V, I, Y, F, N, W, S, T, C, M, G, Q, D, E, K, R, H; preferably, N.

[0026] In one embodiment, the amino acid at position 159 is mutated to a non-V amino acid, for example, L, A, P, I, Y, F, N, W, S, T, C, M, G, Q, D, E, K, R, H; preferably, A, H, K or R; more preferably, H or R.

[0027] In one embodiment, the amino acid at position 160 is mutated to an amino acid other than F, e.g., L, A, V, I, Y, P, N, W, S, T, C, M, G, Q, D, E, K, R, H; preferably, Q.

[0028] In one embodiment, the amino acid at position 161 is mutated to a non-N amino acid, for example, L, A, V, I, P, F, Y, W, S, T, C, M, G, Q, D, E, K, R, H; preferably, R.

[0029] In one embodiment, the amino acid at position 167 is mutated to an amino acid other than E, for example, G, A, L, I, P, F, Y, W, S, T, C, M, N, Q, D, V, K, R, H; preferably, R.

[0030] In one embodiment, the adenosine deaminase is a mutant adenosine deaminase, and the mutant adenosine deaminase has a mutation (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids) at any one or several of the following amino acid positions corresponding to the amino acid sequence shown in SEQ ID No.7 compared with the amino acid sequence of the parent adenosine deaminase: position 55, position 110, position 113, position 123, position 126, position 127, position 153, position 156, position 159, position 160, position 161, and position 167.

[0031] In one embodiment, the mutant adenosine deaminase has mutations at amino acid positions 110, 153, 161 and 167 corresponding to the amino acid sequence shown in SEQ ID No.7 compared to the amino acid sequence of the parent adenosine deaminase.

[0032] In one embodiment, the mutant adenosine deaminase has mutations at amino acid positions 110, 153, 161, 167 and 113 corresponding to the amino acid sequence shown in SEQ ID No.7 compared to the amino acid sequence of the parent adenosine deaminase.

[0033] In one embodiment, the mutant adenosine deaminase has mutations at amino acid positions 110, 153, 161, 167 and 126 corresponding to the amino acid sequence shown in SEQ ID No.7 compared to the amino acid sequence of the parent adenosine deaminase.

[0034] In one embodiment, the mutant adenosine deaminase has mutations at amino acid positions 110, 153, 161, 167 and 127 corresponding to the amino acid sequence shown in SEQ ID No.7 compared to the amino acid sequence of the parent adenosine deaminase.

[0035] In one embodiment, the mutant adenosine deaminase has mutations at amino acid positions 110, 153, 161, 167 and 159 corresponding to the amino acid sequence shown in SEQ ID No.7 compared to the amino acid sequence of the parent adenosine deaminase.

[0036] In one embodiment, the mutant adenosine deaminase has mutations at amino acid positions 110, 153, 161, 167, 55, 113, 123, 126, 127, 156, 159 and 160 corresponding to the amino acid sequence shown in SEQ ID No.7 compared to the amino acid sequence of the parent adenosine deaminase.

[0037] In one embodiment, the amino acid sequence of the adenosine deaminase is compared with SEQ ID No. 2, and mutations occur at the following amino acid positions corresponding to the amino acid sequence shown in SEQ ID No. 2: position 106 and / or position 157.

[0038] In one embodiment, the mutant adenosine deaminase has a mutation at the following amino acid positions corresponding to the amino acid sequence shown in SEQ ID No. 2: position 106 and / or position 157 compared to the amino acid sequence of the parent adenosine deaminase.

[0039] In one embodiment, the amino acid at position 106 is mutated to a non-V amino acid, for example, G, A, L, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, R, H; preferably, W, K or H; more preferably, H.

[0040] In one embodiment, the amino acid at position 157 is mutated to a non-N amino acid, e.g., L, A, V, I, P, F, Y, W, S, T, C, M, G, Q, D, E, K, R, H; preferably, R.

[0041] In one embodiment, the parent adenosine deaminase is a natural wild-type adenosine deaminase; in other embodiments, the parent adenosine deaminase is an engineered adenosine deaminase.

[0042] In one embodiment, the parent adenosine deaminase is TadA, GZ-71, GZ-68, GZ-65, GZ-64, GZ-46, GZ-74, GZ-67, GZ-13 or GZ-76.

[0043] In one embodiment, the amino acid sequence of the parent adenosine deaminase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity compared to the sequence shown in any one of SEQ ID No.2-11.

[0044] In one embodiment, the amino acid sequence of the parent adenosine deaminase is shown in any one of SEQ ID No. 2-11.

[0045] In one embodiment, the amino acid sequence of the parent adenosine deaminase is shown in either SEQ ID No. 2 or SEQ ID No. 7.

[0046] In one embodiment, the adenosine deaminase is selected from any one of the following groups I-III:

[0047] I. An adenosine deaminase obtained by generating mutations at any one or more of the following amino acid positions of the amino acid sequence shown in SEQ ID No.7: 55, 110, 113, 123, 126, 127, 153, 156, 159, 160, 161, 167; or, an adenosine deaminase obtained by generating mutations at the following amino acid positions of the amino acid sequence shown in SEQ ID No.2: 106 and / or 157;

[0048] II. Compared with the adenosine deaminase described in I, it has the mutation site described in I; and compared with the adenosine deaminase described in I, it has an adenosine deaminase with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

[0049] III. Compared with the adenosine deaminase described in I, it has the mutation site described in I; and, compared with the adenosine deaminase described in I, it has a sequence of one or more amino acid substitutions, deletions or additions; the one or more amino acids include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions.

[0050] In the present invention, adenosine deaminase, also known as adenine deaminase, catalyzes the hydrolytic deamination of adenine or adenosine. The adenosine deaminase provided herein (e.g., engineered adenosine deaminase, evolved adenosine deaminase) can be from any organism, such as bacteria.

[0051] In some embodiments, the adenosine deaminase is a naturally occurring adenosine deaminase, or a variant that is mutated but still has adenosine deaminase activity.

[0052] In some embodiments, the adenosine deaminase is from a prokaryotic organism. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from Escherichia coli, Escherichia, Citrobacter rodentium or Citrobacter, Rahnella aceris, Shigella flexneri, or Salmonella enterica subsp.

[0053] In a preferred embodiment, the adenosine deaminase described in the present invention is TadA, and the TadA includes any TadA or its variants in WO2021158921A2, such as TadA7.10, TadA-8e, and TadA-8eV106W.

[0054] In a preferred embodiment, the adenosine deaminase of the present invention is TadA8e, and the amino acid sequence is shown in SEQ ID No.2.

[0055] In a preferred embodiment, the adenosine deaminase GZ-71 of the present invention has an amino acid sequence as shown in SEQ ID No. 3, and is derived from Enterobacteriaceae.

[0056] In a preferred embodiment, the adenosine deaminase GZ-68 of the present invention has an amino acid sequence as shown in SEQ ID No. 4, and is derived from Escherichia coli.

[0057] In a preferred embodiment, the adenosine deaminase GZ-65 of the present invention has an amino acid sequence as shown in SEQ ID No. 5, and is derived from Citrobacter rodentium.

[0058] In a preferred embodiment, the adenosine deaminase GZ-64 of the present invention has an amino acid sequence as shown in SEQ ID No. 6, and is derived from Citrobacter.

[0059] In a preferred embodiment, the adenosine deaminase GZ-46 of the present invention has an amino acid sequence as shown in SEQ ID No. 7, and is derived from Rahnella aceris.

[0060] In a preferred embodiment, the adenosine deaminase GZ-74 of the present invention has an amino acid sequence as shown in SEQ ID No. 8, and is derived from Shigella flexneri.

[0061] In a preferred embodiment, the adenosine deaminase GZ-67 of the present invention has an amino acid sequence as shown in SEQ ID No. 9, and is derived from Salmonella enterica subsp.

[0062] In one aspect, the present invention provides a fusion protein comprising a DNA binding protein and the above-mentioned adenosine deaminase.

[0063] In one embodiment, the DNA binding protein has a nucleic acid programmable DNA binding protein (napDNAbp) domain, and the "DNA binding protein" or "DNA binding protein domain" refers to any protein that can locate and bind to a specific target DNA nucleotide sequence (e.g., a genomic locus). In one embodiment, the DNA binding protein is a Cas protein, and the Cas protein is selected from any one of Cas9, Cas9n, dCas9, CasX, CasY, C2cl, C2c2, C2c3, GeoCas9, CjCas9, Casl2a, Casl2b, Casl2g, Casl2h, Casl2i, Cas12j, Casl3b, Casl3c, Casl3d, Casl4, Csn2, xCas9, Cas9-NG, LbCasl2a, enAsCasl2a, Cas9-KKH, circular displacement Cas9, Argonaute (Ago) domain, SmacCas9, Spy-macCas9, SpGas9-NRRH, SpaCas9-NRTH, SpaCas9-NRCH, Cas9-NG-CP1041, Cas9-NG-VRQR, dCas12i, nCas12i and variants thereof.

[0064] In one embodiment, the Cas protein is Cas12i.

[0065] In one embodiment, the Cas protein may be naturally occurring or non-naturally occurring and engineered.

[0066] In one embodiment, the Cas protein is a nuclease-inactivated Cas protein (dCas).

[0067] In one embodiment, the Cas protein is a dCas12i3 protein, and the amino acid sequence of the wild-type Cas12i is shown in SEQ ID No.1; compared with SEQ ID No.1, the dCas12i protein has an E mutation at position 844 mutated to A or a D mutation at position 619 mutated to A.

[0068] In one embodiment, the Cas protein is a Cas12i mutant protein, and the amino acid sequence of the wild-type Cas12i is shown in SEQ ID No.1; compared with SEQ ID No.1, the Cas12i mutant protein has S at position 7 mutated to R, D at position 233 mutated to R, D at position 267 mutated to R, N at position 369 mutated to R, and S at position 433 mutated to R.

[0069] In one embodiment, the Cas protein is a dCas12i mutant protein, and the amino acid sequence of the wild-type Cas12i is shown in SEQ ID No.1; compared with SEQ ID No.1, the dCas12i mutant protein has E at position 844 mutated to A, S at position 7 mutated to R, D at position 233 mutated to R, D at position 267 mutated to R, N at position 369 mutated to R, and S at position 433 mutated to R.

[0070] In one embodiment, the DNA binding protein in the fusion protein is fused to the N-terminus of adenosine deaminase. In other embodiments, the DNA binding protein in the fusion protein is fused to the C-terminus of adenosine deaminase.

[0071] In one embodiment, the DNA binding protein and adenosine deaminase in the fusion protein are connected via a linker.

[0072] In one embodiment, the linker is an XTEN linker.

[0073] In one embodiment, the fusion protein of the present invention further comprises a nuclear localization sequence (NLS). In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In other embodiments, both the N-terminus and the C-segment of the fusion protein are connected to the NLS.

[0074] In some embodiments, the NLS is fused to the N-terminus of the Cas protein. In some embodiments, the NLS is fused to the C-terminus of the Cas protein. In some embodiments, the NLS is fused to the N-terminus of the deaminase. In some embodiments, the NLS is fused to the C-terminus of the deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker.

[0075] It is clear to those skilled in the art that the structure of a protein can be changed without adversely affecting its activity and functionality, for example, one or more conservative amino acid substitutions can be introduced into the amino acid sequence of a protein without adversely affecting the activity and / or three-dimensional structure of the protein molecule. Examples and embodiments of conservative amino acid substitutions are clear to those skilled in the art. Specifically, the amino acid residue can be replaced with another amino acid residue belonging to the same group as the site to be replaced, that is, a non-polar amino acid residue can be substituted for another non-polar amino acid residue, a polar uncharged amino acid residue can be substituted for another polar uncharged amino acid residue, a basic amino acid residue can be substituted for another basic amino acid residue, and an acidic amino acid residue can be substituted for another acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. As long as the substitution does not result in the inactivation of the biological activity of the protein, a conservative substitution in which an amino acid is replaced by other amino acids belonging to the same group falls within the scope of the present invention. Therefore, the protein of the present invention may contain one or more conservative substitutions in the amino acid sequence, and these conservative substitutions are preferably generated by substitution according to Table 1. In addition, the present invention also covers proteins that also contain one or more other non-conservative substitutions, as long as the non-conservative substitutions do not significantly affect the desired function and biological activity of the protein of the present invention.

[0076] Conservative amino acid substitutions can be performed at one or more predicted non-essential amino acid residues. "Non-essential" amino acid residues are amino acid residues that can be changed (deleted, substituted or replaced) without changing biological activity, while "essential" amino acid residues are required for biological activity. "Conservative amino acid substitutions" are substitutions in which amino acid residues are replaced by amino acid residues with similar side chains. Amino acid substitutions can be performed in the non-conserved regions of the above-mentioned Cas mutant proteins or fusion proteins. In general, such substitutions are not performed on conserved amino acid residues, or are not performed on amino acid residues located in conserved motifs, where such residues are required for protein activity. However, it will be appreciated by those skilled in the art that functional variants may have fewer conservative or non-conservative changes in conserved regions.

[0077] Table 1

[0078]

[0079]

[0080] It is well known in the art that one or more amino acid residues can be changed (replaced, deleted, truncated or inserted) from the N and / or C terminus of a protein while still retaining its functional activity. Therefore, proteins in which one or more amino acid residues are changed from the N and / or C terminus of a Cas mutant protein while retaining its desired functional activity are also within the scope of the present invention. These changes may include changes introduced by modern molecular methods such as PCR, which includes PCR amplification of a protein coding sequence by means of including an amino acid coding sequence in an oligonucleotide used in PCR amplification to change or extend the protein coding sequence.

[0081] It will be appreciated that proteins can be altered in a variety of ways, including amino acid substitutions, deletions, truncations and insertions, and methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the above-mentioned proteins can be prepared by mutations in the DNA. Other forms of mutagenesis and / or directed evolution can also be accomplished, for example, using known mutagenesis, recombination and / or shuffling methods, in combination with related screening methods, to perform single or multiple amino acid substitutions, deletions and / or insertions.

[0082] Those skilled in the art will appreciate that these minor amino acid changes in the Cas proteins of the present invention can occur (e.g., naturally occurring mutations) or be generated (e.g., using r-DNA technology) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site, or other functional domain of the protein, the properties of the polypeptide may be changed, but the polypeptide may retain its activity. If the mutations present are not close to the catalytic domain, active site, or other functional domain, lesser effects may be expected.

[0083] Those skilled in the art can identify the essential amino acids of the Cas mutant protein of the present invention according to methods known in the art, such as site-directed mutagenesis or protein evolution or analysis of a bioinformatics system. The catalytic domain, active site or other functional domain of the protein can also be determined by physical analysis of the structure, such as by the following techniques: such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, combined with mutations of amino acids at putative key sites.

[0084] In the present invention, amino acid residues can be represented by single letters or three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamine (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), arginine (Arg, R).

[0085] The term "AxxB" means that the amino acid A at position xx is changed to amino acid B, for example, E844A means that E at position 844 is mutated to A. When multiple amino acid positions are mutated at the same time, a similar form of S7R-D233R-D267R-N369R-S433R can be used to express it, for example, S7R-D233R-D267R-N369R-S433R means that S at position 7 is mutated to R, D at position 233 is mutated to R, D at position 267 is mutated to R, N at position 369 is mutated to R and S at position 433 is mutated to R.

[0086] The specific amino acid position (number) within the protein of the invention is determined by aligning the amino acid sequence of the target protein with the target sequence (e.g., SEQ ID No. 1) using standard sequence alignment tools, such as using the Smith-Waterman algorithm or using the CLUSTALW2 algorithm to align the two sequences, wherein the sequences are considered aligned when the alignment score is the highest. The alignment score can be calculated according to the method described in Wilbur, WJ and Lipman, DJ (1983) Rapid similarity searches of nucleic acid and protein databanks. Proc. Natl. Acad. Sci. USA, 80: 726-730. Preferably, the default parameters are used in the ClustalW2 (1.82) algorithm: protein gap opening penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein / DNA end gap = -1; protein / DNAGAPDIST = 4. Preferably, the AlignX program (part of the vectorNTI group) is used with default parameters suitable for multiple alignments (gap opening penalty: 10og gap extension penalty 0.05) to determine the position of specific amino acids in the protein of the present invention by comparing the amino acid sequence of the protein with SEQ ID No. 1. Those skilled in the art can use software commonly used in the art, such as Clustal Omega, to compare and align the amino acid sequence of any parent Cas protein with SEQ ID NO. 1 for sequence identity, and then obtain the amino acid sites in the parent Cas protein corresponding to the amino acid sites defined based on SEQ ID NO. 1 described in the present application.

[0087] The fusion protein of the present invention is not limited by the production method thereof, for example, it can be produced by genetic engineering methods (recombinant technology) or by chemical synthesis methods.

[0088] The present invention also provides a base editing tool comprising the above-mentioned fusion protein, for example, a single base editing tool.

[0089] Nucleic Acids

[0090] In another aspect, the present invention provides an isolated polynucleotide comprising:

[0091] (a) a polynucleotide sequence encoding an adenosine deaminase or fusion protein of the present invention;

[0092] (b) a polynucleotide whose sequence is shown in any one of SEQ ID No. 12-20;

[0093] (c) a sequence having one or more base substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 base substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID No. 12-20;

[0094] (d) a polynucleotide having a nucleotide sequence homology of ≥80% (preferably ≥90%, more preferably ≥95%, and most preferably ≥98%) to any one of SEQ ID Nos. 12-20, and encoding a polypeptide as shown in any one of SEQ ID Nos. 3-11; or,

[0095] (e) A polynucleotide complementary to the polynucleotide described in any one of (a) to (d).

[0096] In one embodiment, the nucleotide sequence is codon optimized for expression in prokaryotes. In one embodiment, the nucleotide sequence is codon optimized for expression in eukaryotic cells.

[0097] In one embodiment, the cell is an animal cell, eg, a mammalian cell.

[0098] In one embodiment, the cell is a human cell.

[0099] In one embodiment, the cell is a plant cell, such as a cell of a cultivated plant (such as cassava, corn, sorghum, wheat, or rice), algae, tree, or vegetable.

[0100] In one embodiment, the polynucleotide is preferably single-stranded or double-stranded.

[0101] Guide RNA (gRNA)

[0102] On the other hand, the present invention provides a gRNA, which includes a protein binding sequence and a targeting sequence that targets a target nucleic acid.

[0103] The protein binding sequence of the gRNA can interact with the DNA binding protein (Cas protein) in the fusion protein of the present invention, thereby forming a complex between the DNA binding protein (Cas protein) and the gRNA.

[0104] Designing gRNAs that interact with the Cas proteins of the present invention is common technical knowledge in the art. For example, for Cas9 proteins and Cas proteins of the Cas12 family (including but not limited to Cas12a, Cas12b, Cas12i, etc.), designing gRNAs that interact with them does not require creative labor.

[0105] The targeting sequence of the targeting nucleic acid of the present invention comprises a nucleotide sequence complementary to the sequence in the target nucleic acid. In other words, the targeting sequence of the targeting nucleic acid of the present invention or the targeting segment of the targeting nucleic acid interacts with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). Therefore, the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid can be changed, or can be modified to hybridize any desired sequence in the target nucleic acid. The nucleic acid is selected from DNA or RNA.

[0106] The percent complementarity between the targeting sequence of a targeting nucleic acid or the targeting segment of a targeting nucleic acid and the target sequence of a target nucleic acid can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%).

[0107] The "protein binding sequence" of the gRNA of the present invention can interact with the CRISPR protein (or, Cas protein). The gRNA of the present invention guides its interacting DNA binding protein to a specific nucleotide sequence in the target nucleic acid through the action of the targeting sequence of the targeting nucleic acid.

[0108] The gRNA of the present invention can form a complex with the DNA binding protein (Cas protein).

[0109] Carrier

[0110] The present invention also provides a vector, which comprises the adenosine deaminase, fusion protein, isolated nucleic acid molecule or polynucleotide as described above; preferably, it also comprises a regulatory element operably linked thereto.

[0111] In one embodiment, the regulatory element is selected from one or more of the following groups: enhancer, transposon, promoter, terminator, leader sequence, polyadenylation sequence, marker gene.

[0112] In one embodiment, the vector includes a cloning vector, an expression vector, a shuttle vector, and an integration vector.

[0113] In some embodiments, the vector included in the system is a viral vector (e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated vector, and a herpes simplex vector), and can also be a plasmid, a virus, a cosmid, a phage, etc., which are well known to those skilled in the art.

[0114] Base editing system

[0115] The present invention provides an engineered non-naturally occurring base editing system, which comprises the above-mentioned fusion protein or a nucleic acid sequence encoding the fusion protein and a nucleic acid encoding one or more of the above-mentioned guide RNAs, wherein the DNA binding protein in the fusion protein is a Cas protein, and the gRNA can bind to the Cas protein.

[0116] In one embodiment, the nucleic acid sequence encoding the fusion protein and the nucleic acid encoding one or more guide RNAs are artificially synthesized.

[0117] In one embodiment, the nucleic acid sequence encoding the fusion protein and the nucleic acid encoding one or more guide RNAs do not naturally co-occur.

[0118] The one or more guide RNAs target one or more target sequences in the cell. The one or more target sequences hybridize with the genomic loci of the DNA molecules encoding the one or more gene products, and guide the fusion protein to the genomic loci of the DNA molecules encoding the one or more gene products. After the fusion protein reaches the target sequence position, the target sequence is modified and edited, thereby changing or modifying the expression of the one or more gene products.

[0119] The cells of the present invention include one or more of animals, plants or microorganisms.

[0120] In some embodiments, the fusion protein is codon optimized for expression in a cell.

[0121] The present invention also provides an engineered non-naturally occurring vector system, which may include one or more vectors, wherein the one or more vectors include:

[0122] a) a first regulatory element, which is operably linked to the gRNA,

[0123] b) a second regulatory element, which is operably linked to the fusion protein;

[0124] Components (a) and (b) are located on the same or different carriers of the system.

[0125] The first and second regulatory elements include a promoter (e.g., a constitutive promoter or an inducible promoter), an enhancer (e.g., a 35S promoter or a 35S enhanced promoter), an internal ribosome entry site (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).

[0126] In some embodiments, the vector in the system is a viral vector (e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated vector, and a herpes simplex vector), and can also be a plasmid, a virus, a cosmid, a phage, etc., which are well known to those skilled in the art.

[0127] In some embodiments, the systems provided herein are in a delivery system. In some embodiments, the delivery system is a nanoparticle, a liposome, an exosome, a microbubble, and a gene gun.

[0128] In one embodiment, the target sequence is a DNA or RNA sequence from a prokaryotic cell or a eukaryotic cell. In one embodiment, the target sequence is a non-naturally occurring DNA or RNA sequence.

[0129] In one embodiment, the target sequence is present in a cell. In one embodiment, the target sequence is present in the nucleus or in the cytoplasm (e.g., an organelle). In one embodiment, the cell is a eukaryotic cell. In other embodiments, the cell is a prokaryotic cell.

[0130] Protein-nucleic acid complexes / compositions

[0131] In another aspect, the present invention provides a compound or composition comprising:

[0132] (i) a protein component selected from: the above-mentioned fusion protein, wherein the DNA binding protein in the fusion protein is a Cas protein;

[0133] (ii) a nucleic acid component comprising (a) a guide sequence capable of hybridizing to a target sequence; and (b) a protein binding sequence capable of binding to a DNA binding protein in a fusion protein of the present invention.

[0134] The protein component and the nucleic acid component can bind to each other to form a complex.

[0135] In one embodiment, the nucleic acid component is a guide RNA in a CRISPR-Cas system or a base editing system.

[0136] In one embodiment, the complex or composition is non-naturally occurring or modified. In one embodiment, at least one component of the complex or composition is non-naturally occurring or modified. In one embodiment, the first component is non-naturally occurring or modified; and / or, the second component is non-naturally occurring or modified.

[0137] Delivery and delivery compositions

[0138] The fusion proteins, gRNAs, nucleic acid molecules, vectors, systems, complexes and compositions of the present invention can be delivered by any method known in the art. Such methods include, but are not limited to, electroporation, lipofection, nuclear transfection, microinjection, sonoporation, gene guns, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendritic transfection, heat shock transfection, nuclear transfection, magnetofection, lipofection, puncture transfection, optical transfection, agent-enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, viral particles, artificial virions, etc.

[0139] Therefore, in another aspect, the present invention provides a delivery composition comprising a delivery vector and one or more selected from the following: a fusion protein, a gRNA, a nucleic acid molecule, a vector, a system, a complex and a composition of the present invention.

[0140] In one embodiment, the delivery vehicle is a particle.

[0141] In one embodiment, the delivery vehicle is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns or viral vectors (e.g., replication-defective retroviruses, lentiviruses, adenoviruses or adeno-associated viruses).

[0142] Host cells

[0143] The present invention also relates to an in vitro, ex vivo or in vivo cell or cell line or their progeny, wherein the cell or cell line or their progeny comprises: the fusion protein, nucleic acid molecule, protein-nucleic acid complex, vector, and delivery composition of the present invention.

[0144] In certain embodiments, the cell is a prokaryotic cell.

[0145] In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is a non-human mammalian cell, such as a cell of a non-human primate, a cow, a sheep, a pig, a dog, a monkey, a rabbit, a rodent (such as a rat or a mouse). In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as a cell of a poultry bird (such as a chicken), a fish or a crustacean (such as a clam, a shrimp). In certain embodiments, the cell is a plant cell, such as a cell or a cultivated plant or a food crop such as cassava, corn, sorghum, soybean, wheat, oat or rice, such as algae, a tree or a production plant, a fruit or a vegetable (for example, a tree such as a citrus tree, a nut tree; Solanum, cotton, tobacco, tomato, grape, coffee, cocoa, etc.).

[0146] In certain embodiments, the cell is a stem cell or a stem cell line.

[0147] In certain cases, the host cells of the invention contain genetic or genomic modifications that are not present in their wild type.

[0148] Gene Editing Methods and Applications

[0149] The fusion protein, nucleic acid, composition, base editing system, vector system, delivery composition or host cell of the present invention can be used for any one or more of the following purposes: gene editing; targeting and / or editing target nucleic acid; specifically editing double-stranded nucleic acid; base editing double-stranded nucleic acid; base editing single-stranded nucleic acid. In other embodiments, it can also be used to prepare reagents or kits for any one or more of the above purposes.

[0150] The present invention also provides a method for editing nucleic acid, the method comprising the step of contacting a target region of a nucleic acid (e.g., a double-stranded DNA sequence) with a complex comprising the above-mentioned fusion protein and gRNA; wherein the target region comprises a targeted base pair, and the targeted base pair in the target region is base replaced. In one embodiment, the deaminase in the fusion protein is adenosine deaminase, and the targeted base pair is replaced by A:T to G:C.

[0151] The above A:T means that the bases paired in the base pair are A and T; similarly, G:C means that the bases paired in the base pair are G and C.

[0152] The present invention also provides the use of the fusion protein, the nucleic acid, the above-mentioned composition, the above-mentioned base editing system, the above-mentioned vector system, the above-mentioned delivery composition or the above-mentioned host cell in gene editing; or, the use in preparing a reagent or kit for gene editing.

[0153] In one embodiment, the gene editing is performed inside and / or outside the cell.

[0154] In one embodiment, the gene editing is single base editing of the target gene.

[0155] The present invention also provides a method for editing a target nucleic acid, the method comprising contacting the target nucleic acid with the above-mentioned fusion protein, nucleic acid, composition, base editing system, vector system or delivery composition. In one embodiment, the method is to edit the target nucleic acid inside or outside the cell.

[0156] The gene editing or editing of the target nucleic acid includes the step of editing a single base of the target gene.

[0157] The editing can be performed in prokaryotic cells and / or eukaryotic cells.

[0158] On the other hand, the present invention also provides a kit for gene editing, which comprises the above-mentioned adenosine deaminase, fusion protein, gRNA, nucleic acid, the above-mentioned composition, the above-mentioned base editing system, the above-mentioned vector system, the above-mentioned delivery composition or the above-mentioned host cell.

[0159] On the other hand, the invention provides the use of the adenosine deaminase, the fusion protein, the nucleic acid, the composition, the base editing system, the vector system, the delivery composition or the host cell in the preparation of a preparation or a kit, wherein the preparation or the kit is used for:

[0160] (i) gene or genome editing;

[0161] (ii) editing a target sequence in a target locus to modify an organism;

[0162] (iii) single base editing;

[0163] (iv) Treatment of disease.

[0164] Preferably, the above-mentioned gene or genome editing is performed inside or outside the cell.

[0165] Preferably, the treatment of the disease is the treatment of a condition caused by a defect in the target sequence in the target locus.

[0166] Method for specifically modifying target nucleic acid

[0167] On the other hand, the present invention also provides a method for specifically modifying a target nucleic acid, the method comprising: contacting the target nucleic acid with the above-mentioned fusion protein, nucleic acid, the above-mentioned composition, the above-mentioned base editing system, the above-mentioned vector system or the above-mentioned delivery composition.

[0168] The specific modification can occur in vivo or in vitro.

[0169] The specific modification can occur inside or outside the cell.

[0170] In some cases, the cell is selected from a prokaryotic cell or a eukaryotic cell, for example, an animal cell, a plant cell, or a microbial cell.

[0171] Adenosine deaminase

[0172] As used herein, the term "adenosine deaminase" catalyzes the hydrolytic deamination of the nucleobase adenine. The adenosine deaminase provided herein (such as an engineered adenosine deaminase or an optimized adenosine deaminase) can be an enzyme that converts adenosine (A) in DNA into inosine (I), and such an adenosine deaminase can cause the conversion of A:T to G:C base pairs. For example, in some embodiments, the amino acid sequence of the adenosine deaminase has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identity compared to any one of the sequences of SEQ ID No.2-11.

[0173] DNA Binding Proteins

[0174] As used herein, the term "DNA binding protein" or "DNA binding protein domain" is any protein designated to be located and bound to a specific target DNA nucleotide sequence (e.g., a locus of a genome). The term includes RNA-programmable proteins that bind (e.g., form a complex) to one or more nucleic acid molecules (i.e., including, for example, guide RNAs in the case of a Cas system) that guide or otherwise program the protein to localize to a specific target nucleotide sequence (e.g., a DNA sequence) that is complementary to the one or more nucleic acid molecules (or portions or regions thereof) to which the protein binds. Exemplary RNA programmable proteins are CRISPR-Cas9 proteins, as well as Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or modified), and can include Cas9 equivalents from any type of CRISPR system (e.g., type II, type V, type VI), including Cpf1 (type V CRISPR-Cas system), C2cl (type V CRISPR-Cas system), C2c2 (type VI CRISPR-Cas system), C2c3 (type V CRISPR-Cas system), dCas9, GeoCas9, CjCas9, Cas 12a, Cas 12b, Cas12c, Cas12d, Cas12g, Cassi2h, Cas12i, dCas12i, and other further Cas equivalents.

[0175] CRISPR system

[0176] As used herein, the term "Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated (Cas) (CRISPR-Cas) System" or "CRISPR System" is used interchangeably and has the meaning generally understood by those skilled in the art, which generally includes a transcription product or other element related to the expression of a CRISPR-associated ("Cas") gene, or a transcription product or other element capable of directing the activity of the Cas gene. The Cas protein in the present invention is Crisprassociated protein.

[0177] CRISPR / Cas complex

[0178] As used herein, the term "CRISPR / Cas complex" refers to a complex formed by the binding of a guide RNA or mature crRNA to a Cas protein, which comprises a direct repeat sequence that hybridizes to a guide sequence of a target sequence and binds to the Cas protein, and the complex is capable of recognizing and cleaving a polynucleotide that can hybridize to the guide RNA or mature crRNA.

[0179] Guide RNA (gRNA)

[0180] As used herein, the terms "guide RNA (gRNA)", "mature crRNA", "guide sequence" are used interchangeably and have meanings generally understood by those skilled in the art. In general, the guide RNA may comprise a direct repeat sequence and a guide sequence, or may consist essentially of or consist of a direct repeat sequence and a guide sequence.

[0181] In some cases, the guide sequence is any polynucleotide sequence that has sufficient complementarity with the target sequence to hybridize with the target sequence and guide the specific binding of the CRISPR / Cas complex to the target sequence. In one embodiment, when optimally aligned, the degree of complementarity between the guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Determining the optimal alignment is within the capabilities of ordinary technicians in this field. For example, there are publicly available and commercially available alignment algorithms and programs, such as, but not limited to, ClustalW, Smith-Waterman algorithm in matlab, Bowtie, Geneious, Biopython, and SeqMan.

[0182] Target sequence

[0183] "Target sequence" refers to a polynucleotide targeted by a guide sequence in a gRNA, such as a sequence having complementarity with the guide sequence, wherein hybridization between the target sequence and the guide sequence will promote the formation of a CRISPR / Cas complex (including Cas protein and gRNA). Complete complementarity is not required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR / Cas complex.

[0184] The target sequence can comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located inside or outside the cell. In some cases, the target sequence is located in the nucleus or cytoplasm of the cell. In some cases, the target sequence may be located in an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. A sequence or template that can be used to recombine into a target locus comprising the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In one embodiment, the editing template is an exogenous nucleic acid. In one embodiment, the recombination is homologous recombination.

[0185] In the present invention, "target sequence" or "target polynucleotide" or "target nucleic acid" can be any endogenous or exogenous polynucleotide to a cell (e.g., a eukaryotic cell). For example, the target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). In some cases, the target sequence should be associated with a protospacer adjacent motif (PAM).

[0186] Base editing

[0187] The term "base editing" refers to a genome editing technique, which involves converting a specific nucleic acid base into another base at a targeted genomic locus. In certain embodiments, this can be achieved without the need for double-stranded DNA breaks (DSBs) or single-strand breaks (nicking). So far, other genome editing techniques including systems based on CRISPR have started with the introduction of DSBs at the seat of interest. Subsequently, cellular DNA repair enzymes repair the break, typically resulting in random insertions or deletions (indels) of bases at the DSB site. However, when it is desired to introduce or correct point mutations at the target locus rather than random destruction of the entire gene, these genome editing techniques are not suitable because the correction rate is low (usually 0.1% to 5%), and the main genome editing product is indel. In order to increase the efficiency of gene correction without introducing Rando indel, the inventors use adenosine deaminase in combination with the CRISPR system to directly convert a DNA base into another DNA base without forming DSBs.

[0188] wild type

[0189] As used herein, the term "wild type" has the meaning generally understood by those skilled in the art, which refers to the typical form of an organism, strain, gene, or the characteristics that distinguish it from mutant or variant forms when it exists in nature, which can be isolated from a source in nature and has not been intentionally modified by man.

[0190] Not naturally occurring

[0191] As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably and indicate the involvement of human effort. When these terms are used to describe a nucleic acid molecule or polypeptide, it means that the nucleic acid molecule or polypeptide is at least substantially free from at least one other component with which it is associated in nature or as found in nature.

[0192] Identity

[0193] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0194] Carrier

[0195] The term "vector" refers to a nucleic acid molecule that is capable of transporting another nucleic acid molecule to which it is attached. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules including one or more free ends, no free ends (e.g., circular); nucleic acid molecules including DNA, RNA, or both; and other various polynucleotides known in the art. The vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material elements it carries are expressed in the host cell. A vector can be introduced into a host cell to produce transcripts, proteins, or peptides, including proteins, fusion proteins, isolated nucleic acid molecules, etc. as described herein (e.g., CRISPR transcripts, such as nucleic acid transcripts, proteins, or enzymes). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription start sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication initiation site.

[0196] One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.

[0197] Another type of vector is a viral vector, wherein a virally derived DNA or RNA sequence is present in a vector for packaging a virus (e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an adeno-associated virus). The viral vector also comprises a polynucleotide carried by a virus for transfection into a host cell. Some vectors (e.g., bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) can replicate autonomously in the host cell into which they are introduced.

[0198] Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell, and are replicated together with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to as "expression vectors" herein.

[0199] Host cells

[0200] As used herein, the term "host cell" refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, eukaryotic cells such as microbial cells, fungal cells, animal cells and plant cells.

[0201] Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the level of expression desired, and the like.

[0202] Regulatory elements

[0203] As used herein, the term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences), which are described in detail in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, California (1990). In some cases, regulatory elements include those sequences that direct the constitutive expression of a nucleotide sequence in many types of host cells and those sequences that direct the nucleotide sequence to be expressed only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in desired tissues of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or special cell types (e.g., lymphocytes). In some cases, the regulatory element can also direct expression in a timing-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue or cell type specific. In some cases, the term "regulatory element" encompasses enhancer elements such as WPRE; CMV enhancer; R-U5' fragment in the LTR of HTLV-I ((Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); SV40 enhancer; and intron sequences between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

[0204] Promoter

[0205] As used herein, the term "promoter" has a meaning well known to those skilled in the art, and refers to a non-coding nucleotide sequence located upstream of a gene that can initiate expression of a downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of a gene product in a cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell essentially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of a gene product in the cell essentially only when the cell is a cell of the tissue type corresponding to the promoter.

[0206] NLS

[0207] A "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" a protein for import into the cell nucleus by nuclear transport, i.e., a protein with an NLS is transported to the cell nucleus. Typically, an NLS comprises a positively charged Lys or Arg residue exposed on the surface of the protein. Exemplary nuclear localization sequences include, but are not limited to, NLSs from: SV40 large T antigen, EGL-13, c-Myc, and TUS proteins. In some embodiments, the NLS comprises the sequence MKRTADGSEFESPKKKRKV. In some embodiments, the NLS comprises the sequence KRPAATKKAGQAKKKK. Other nuclear localization sequences include, but are not limited to, the acidic M9 domain of hnRNPA1, the sequence KIPIK in the yeast transcription repressor Matα2, and PY-NLS.

[0208] Operatively connected

[0209] As used herein, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0210] Complementarity

[0211] As used herein, the term "complementarity" refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence by means of traditional Watson-Crick or other non-traditional types. The percentage of complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Complete complementarity" means that all consecutive residues of a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or to two nucleic acids that hybridize under stringent conditions.

[0212] Strict conditions

[0213] As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity with a target sequence predominantly hybridizes to the target sequence and substantially does not hybridize to non-target sequences. Stringent conditions are typically sequence-dependent and vary depending on many factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence.

[0214] Hybridization

[0215] The terms "hybridize" or "complementary" or "substantially complementary" refer to a nucleic acid (e.g., RNA, DNA) comprising a nucleotide sequence that enables it to non-covalently bind, i.e., form base pairs and / or G / U base pairs, "anneal" or "hybridize" with another nucleic acid in a sequence-specific, anti-parallel manner (i.e., nucleic acids specifically bind to complementary nucleic acids).

[0216] Hybridization requires that the two nucleic acids contain complementary sequences, although there may be mismatches between the bases. Suitable conditions for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, which are variables well known in the art. Typically, the length of a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

[0217] It should be understood that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to specifically hybridize. A polynucleotide may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to the target region in the target nucleic acid sequence with which it hybridizes.

[0218] The hybridization of the target sequence and the gRNA represents that at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the nucleic acid sequences of the target sequence and the gRNA can hybridize to form a complex; or represents that at least 12, 15, 16, 17, 18, 19, 20, 21, 22 or more bases of the nucleic acid sequences of the target sequence and the gRNA can complementarily pair and hybridize to form a complex.

[0219] Express

[0220] As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

[0221] Connectors

[0222] As used herein, the term "joint" refers to a linear polypeptide formed by connecting a plurality of amino acid residues via peptide bonds. The joint of the present invention can be an artificially synthesized amino acid sequence, or a naturally occurring polypeptide sequence, such as a polypeptide having a hinge region function. Such joint polypeptides are well known in the art (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ et al. (1994) Structure 2: 1121-1123).

[0223] treat

[0224] As used herein, the term "treat" refers to treating or curing a disorder, delaying the onset of symptoms of a disorder, and / or delaying the progression of a disorder.

[0225] Subjects

[0226] As used herein, the term "subject" includes, but is not limited to, various animals, plants, and microorganisms.

[0227] animal

[0228] For example, mammals, such as bovines, equines, ovines, porcines, canines, felines, lagomorphs, rodents (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys), or humans. In certain embodiments, the subject (e.g., a human) suffers from a disorder (e.g., a disorder caused by a disease-related gene defect).

[0229] plant

[0230] The term "plant" is to be understood as any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops, including artichokes, Brussels sprouts, rocket, leeks, asparagus, lettuce (e.g., head lettuce, leaf lettuce, romaine lettuce), bok choy, yellow taro, melons (e.g., melon, watermelon, Crenshaw melon, honeydew melon, cantaloupe), rapeseed crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, kale, Chinese cabbage, pak choi), cardoon, carrot, napa, okra, onion, celery, parsley, chickpeas, parsnips, endive, peppers, potatoes, cucurbits (e.g., zucchini, cucumber, courgette, squash, pumpkin), radish, bulb onions, rutabagas, eggplant (also called eggplant), salsify, lettuce, shallots, endive, garlic, spinach, green onions, squash, greens, beets (sugar beets and fodder beets), sweet potatoes, Swiss chard, horseradish, tomatoes, turnips, and spices; fruits and / or vines such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, citrus, blueberries, boysenberries, erry), cranberries, currants, loganberries, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwis, persimmons, pomegranates, pineapples, tropical fruits, pomegranates, melons, mangoes, papayas, and lychees; field crops such as clover, alfalfa, evening primrose, silver grass, corn / maize (fodder corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grain cereals (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas beans, soybeans), oil plants (rapeseed, mustard, olives, sunflowers, coconuts, castor oil plants, cocoa beans, peanuts), Arabidopsis, fiber plants (cotton, flax, jute), Lauraceae (cinnamon, camphor), or a plant such as coffee, sugar cane, tea, and natural rubber plants; and / or bedding plants, such as flowering plants, cacti, succulents and / or ornamental plants, and trees such as forests (broadleaf trees and evergreen trees, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, as well as shrubs and other seedlings.

[0231] Advantageous Effects of the Invention

[0232] The present invention obtains a variety of adenosine deaminases by screening and fuses them with DNA binding proteins, which can be used for single-base editing of target nucleic acids and have broad application prospects.

[0233] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments.

[0234] The sequence information involved in this application is as follows:

[0235]

[0236]

[0237] BRIEF DESCRIPTION OF THE DRAWINGS

[0238] Figure 1 .Schematic diagram of the structure of the ABE single-base editing tool.

[0239] Figure 2 .Comparison of editing efficiency of ABE single-base editing tools composed of different adenosine deaminases and dCas12i3.

[0240] Figure 3 .Schematic diagram of the structure of the ABE single-base editing tool.

[0241] Figure 4 .Verification results of base editing efficiency of ABE vectors composed of different adenosine deaminases GZ-46 and dCas12i3.

[0242] Figure 5 .Verification results of base editing efficiency of ABE vector composed of adenosine deaminase with different combinations of mutations and dCas12i3.

[0243] Figure 6 .Verification results of base editing efficiency of ABE vector composed of adenosine deaminase with different combinations of mutations and dCas12i3.

[0244] Figure 7 .Verification results of base editing efficiency of ABE vectors composed of different adenosine deaminases and dCas12i3. DETAILED DESCRIPTION

[0245] The following examples are only used to describe the present invention, but not to limit the present invention. Unless otherwise specified, the experiment and method described in the embodiment are carried out basically according to the conventional methods well known in the art and described in various references. For example, the conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, "Molecular cloning: laboratory manual" (MOLECULAR CLONING: A LABORATORY MANUAL), 2nd edition (1989); "Contemporary molecular biology experimental manual" (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (FM Ausubel (FM Ausubel) et al. edited, (1987)); "Enzymology" (METHODS IN ENZYMOLOGY) series (Academic Publishing Company): "PCR 2: Practical method" (PCR 2: A PRACTICAL METHOD) APPROACH) (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)), ANTIBODIES, A LABORATORY MANUAL, eds. Harlow and Lane, eds. (1988), and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)).

[0246] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0247] Example 1. Editing activity of single-base editing tools constructed with different deaminases

[0248] The applicant used the dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein with inactivated nuclease activity and multiple adenosine deaminases (GZ-71, GZ-68, GZ-65, GZ-64, GZ-46, GZ-74, GZ-67, GZ-13, GZ-76) to construct the ABE single-base editing tool. Among them, dCas12i3 is Cas12i3 with E844A mutation (Cas12i3 is Cas12f.4 in CN111757889B, and the amino acid sequence of wild-type Cas12i3 is shown in SEQ ID No.1), and its nuclease activity is inactivated, that is, the amino acid sequence of dCas12i3 is mutated to A at position 844 compared with SEQ ID No.1; dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein is Cas12i3 protein with E844A, S7R, D233R, D267R, N369R and S433R mutations, and the amino acid sequence of dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein is the same as SEQ ID Compared with No.1, E at position 844 mutates to A, S at position 7 mutates to R, D at position 233 mutates to R, D at position 267 mutates to R, N at position 369 mutates to R, and S at position 433 mutates to R. In other embodiments, the D at position 619 of the Cas12i3 protein corresponding to the sequence shown in SEQ ID No.1 may be mutated to A to obtain a Cas mutant protein (i.e., dCas12i3) with inactivated nuclease activity; in other embodiments, the D at position 619 of the Cas12i3 protein corresponding to the sequence shown in SEQ ID No.1 may be mutated to A to obtain a Cas mutant protein (i.e., dCas12i3) with inactivated nuclease activity; in other embodiments, the dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein may be replaced with other Cas mutant proteins with inactivated nuclease activity, such as dCas9 and nCas9.

[0249] The amino acid sequence of adenosine deaminase TadA8e is shown in SEQ ID No.2; the amino acid sequence of adenosine deaminase GZ-71 is shown in SEQ ID No.3, and the nucleotide sequence is shown in SEQ ID No.12; the amino acid sequence of adenosine deaminase GZ-68 is shown in SEQ ID No.4, and the nucleotide sequence is shown in SEQ ID No.13; the amino acid sequence of adenosine deaminase GZ-65 is shown in SEQ ID No.5, and the nucleotide sequence is shown in SEQ ID No.14; the amino acid sequence of adenosine deaminase GZ-64 is shown in SEQ ID No.6, and the nucleotide sequence is shown in SEQ ID No.15; the amino acid sequence of adenosine deaminase GZ-46 is shown in SEQ ID No.7, and the nucleotide sequence is shown in SEQ ID No.16; the amino acid sequence of adenosine deaminase GZ-74 is shown in SEQ ID No.8, and the nucleotide sequence is shown in SEQ ID No.17; the amino acid sequence of adenosine deaminase GZ-67 is shown in SEQ ID No.9, and the nucleotide sequence is shown in SEQ ID The amino acid sequence of adenosine deaminase GZ-13 is shown in SEQ ID No.10, and the nucleotide sequence is shown in SEQ ID No.19; the amino acid sequence of adenosine deaminase GZ-76 is shown in SEQ ID No.11, and the nucleotide sequence is shown in SEQ ID No.20.

[0250] Schematic diagram of the ABE single-base editing tool constructed using dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein and adenosine deaminase Figure 1 As shown, adenosine deaminase ( Figure 1 TadA in dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein ( Figure 1 The N-terminus of the dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein is connected through an XTEN linker, and the amino acid sequence of the XTEN linker is SGGSSGGSSGSETPGTSESATPESSGGSSGGS; the other ends of adenosine deaminase and dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein are also connected to NLS, and the amino acid sequence of the NLS at the N-terminus of adenosine deaminase is MKRTADGSEFESPKKKRKV, and the amino acid sequence of the NLS at the C-terminus of dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein is KRPAATKKAGQAKKKK, and EGFP is a label designed for screening positive cells. Figure 1It is just an exemplary connection method of adenosine deaminase and dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein. In order to verify the editing activity of single-base editing tools constructed with different adenosine deaminases, dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein can be constructed with different adenosine deaminases in the same connection method, that is, Figure 1 The adenosine deaminase TadA in the above-mentioned embodiment can be replaced by other adenosine deaminase. In other embodiments, those skilled in the art can adjust the position or connection order of the above-mentioned elements.

[0251] In order to verify the activity of the above-mentioned ABE single-base editing tool, referring to the experimental method familiar to those skilled in the art, the target DNA sequence is set to the tga terminator region between the EGFP gene and the dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein, whose PAM is TTG, and EGFP cannot emit light normally. Only when the ABE reporter system converts the tga terminator to cga (R) through single-base editing can EGFP emit green fluorescence. The editing efficiency of the ABE base editing tool can be evaluated by detecting the ratio of green fluorescence. The constructed expression vector is transfected into the 293T cell line, and the EGFP ratio is calculated by flow cytometry fluorescence detection after 48 hours; the single-base editing efficiency is calculated as the number of EGFP-positive cells / total number of cells.

[0252] The editing activity of ABE single-base editing tools constructed with dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein and different adenosine deaminases is shown in Figure 2. Figure 2 As shown: using adenosine deaminase GZ-71 ( Figure 2 The editing efficiency of the ABE single-base editing tool constructed by using adenosine deaminase GZ-68 ( Figure 2 The editing efficiency of the ABE single-base editing tool constructed by using adenosine deaminase GZ-65 ( Figure 2 The editing efficiency of the ABE single-base editing tool constructed by using adenosine deaminase GZ-64 ( Figure 2 The editing efficiency of the ABE single-base editing tool constructed by using adenosine deaminase GZ-46 ( Figure 2 The editing efficiency of the ABE single-base editing tool constructed by using adenosine deaminase GZ-74 ( Figure 2The editing efficiency of the ABE single-base editing tool constructed by using adenosine deaminase GZ-67 ( Figure 2 The editing efficiency of the ABE single-base editing tool constructed by using adenosine deaminase GZ-13 ( Figure 2 The editing efficiency of the ABE single-base editing tool constructed by using adenosine deaminase GZ-76 ( Figure 2 The editing efficiency of the ABE single-base editing tool constructed by GZ0112253-76 in the genome was 3.23%; Figure 2 The GFP is the positive control group, specifically, the fluorescent reporter system does not contain the tga terminator region, that is, EGFP can emit light normally without single base editing; Figure 2 NC is the negative control group, which is an experiment using the ABE base editing tool that does not contain any adenosine deaminase. Due to the absence of adenosine deaminase, base editing cannot be performed and fluorescence cannot be emitted.

[0253] From the above results, it can be seen that the single-base editing tools constructed using the above-mentioned multiple adenosine deaminases can all achieve single-base editing.

[0254] Example 2. Screening of mutant adenosine deaminase GZ-46 and verification of editing activity

[0255] The structure of adenosine deaminase GZ-46 (amino acid sequence as shown in SEQ ID No.7, nucleotide sequence as shown in SEQ ID No.16) in Example 1 was predicted, and the applicant predicted the key amino acid sites that may affect its biological function through bioinformatics, selected relevant sites to establish a mutant library, and screened using a fluorescent reporter system. In this embodiment, amino acid site mutations were performed at positions 110, 153, 161, and 167 of SEQ ID No.7 from the N-terminus.

[0256] Variants of adenosine deaminase GZ-46 were generated by PCR-based site-directed mutagenesis. The specific method is to divide the DNA sequence of adenosine deaminase into two parts with the mutation site as the center, design primers for the mutation site, and each two pairs of primers correspond to an amino acid mutation. Two pairs of primers are designed to amplify the two parts of the DNA sequence respectively, and the sequences that need to be mutated are introduced into the primers. Finally, the two fragments are loaded into the pcDNA3.3-eGFP vector by Gibson cloning. The combination of mutants is constructed by splitting the DNA of adenosine deaminase into multiple segments and using PCR and Gibson clone. Fragment amplification kit: TransStart FastPfu DNA Polymerase (containing 2.5mM dNTPs), please refer to the instructions for the specific experimental process. Gel recovery kit: Gel DNA Extraction Mini Kit, please refer to the instruction manual for the specific experimental process. Kit used for vector construction: pEASY-Basic Seamless Cloning and Assembly Kit (CU201-03), please refer to the instruction manual for the specific experimental process.

[0257] Based on the above amino acid mutation sites, the wild-type protein of adenosine deaminase GZ-46 and adenosine deaminases with single-point mutations at amino acids 110, 153, 161 and 167 (named after mutation types) were obtained respectively: V110K, V110H, Y153F, N161R, E167R; compared with SEQ ID No.7, the mutated adenosine deaminase V110K mutated to K at amino acid 110; compared with SEQ ID No.7, the mutated adenosine deaminase V110H mutated to H at amino acid 110; compared with SEQ ID No.7, the mutated adenosine deaminase Y153F mutated to F at amino acid 153; compared with SEQ ID No.7, the mutated adenosine deaminase N161R mutated to R at amino acid 161; compared with SEQ ID No.7, the mutated adenosine deaminase E167R mutated to R at amino acid 167.

[0258] In addition, an adenosine deaminase with a combination of mutations in amino acids at positions 110, 153, 161 and 167 (named after the mutation type) was obtained: V110K-Y153F-N161R-E167R; compared with SEQ ID No. 7, the mutant adenosine deaminase V110K-Y153F-N161R-E167R had the amino acid at position 110 mutated to K, the amino acid at position 153 mutated to F, the amino acid at position 161 mutated to R and the amino acid at position 167 mutated to R.

[0259] The applicant used the nuclease-inactivated dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein in Example 1 and the adenosine deaminase obtained above to construct an ABE single-base editing tool. The schematic diagram of the ABE single-base editing tool constructed using dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein and adenosine deaminase is shown in FIG. Figure 3 As shown, adenosine deaminase is expressed in dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein ( Figure 3 The N-terminus of the dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein is connected through an XTEN linker, and the amino acid sequence of the XTEN linker is SGGSSGGSSGSETPGTSESATPESSGGSSGGS; the other ends of adenosine deaminase and dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein are also connected to NLS, and the amino acid sequence of the NLS at the N-terminus of adenosine deaminase is MKRTADGSEFESPKKKRKV, and the amino acid sequence of the NLS at the C-terminus of dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein is KRPAATKKAGQAKKKK, and EGFP is a label designed for screening positive cells. Figure 3 It is just an exemplary connection method of adenosine deaminase and dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein. In order to verify the editing activity of single-base editing tools constructed with different adenosine deaminases, dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein can be constructed with different adenosine deaminases in the same connection method, that is, Figure 3 The adenosine deaminase in the above-mentioned components can be replaced with other adenosine deaminase. In other embodiments, those skilled in the art can adjust the position or connection order of the above-mentioned components.

[0260] Referring to the method for verifying the activity of the ABE single-base editing tool in Example 1, the editing activity of the above-mentioned single-base editing tool was verified.

[0261] The editing activity of ABE single-base editing tools constructed with dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein and different adenosine deaminases is shown in Figure 2. Figure 4 shown. Figure 4 NC is the negative control group, which is an experiment using the ABE base editing tool that does not contain any adenosine deaminase. Due to the absence of adenosine deaminase, base editing cannot be performed and fluorescence cannot be emitted.

[0262] exist Figure 4 In the experiment, wild-type adenosine deaminase GZ-46 ( Figure 4 The editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase 46RAHSY) was 14.4%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase V110K was 44.1%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase V110H was 31.5%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase Y153F was 20.2%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase N161R was 19.8%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase E167R was 24.2%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase V110K-Y153F-N161R-E167R ( Figure 4 The editing efficiency of the ABE single-base editing tool constructed with V110K Y153F N161RE167R in was 61.4%.

[0263] From the above results, it can be seen that the editing efficiency is significantly improved after single-site mutation and combined site mutation of amino acid at position 110, 153, 161 or 167 of adenosine deaminase GZ-46.

[0264] Example 3: Based on the mutant adenosine deaminase GZ-46 (V110K-Y153F-N161R-E167R), Step 1: Obtaining a mutant adenosine deaminase with enhanced activity

[0265] Based on the mutant adenosine deaminase GZ-46 (V110K-Y153F-N161R-E167R) obtained in Example 2, this example refers to it as KFRR (compared with SEQ ID No. 7, the amino acid at position 110 mutated to K, the amino acid at position 153 mutated to F, the amino acid at position 161 mutated to R and the amino acid at position 167 mutated to R).

[0266] In this example, the following sites were mutated based on the adenosine deaminase KFRR. The mutation method was referred to Example 2: amino acids 55, 113, 123, 126, 127, 156, 159, and 160, and the following mutant adenosine deaminase were obtained: KFRR (S113N), KFRR (S113R), KFRR (N126Q), KFRR (Y127H), KFRR (Y127L), KFRR (Y127M), KFRR (V159A), KFRR (V159H), KFRR (V159K), KFRR (V159R), TKNDQHFNHQRR, and TKNDQHFNRQRR.

[0267] Among them, the mutant adenosine deaminase KFRR (S113N) is compared with SEQ ID No. 7, the amino acid at position 110 is mutated to K, the amino acid at position 153 is mutated to F, the amino acid at position 161 is mutated to R, the amino acid at position 167 is mutated to R and the amino acid at position 113 is mutated to N;

[0268] Compared with SEQ ID No. 7, the mutant adenosine deaminase KFRR (S113R) has amino acid 110 mutated to K, amino acid 153 mutated to F, amino acid 161 mutated to R, amino acid 167 mutated to R and amino acid 113 mutated to R;

[0269] Compared with SEQ ID No. 7, the mutant adenosine deaminase KFRR (N126Q) has amino acid 110 mutated to K, amino acid 153 mutated to F, amino acid 161 mutated to R, amino acid 167 mutated to R and amino acid 126 mutated to Q;

[0270] Compared with SEQ ID No. 7, the mutant adenosine deaminase KFRR (Y127H) has amino acid 110 mutated to K, amino acid 153 mutated to F, amino acid 161 mutated to R, amino acid 167 mutated to R and amino acid 127 mutated to H;

[0271] Compared with SEQ ID No. 7, the mutant adenosine deaminase KFRR (Y127L) has amino acid 110 mutated to K, amino acid 153 mutated to F, amino acid 161 mutated to R, amino acid 167 mutated to R and amino acid 127 mutated to L;

[0272] Compared with SEQ ID No. 7, the mutant adenosine deaminase KFRR (Y127M) has amino acid 110 mutated to K, amino acid 153 mutated to F, amino acid 161 mutated to R, amino acid 167 mutated to R and amino acid 127 mutated to M;

[0273] Compared with SEQ ID No. 7, the mutant adenosine deaminase KFRR (V159A) has amino acid 110 mutated to K, amino acid 153 mutated to F, amino acid 161 mutated to R, amino acid 167 mutated to R and amino acid 159 mutated to A;

[0274] Compared with SEQ ID No. 7, the mutant adenosine deaminase KFRR (V159H) has amino acid 110 mutated to K, amino acid 153 mutated to F, amino acid 161 mutated to R, amino acid 167 mutated to R and amino acid 159 mutated to H;

[0275] Compared with SEQ ID No. 7, the mutant adenosine deaminase KFRR (V159K) has amino acid 110 mutated to K, amino acid 153 mutated to F, amino acid 161 mutated to R, amino acid 167 mutated to R and amino acid 159 mutated to K;

[0276] Compared with SEQ ID No. 7, the mutant adenosine deaminase KFRR (V159R) has amino acid 110 mutated to K, amino acid 153 mutated to F, amino acid 161 mutated to R, amino acid 167 mutated to R and amino acid 159 mutated to R;

[0277] Compared with SEQ ID No. 7, the mutant adenosine deaminase TKNDQHFNHQRR has amino acid 55 mutated to T, amino acid 110 mutated to K, amino acid 113 mutated to N, amino acid 123 mutated to D, amino acid 126 mutated to Q, amino acid 127 mutated to H, amino acid 153 mutated to F, amino acid 156 mutated to N, amino acid 159 mutated to H, amino acid 160 mutated to Q, amino acid 161 mutated to R and amino acid 167 mutated to R;

[0278] Compared with SEQ ID No.7, the mutant adenosine deaminase TKNDQHFNRQRR has the 55th amino acid mutated to T, the 110th amino acid mutated to K, the 113th amino acid mutated to N, the 123rd amino acid mutated to D, the 126th amino acid mutated to Q, the 127th amino acid mutated to H, the 153rd amino acid mutated to F, the 156th amino acid mutated to N, the 159th amino acid mutated to R, the 160th amino acid mutated to Q, the 161st amino acid mutated to R and the 167th amino acid mutated to R.

[0279] The ABE single-base editing tool was constructed using the dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein with inactivated nuclease activity in Example 1 and the adenosine deaminase obtained above, and the construction method of the ABE single-base editing tool was the same as that in Example 2. And refer to the method for verifying the activity of the ABE single-base editing tool in Example 1 to verify the editing activity of the above-mentioned single-base editing tool.

[0280] The editing activity of ABE single-base editing tools constructed with dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein and different adenosine deaminases is shown in Figure 2. Figure 5 and Figure 6 shown. Figure 5 and Figure 6NC is the negative control group, which is an experiment using the ABE base editing tool that does not contain any adenosine deaminase. Due to the absence of adenosine deaminase, base editing cannot be performed and fluorescence cannot be emitted.

[0281] exist Figure 5 In the experiment, wild-type adenosine deaminase GZ-46 ( Figure 5 The editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase KFRR (46RAHSY) was 13.09%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase KFRR was 57.70%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase KFRR (S113N) was 68.02%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase KFRR (S113R) was 61.95%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase KFRR (N126Q) was 62.62%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase KFRR (Y127H) was 63.39%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase KFRR (S113N) was 68.02%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase KFRR (S113R) was 61.95%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase KFRR (N126Q) was 62.62%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase KFRR (Y127H) was 63.39%; The editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase KFRR (Y127L) is 60.63%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase KFRR (Y127M) is 67.72%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase KFRR (V159A) is 67.00%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase KFRR (V159H) is 68.50%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase KFRR (V159K) is 72.49%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase KFRR (V159R) is 73.57%.

[0282] exist Figure 6 In the experiment, wild-type adenosine deaminase GZ-46 ( Figure 6 The editing efficiency of the ABE single-base editing tool constructed by using the adenosine deaminase TadA8e.1 ( Figure 6 The editing efficiency of the ABE single-base editing tool constructed by using Tad8e.1 ​​in SEQ ID No. 2 is 54.22%; the editing efficiency of the ABE single-base editing tool constructed by using the mutant adenosine deaminase TKNDQHFNHQRR is 60.69%; the editing efficiency of the ABE single-base editing tool constructed by using the mutant adenosine deaminase TKNDQHFNRQRR is 56.11%. Among them, the amino acid sequence of TadA8e.1 is compared with SEQ ID No. 2, and the amino acid V at position 106 is mutated to W.

[0283] From the above results, it can be seen that on the basis of the combined site mutations (V110K-Y153F-N161R-E167R) of the amino acids at positions 110, 153, 161 and 167 of adenosine deaminase GZ-46, the editing efficiency was significantly improved after single site mutations and combined site mutations of the amino acids at positions 55, 113, 123, 126, 127, 156, 159 or 160 of adenosine deaminase GZ-46, which was even higher than that of adenosine deaminase TadA8e.1.

[0284] That is, after single-site mutation and combined-site mutation of amino acids at positions 55, 110, 113, 123, 126, 127, 153, 156, 159, 160, 161 or 167 of adenosine deaminase GZ-46, the editing efficiency was significantly improved.

[0285] Example 4. Screening of mutant adenosine deaminase and verification of editing activity

[0286] The adenosine deaminase mutation method in Example 2 was used to mutate adenosine deaminase TadA8e (amino acid sequence as shown in SEQ ID No. 2), and adenosine deaminases (named according to the mutation type) with single-point mutations at amino acid positions 106 (homologous to amino acid position 110 of adenosine deaminase GZ-46) and 157 (homologous to amino acid position 161 of adenosine deaminase GZ-46) of adenosine deaminase TadA8e were obtained: TadA8e (V106W), TadA8e (V106K), TadA8e (V106H), and TadA8e (N157R); compared with SEQ ID No. 2, the mutated adenosine deaminase TadA8e (V106W) had the amino acid position 106 mutated to W; compared with SEQ ID No. 2, the mutated adenosine deaminase TadA8e (V106K) had the amino acid position 157 mutated to W. Compared with SEQ ID No.2, the 106th amino acid mutated to K; compared with SEQ ID No.2, the 106th amino acid mutated to H in the mutant adenosine deaminase TadA8e (V106H); compared with SEQ ID No.2, the 157th amino acid mutated to R in the mutant adenosine deaminase TadA8e (N157R).

[0287] The ABE single-base editing tool was constructed using the dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein with inactivated nuclease activity in Example 1 and the adenosine deaminase obtained above, respectively. The schematic diagram and construction method of the ABE single-base editing tool are the same as those in Example 2. And refer to the method for verifying the activity of the ABE single-base editing tool in Example 1 to verify the editing activity of the above-mentioned single-base editing tool.

[0288] The editing activity of the ABE single-base editing tool constructed by dCas12i3 (S7R-D233R-D267R-N369R-S433R) protein and different adenosine deaminases TadA8e is shown in Figure 2. Figure 7 shown. Figure 7 NC is the negative control group, which is an experiment using the ABE base editing tool that does not contain any adenosine deaminase. Due to the absence of adenosine deaminase, base editing cannot be performed and fluorescence cannot be emitted.

[0289] exist Figure 7 In the study, adenosine deaminase TadA8e ( Figure 7 The editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase TadA8e (V106W) is 48.86%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase TadA8e (V106K) is 56.32%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase TadA8e (V106H) is 63.08%; the editing efficiency of the ABE single-base editing tool constructed using the mutant adenosine deaminase TadA8e (N157R) is 52.26%.

[0290] From the above results, it can be seen that the editing efficiency is significantly improved after single-site mutation and combined site mutation at the 106th or 157th amino acid of adenosine deaminase TadA8e.

[0291] Since the 106th amino acid of adenosine deaminase TadA8e and the 110th amino acid of adenosine deaminase GZ-46 are homologous sites, and the 157th amino acid of adenosine deaminase TadA8e and the 161st amino acid of adenosine deaminase GZ-46 are homologous sites, this indicates that mutations at the homologous sites of adenosine deaminase TadA8e or adenosine deaminase GZ-46 can improve the editing efficiency or activity of the deaminase.

[0292] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. An adenosine deaminase, characterized in that The adenosine deaminase is an adenosine deaminase described in any one of the following IV: I. The amino acid sequence of the adenosine deaminase has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with any of SEQ ID No.7, SEQ ID No.3-6, or SEQ ID No.8-11, and substantially retains the biological function of the sequence from which it is derived; preferably, the adenosine deaminase is derived from Enterobacteriaceae, Escherichia, Citrobacter, Rahnella aceris, Shigella flexneri flexneri or Salmonella enterica subsp. II. The amino acid sequence of the adenosine deaminase has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids) amino acid substitutions, deletions or additions compared to any of SEQ ID No.7, SEQ ID No.3-6 or SEQ ID No.8-11, and substantially retains the biological function of the sequence from which it is derived; preferably, the adenosine deaminase is derived from Enterobacteriaceae, Escherichia, Citrobacter, Rahnella aceris, Shigella flexneri or Salmonella enterica subsp. III. The adenosine deaminase comprises an amino acid sequence shown in any one of SEQ ID No.3-11; IV. The adenosine deaminase is a mutant adenosine deaminase, and the mutant adenosine deaminase has a mutation (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids) at any one or more of the following amino acid positions corresponding to the amino acid sequence of SEQ ID No. 7 compared to the parent adenosine deaminase: position 55, position 110, position 113, position 123, position 126, position 127, position 153, position 156, position 159, position 160, position 161, position 167; V. The adenosine deaminase is a mutant adenosine deaminase, and compared with the parent adenosine deaminase, the mutant adenosine deaminase has mutations at the following amino acid sites corresponding to the amino acid sequence shown in SEQ ID No. 2: position 106 and / or position 157.

2. The adenosine deaminase according to claim 1, characterized in that The adenosine deaminase is a mutant adenosine deaminase, and the mutant adenosine deaminase is selected from any one or any several of the following (1)-(6): (1) The mutant adenosine deaminase has mutations at the 110th, 153rd, 161st and 167th amino acid positions corresponding to the amino acid sequence shown in SEQ ID No. 7 compared with the amino acid sequence of the parent adenosine deaminase; (2) compared with the amino acid sequence of the parent adenosine deaminase, the mutant adenosine deaminase has mutations at the 110th, 153rd, 161st, 167th and 113th amino acid positions corresponding to the amino acid sequence shown in SEQ ID No. 7; (3) The mutant adenosine deaminase has mutations at the 110th, 153rd, 161st, 167th and 126th amino acid positions corresponding to the amino acid sequence shown in SEQ ID No. 7 compared with the amino acid sequence of the parent adenosine deaminase; (4) The mutant adenosine deaminase has mutations at the 110th, 153rd, 161st, 167th and 127th amino acid positions corresponding to the amino acid sequence shown in SEQ ID No. 7 compared with the amino acid sequence of the parent adenosine deaminase; (5) The mutant adenosine deaminase has mutations at the 110th, 153rd, 161st, 167th and 159th amino acid positions corresponding to the amino acid sequence shown in SEQ ID No. 7 compared with the amino acid sequence of the parent adenosine deaminase; (6) Compared with the amino acid sequence of the parent adenosine deaminase, the mutant adenosine deaminase has mutations at the 110th, 153rd, 161st, 167th, 55th, 113th, 123rd, 126th, 127th, 156th, 159th and 160th amino acid positions corresponding to the amino acid sequence shown in SEQ ID No.

7.

3. The adenosine deaminase according to claim 1 or 2, characterized in that The adenosine deaminase is a mutant adenosine deaminase, and the amino acid sequence of the parent adenosine deaminase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity compared to the sequence shown in any one of SEQ ID No.2-11.

4. An adenosine deaminase, characterized in that The adenosine deaminase is selected from any one of the following groups I-III: I. An adenosine deaminase obtained by generating mutations at any one or more of the following amino acid positions of the amino acid sequence shown in SEQ ID No.7: 55, 110, 113, 123, 126, 127, 153, 156, 159, 160, 161, 167; or, an adenosine deaminase obtained by generating mutations at the following amino acid positions of the amino acid sequence shown in SEQ ID No.2: 106 and / or 157; II, compared with the adenosine deaminase described in I, having the mutation site described in I; and an adenosine deaminase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the adenosine deaminase of claim 1; III. Compared with the adenosine deaminase described in I, it has the mutation site described in I; Furthermore, compared with the adenosine deaminase described in I, it has a sequence with one or more amino acid substitutions, deletions or additions; the one or more amino acids include substitutions, deletions or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

5. A fusion protein, characterized in that The fusion protein comprises a DNA binding protein and the adenosine deaminase according to any one of claims 1 to 4; Preferably, the DNA binding protein is a Cas protein; preferably, the Cas protein is selected from any one of Cas9, Cas9n, dCas9, CasX, CasY, C2cl, C2c2, C2c3, GeoCas9, CjCas9, Cas12a, Cas12b, Cas12g, Cas12h, Cas12i, Cas12j, Cas13b, Cas13c, Cas13d, Cas14, Csn2, xCas9, Cas9-NG, LbCas12a, enAsCas12a, Cas9-KKH, circular displacement Cas9, Argonaute (Ago) domain, SmacCas9, Spy-macCas9, SpGas9-NRRH, SpaCas9-NRTH, SpaCas9-NRCH, Cas9-NG-CP1041, Cas9-NG-VRQR, dCas12i, nCas12i and variants thereof; More preferably, the DNA binding protein is a nuclease-inactivated Cas protein.

6. An isolated polynucleotide, characterized in that The polynucleotide encodes the adenosine deaminase according to any one of claims 1 to 4, or the polynucleotide encodes the fusion protein according to claim 5.

7. A carrier, characterized in that The vector comprises the polynucleotide according to claim 6 and a regulatory element operably linked thereto.

8. A base editing system, characterized in that: The system comprises the fusion protein of claim 5 and at least one gRNA; the DNA binding protein in the fusion protein is a Cas protein, and the gRNA can bind to the Cas protein.

9. A composition, characterized in that The composition comprises: (i) a protein component selected from: the fusion protein of claim 5, wherein the DNA binding protein in the fusion protein is a Cas protein; (ii) a nucleic acid component, which is a gRNA, and the gRNA is capable of binding to the Cas protein.

10. An engineered host cell, characterized in that The host cell comprises the adenosine deaminase of any one of claims 1-4, or the fusion protein of claim 5, or the polynucleotide of claim 6, or the vector of claim 7, or the base editing system of claim 8, or the composition of claim 9.

11. Use of the adenosine deaminase according to any one of claims 1 to 4, or the fusion protein according to claim 5, or the polynucleotide according to claim 6, or the vector according to claim 7, or the base editing system according to claim 8, or the composition according to claim 9, or the host cell according to claim 10 in gene editing; or, use in the preparation of a reagent or kit for gene editing; Preferably, the gene editing is single base editing of the target gene.

12. A kit for gene editing, characterized in that: The kit comprises the adenosine deaminase according to any one of claims 1 to 4, or the fusion protein according to claim 5, or the polynucleotide according to claim 6, or the vector according to claim 7, or the base editing system according to claim 8, or the composition according to claim 9, or the host cell according to claim 10.

13. A method for editing a nucleic acid, comprising the step of contacting a target region of the nucleic acid with the fusion protein of claim 5 and a gRNA, wherein the gRNA comprises a sequence capable of binding to the Cas protein in the fusion protein of claim 5 and a sequence capable of binding to the target region of the nucleic acid; wherein, The target region comprises a targeted base pair, and the fusion protein is capable of performing base substitution on the targeted base pair; Preferably, the targeted base pair is replaced by A:T to G:C.

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