WO2023169454A1 - Adenine deaminase and use thereof in base editing - Google Patents

Abstract

An adenine deaminase and the use thereof in base editing. More specifically: a base editing system based on newly identified adenine deaminase; a method of using said system to carry out base editing of a target sequence in the genome of an organism (such as a plant); a genetically modified organism (such as a plant) produced by said method; and progeny of said organism.

Description

Adenine deaminase and its use in base editing Technical field

The present invention relates to the field of genetic engineering. In particular, the present invention relates to adenine deaminase and its use in base editing. More specifically, the present invention relates to a base editing system based on a newly identified adenine deaminase, and a method for base editing a target sequence in the genome of an organism (such as a plant) using the base editing system, As well as genetically modified organisms (eg plants) produced by such methods and their progeny.

Background of the invention

Sequence-specific modifications to an organism's genome can give the organism new, stably heritable traits. Among them, single nucleotide variations at specific sites may lead to changes in the amino acid sequence of the gene or early termination, or may lead to changes in the regulatory sequence, leading to the production of excellent traits. Genome editing technologies, such as the CRISPR/Cas9 system, can achieve targeting functions to target sequences. The base editing system developed by combining the characteristics of the genome editing system with the target sequence and combining it with deaminase can achieve the function of accurately deaminating target sites on the genome. Currently, the most commonly used large-volume base editing systems include cytosine base editing systems and adenine base editing systems. Among them, by fusing a variant of E. coli TadA (tRNA-specific adenosine deaminase), the conversion of adenine (A) to hypoxanthine (I) at the target site can be achieved. The I on DNA can be recognized by cells as guanine (G), and the I is replaced by G during replication. Therefore, A at the target site can eventually transform into G. In addition, the efficiency of base editing can be significantly improved by introducing a nick into the single strand that has not undergone deamination on the opposite side to break it. Since there is no adenine deaminase in nature that can directly deaminate adenine (A) in DNA, the only system that can be used for adenine deaminase at the DNA level has been evolved by David R. Liu’s team. A series of ecTadA variants derived from E. coli. Therefore, the search for new adenine deaminase is of great significance for expanding the existing adenine base editing system and improving the ability to precisely manipulate target DNA sequences.

Brief description of the drawings

Figure 1: Sequence similarity between No.135 potential adenine deaminase and E. coli ecTadA.

Figure 2: Adenine base editing can be achieved in the reporter system after modification of the key site of potential deaminase NO.135.

Figure 3: Structural similarity between randomly selected proteins with VnxN10xHAEnxPCxMC characteristic sequences and annotated as guanine deaminase, lysine tRNA synthetase, HAD hydrolase and unannotated protein functions and TadA respectively. The light color is E. coli TadA, and the dark color is the candidate protein.

Figure 4: Sequence similarity between No.1299 and No.1417 guanine deaminase and E. coli ecTadA.

Figure 5: After modifying the key site of the potential deaminase of NO.1299, the adenine base can be realized in the reporter system edit.

Figure 6: Adenine base editing can be achieved in the reporter system after modification of the key site of the potential deaminase of NO.1417.

Detailed description of the invention

1. Definition

In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used in this article are terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

As used herein, the term "and/or" encompasses all combinations of the items connected by this term, and each combination shall be deemed to have been individually set forth herein. For example, "A and/or B" encompasses "A", "A and B" and "B". For example, "A, B and/or C" encompasses "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

"Genome" as used herein encompasses not only chromosomal DNA present in the nucleus, but also organellar DNA present in subcellular components of the cell (eg, mitochondria, plastids).

As used herein, "organism" includes any organism suitable for genome editing, preferably eukaryotes. Examples of organisms include, but are not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, cats; poultry such as chickens, ducks, and geese; plants including monocots and dicots, For example, rice, corn, wheat, sorghum, barley, soybean, peanut, Arabidopsis thaliana, etc.

"Genetically modified organism" or "genetically modified cell" means an organism or cell that contains exogenous polynucleotides or modified genes or expression regulatory sequences within its genome. For example, exogenous polynucleotides can be stably integrated into the genome of an organism or cell and inherited for successive generations. Exogenous polynucleotides can be integrated into the genome alone or as part of a recombinant DNA construct. A modified gene or expression control sequence is one in which the sequence contains single or multiple deoxynucleotide substitutions, deletions, and additions in the genome of an organism or cell.

"Foreign" with respect to a sequence means a sequence from an alien species or, if from the same species, a sequence that has undergone significant changes in composition and/or locus from its native form by deliberate human intervention.

"Polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid fragment" are used interchangeably and are single- or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides are referred to by their single-letter names as follows: "A" for adenosine or deoxyadenosine (for RNA or DNA, respectively), "C" for cytidine or deoxycytidine, and "G" for guanosine or Deoxyguanosine, "U" represents uridine, "T" represents deoxythymidine, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, " H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide.

"Polypeptide,""peptide," and "protein" are used interchangeably herein and refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino" "Acid sequence" and "protein" may also include modified forms including, but not limited to, glycosylation, lipid linkage, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

Sequence "identity" has an art-recognized meaning, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. (See, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are many methods of measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988) ).

When the word "comprising" is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleic acids at one or both ends of the protein or nucleic acid. glycosides, but still have the activity described in the present invention. In addition, those skilled in the art know that the methionine encoded by the start codon at the N-terminus of the polypeptide will be retained under certain practical circumstances (such as when expressed in a specific expression system), but will not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the description and claims of this application, although it may not contain the N-terminal methionine encoded by the start codon, it is also encompassed at this time that it contains the methionine. Sequence, correspondingly, its encoding nucleotide sequence may also contain an initiation codon; and vice versa.

In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Generally, those skilled in the art recognize that single amino acid substitutions in non-essential regions of polypeptides do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub .co.,p.224).

As used herein, "expression construct" refers to a vector, such as a recombinant vector, suitable for expression of a nucleotide sequence of interest in an organism. "Expression" refers to the production of a functional product. For example, expression of a nucleotide sequence may refer to transcription of the nucleotide sequence (eg, transcription to produce mRNA or functional RNA) and/or translation of the RNA into a precursor or mature protein.

The "expression construct" of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, an RNA capable of translation (such as mRNA).

An "expression construct" of the present invention may comprise regulatory sequences and nucleotide sequences of interest from different sources, or control sequences and nucleotide sequences of interest from the same source but arranged in a manner different from that which normally occurs in nature.

"Regulatory sequence" and "regulatory element" are used interchangeably and refer to a coding sequence that is located upstream (5' non-coding sequence), intermediate or downstream (3' non-coding sequence) and affects the transcription, RNA processing or Stability or translated nucleotide sequence. Regulatory sequences may include, but are not limited to, promoters, translation leaders, introns, and polyadenylation recognition sequences.

"Promoter" refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the invention, a promoter is a promoter capable of controlling the transcription of a gene in a cell, whether or not it is derived from said cell. The promoter can It is a constitutive promoter or a tissue-specific promoter or a developmentally regulated promoter or an inducible promoter.

A "constitutive promoter" refers to a promoter that will generally cause expression of a gene in most cell types under most circumstances. "Tissue-specific promoter" and "tissue-preferred promoter" are used interchangeably and refer to expression primarily, but not necessarily exclusively, in one tissue or organ, but also in a specific cell or cell type promoter. "Developmentally regulated promoter" refers to a promoter whose activity is determined by developmental events. "Inducible promoters" selectively express operably linked DNA sequences in response to endogenous or exogenous stimuli (environment, hormones, chemical signals, etc.).

Examples of promoters include, but are not limited to, polymerase (pol) I, pol II, or pol III promoters. Examples of pol I promoters include the chicken RNA pol I promoter. Examples of pol II promoters include, but are not limited to, the cytomegalovirus immediate early (CMV) promoter, the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, and the simian virus 40 (SV40) immediate early promoter. Examples of pol III promoters include the U6 and H1 promoters. Inducible promoters such as metallothionein promoters can be used. Other examples of promoters include the T7 phage promoter, the T3 phage promoter, the β-galactosidase promoter, and the Sp6 phage promoter. When used in plants, the promoter may be cauliflower mosaic virus 35S promoter, corn Ubi-1 promoter, wheat U6 promoter, rice U3 promoter, corn U3 promoter, rice actin promoter.

As used herein, the term "operably linked" means that a regulatory element (eg, but not limited to, a promoter sequence, a transcription termination sequence, etc.) is linked to a nucleic acid sequence (eg, a coding sequence or an open reading frame) such that the nucleotide Transcription of the sequence is controlled and regulated by the transcriptional regulatory elements. Techniques for operably linking regulatory element regions to nucleic acid molecules are known in the art.

"Introducing" a nucleic acid molecule (eg, plasmid, linear nucleic acid fragment, RNA, etc.) or protein into an organism means transforming an organism's cells with the nucleic acid or protein so that the nucleic acid or protein can function in the cell. "Transformation" as used in the present invention includes stable transformation and transient transformation.

"Stable transformation" refers to the introduction of foreign nucleotide sequences into the genome, resulting in stable inheritance of foreign genes. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any successive generations thereof.

"Transient transformation" refers to the introduction of nucleic acid molecules or proteins into cells to perform functions without stable inheritance of foreign genes. In transient transformation, the foreign nucleic acid sequence is not integrated into the genome.

2. Adenine deaminase and base editing fusion proteins containing it

In one aspect, the present application provides an adenine deaminase, which

1) Contains the characteristic sequence motif VX _n NX ₁₀ HAEX _n PCXMC; and/or

2) Contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, An amino acid sequence with 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity at the amino acid position corresponding to position 108 of SEQ ID NO: 14 The amino acid is N.

In some embodiments, the amino acid of the adenine deaminase at amino acid position corresponding to amino acid position 106 of SEQ ID NO:14 is A or V. In some embodiments, the amino acid of the adenine deaminase at amino acid position corresponding to amino acid position 107 of SEQ ID NO:14 is L or R. In some embodiments, the adenine The amino acid at the deaminase position corresponding to amino acid position 109 of SEQ ID NO: 14 is K or S.

In some embodiments, the adenine deaminase at the amino acid position corresponding to amino acid positions 106-109 of SEQ ID NO: 14 is VRNS, ALNK, ALNS, ARNK, ARNS, VLNK, VLNS, or VRNK .

In some embodiments, the adenine deaminase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-9, 11, and 13.

In some embodiments, the "adenine deaminase" is capable of accepting a nucleic acid such as single-stranded DNA as a substrate and catalyzing the formation of inosine (I) from adenosine or deoxyadenosine (A).

As used herein, "the amino acid at the amino acid position corresponding to amino acid position 108 of SEQ ID NO: 14" means that after sequence alignment with the amino acid sequence of SEQ ID NO: 14, the adenine deamination described herein is The amino acid in the enzyme that aligns with the amino acid at position 108 of SEQ ID NO:14. Other similar terms/phrases in this article have similar meanings. The correspondence of amino acids in different sequences can be determined according to sequence comparison methods known in the art. For example, amino acid correspondence can be determined through the EMBL-EBI online alignment tool (https://www.ebi.ac.uk/Tools/psa/), where two sequences can be determined using the Needleman-Wunsch algorithm using default parameters. Alignment.

In the characteristic sequence motifs VX _n NX ₁₀ HAEX _n PCXMC in various aspects of this article, X represents any amino acid; n represents any integer, such as any in the range of 1-100, 1-50, 1-20 or 1-10 integer.

In one aspect, the present application relates to the use of an adenine deaminase for gene editing, such as base editing, in an organism or a cell of an organism, wherein the adenine deaminase

1) Contains the characteristic sequence motif VX _n NX ₁₀ HAEX _n PCXMC; and/or

2) Contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, An amino acid sequence with 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity at the amino acid position corresponding to position 108 of SEQ ID NO: 14 The amino acid is N.

In some embodiments, the amino acid at the adenine deaminase enzyme position corresponding to amino acid position 106 of SEQ ID NO: 14 is A or V. In some embodiments, the amino acid at the adenine deaminase enzyme at amino acid position corresponding to position 107 of SEQ ID NO: 14 is L or R. In some embodiments, the amino acid at the adenine deaminase enzyme at amino acid position corresponding to position 109 of SEQ ID NO: 14 is K or S.

In some embodiments, the adenine deaminase at the amino acid position corresponding to amino acid positions 106-109 of SEQ ID NO: 14 is VRNS, ALNK, ALNS, ARNK, ARNS, VLNK, VLNS, or VRNK .

In some embodiments, the adenine deaminase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-9, 11, and 13.

In some embodiments, the "adenine deaminase" is capable of accepting a nucleic acid such as single-stranded DNA as a substrate and catalyzing the formation of inosine (I) from adenosine or deoxyadenosine (A).

In some embodiments, the adenine deaminase is used to prepare base editing fusion proteins or base editing systems. The base editing fusion protein or base editing system is used for base editing in organisms or organism cells.

In another aspect, the invention provides a base editing fusion protein comprising a nucleic acid targeting domain and an adenine deamination domain, wherein the adenine deamination domain comprises at least one (eg, one or two) Adenine deaminase polypeptide, the adenine deaminase

1) Contains the characteristic sequence motif VX _n NX ₁₀ HAEX _n PCXMC; and/or

2) Contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, An amino acid sequence with 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity at the amino acid position corresponding to position 108 of SEQ ID NO: 14 The amino acid is N.

In some embodiments, the amino acid at the adenine deaminase enzyme position corresponding to amino acid position 106 of SEQ ID NO: 14 is A or V. In some embodiments, the amino acid at the adenine deaminase enzyme at amino acid position corresponding to position 107 of SEQ ID NO: 14 is L or R. In some embodiments, the amino acid at the adenine deaminase enzyme at amino acid position corresponding to position 109 of SEQ ID NO: 14 is K or S.

In some embodiments, the adenine deaminase at the amino acid position corresponding to amino acid positions 106-109 of SEQ ID NO: 14 is VRNS, ALNK, ALNS, ARNK, ARNS, VLNK, VLNS, or VRNK .

In some embodiments, the adenine deaminase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-9, 11, and 13.

In some embodiments, the "adenine deaminase" is capable of accepting a nucleic acid such as single-stranded DNA as a substrate and catalyzing the formation of inosine (I) from adenosine or deoxyadenosine (A).

In the embodiments herein, "base editing fusion protein" and "base editor" are used interchangeably to refer to a protein that can mediate one or more nucleotide substitutions of a target sequence in the genome in a sequence-specific manner. protein. The one or more nucleotide substitutions are, for example, A to G substitutions.

As used herein, a "nucleic acid targeting domain" refers to a domain capable of mediating attachment of the base editing fusion protein to a specific target sequence in the genome in a sequence-specific manner (eg, via a guide RNA). In some embodiments, the nucleic acid targeting domain may include one or more zinc finger protein domains (ZFP) or transcription factor effector domains (TALE) directed to a specific target sequence. . In some embodiments, the nucleic acid targeting domain comprises at least one (eg, one) CRISPR effector polypeptide.

"Zinc finger desmin domains (ZFPs)" typically contain 3-6 individual zinc finger repeats, each of which can recognize a unique sequence of, for example, 3 bp. By combining different zinc finger repeats, different genomic sequences can be targeted.

"Transcription activator-like effector domain" is the DNA-binding domain of a transcription activator-like effector (TALE). TALEs can be engineered to bind to almost any desired DNA sequence.

As used herein, the term "CRISPR effector protein" generally refers to a nuclease present in a naturally occurring CRISPR system (CRISPR nuclease) or a functional variant thereof. The term covers CRISPR-based systems capable of Any effector protein that achieves sequence-specific targeting within the cell.

As used herein, a "functional variant" with respect to a CRISPR nuclease means that it retains at least the sequence-specific targeting ability mediated by the guide RNA. Preferably, the functional variant is a nuclease-inactive variant, ie, it lacks double-stranded nucleic acid cleavage activity. However, CRISPR nucleases lacking double-stranded nucleic acid cleavage activity also include nickases, which form nicks in double-stranded nucleic acid molecules but do not completely cut the double-stranded nucleic acid. In some preferred embodiments of the invention, the CRISPR effector protein of the invention has nickase activity. In some embodiments, the functional variant recognizes a different PAM (protospacer adjacent motif) sequence relative to the wild-type nuclease.

"CRISPR effector proteins" can be derived from Cas9 nucleases, including Cas9 nucleases or functional variants thereof. The Cas9 nuclease may be a Cas9 nuclease from a different species, such as spCas9 from S. pyogenes or SaCas9 derived from S. aureus. "Cas9 nuclease" and "Cas9" are used interchangeably herein to refer to an RNA that includes a Cas9 protein or a fragment thereof (e.g., a protein that includes the active DNA cleavage domain of Cas9 and/or the gRNA binding domain of Cas9) Guided nuclease. Cas9 is a component of the CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats and Related Systems) genome editing system. It can target and cut DNA target sequences to form DNA double-strand breaks (DSBs) under the guidance of guide RNA. ). An exemplary amino acid sequence of wild-type SpCas9 is shown in SEQ ID NO: 15.

"CRISPR effector proteins" can also be derived from Cpf1 (i.e., Cas12a) nuclease, including Cpf1 nuclease or functional variants thereof. The Cpf1 nuclease can be a Cpf1 nuclease from different species, such as Cpf1 nuclease from Francisella novicida U112, Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006.

Available "CRISPR effector proteins" can also be derived from Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, Csn2 , Cas4, C2c1 (Cas12b), C2c3, C2c2, Cas12c, Cas12d (i.e. CasY), Cas12e (i.e. CasX), Cas12f (i.e. Cas14), Cas12g, Cas12h, Cas12i, Cas12j (i.e. CasΦ), Cas12k, Cas12l, Cas12m, etc. Nucleases include, for example, these nucleases or functional variants thereof.

In some embodiments, the CRISPR effector protein is nuclease-inactivated Cas9. The DNA cleavage domain of Cas9 nuclease is known to contain two subdomains: the HNH nuclease subdomain and the RuvC subdomain. The HNH subdomain cleaves the strand that is complementary to the gRNA, while the RuvC subdomain cleaves the non-complementary strand. Mutations in these subdomains can inactivate the nuclease activity of Cas9, forming "nuclease-inactive Cas9." The nuclease-inactivated Cas9 still retains the gRNA-guided DNA binding ability.

The nuclease-inactivated Cas9 of the present invention can be derived from Cas9 of different species, for example, derived from S. pyogenes Cas9 (SpCas9), or derived from Staphylococcus aureus (S. aureus) Cas9 (SaCas9 ). Simultaneously mutating the HNH nuclease subdomain and RuvC subdomain of Cas9 (e.g., containing mutations D10A and H840A) renders Cas9 nuclease inactive and becomes nuclease-dead Cas9 (dCas9). Mutational inactivation of one of the subdomains can render Cas9 nickase active, that is, a Cas9 nickase (nCas9) is obtained, for example, nCas9 with only the D10A mutation.

Accordingly, in some embodiments of aspects of the invention, the nuclease-inactivated Cas9 variants of the invention are For wild-type Cas9, the amino acid substitutions D10A and/or H840A are included, where the amino acid numbering refers to SEQ ID NO: 15. In some preferred embodiments, the nuclease-inactivated Cas9 includes the amino acid substitution D10A relative to wild-type Cas9, where the amino acid numbering refers to SEQ ID NO: 15. In some embodiments, the nuclease-inactivated Cas9 comprises the amino acid sequence set forth in SEQ ID NO: 16 (nCas9(D10A)).

When Cas9 nuclease is used for gene editing, it usually requires that the target sequence has a 5'-NGG-3' PAM (protospacer adjacent motif) sequence at the 3' end. However, the inventors surprisingly found that the frequency of this PAM sequence in some species such as rice is very low, which greatly limits gene editing in these species such as rice. To this end, CRISPR effector proteins that recognize different PAM sequences can be used in the present invention, such as functional variants of Cas9 nuclease with different PAM sequences.

In some embodiments of the invention, the adenine deamination domain in the fusion protein is capable of converting adenosine deamination of single-stranded DNA produced in the formation of the CRISPR effector protein-guide RNA-DNA complex into inosine (I) , because DNA polymerase treats inosine (I) as guanine (G), A to G substitution can be achieved through base mismatch repair.

In some embodiments of the invention, the nucleic acid targeting domain and the adenine deamination domain are fused through a linker.

As used herein, a "linker" may be 1-50 in length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, A non-functional amino acid sequence with 18, 19, 20 or 20-25, 25-50) or more amino acids and no secondary or higher structure. For example, the joint may be a flexible joint.

In some embodiments, the base editing fusion protein includes in the following order from N-terminus to C-terminus: an adenine deamination domain and a nucleic acid targeting domain.

In some embodiments of the invention, the fusion protein of the invention may further comprise a nuclear localization sequence (NLS). In general, one or more NLSs in the fusion protein should be of sufficient strength to drive accumulation of the fusion protein in the nucleus of the cell in an amount that enables its base editing function. In general, the strength of nuclear localization activity is determined by the number, location of NLS in the fusion protein, the specific NLS(s) used, or a combination of these factors.

In some embodiments of the invention, the NLS of the fusion protein of the invention can be located at the N-terminus and/or C-terminus. In some embodiments of the invention, the NLS of the fusion protein of the invention can also be located between the adenine deamination domain and the nucleic acid targeting domain. In some embodiments, the fusion protein contains about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS. In some embodiments, the fusion protein contains about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS at or near the N-terminus. In some embodiments, the fusion protein contains about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS at or near the C-terminus. In some embodiments, the polypeptide comprises a combination of these, such as one or more NLS at the N-terminus and one or more NLS at the C-terminus. When more than one NLS is present, each one can be selected to be independent of the others.

Generally, NLS consists of one or more short sequences of positively charged lysine or arginine exposed on the protein surface, but other types of NLS are also known. Non-limiting examples of NLS include: KKRKV, PKKKRKV Or KRPAATKKAGQAKKKK.

In addition, depending on the DNA position to be edited, the fusion protein of the present invention may also include other positioning sequences, such as cytoplasmic positioning sequences, chloroplast positioning sequences, mitochondrial positioning sequences, etc.

3. Base editing system

In another aspect, the invention provides a base editing system for modifying a target nucleic acid region in a genome, comprising:

i) the base editing fusion protein of the present invention and/or the expression construct containing the nucleotide sequence encoding the base editing fusion protein; and/or

ii) at least one guide RNA and/or at least one expression construct comprising a nucleotide sequence encoding said at least one guide RNA,

wherein said at least one guide RNA is directed to at least one target sequence within said target nucleic acid region.

As used herein, a "base editing system" refers to a combination of components required for base editing of a genome in a cell or organism. Each component of the system, such as the base editing fusion protein and one or more guide RNAs, can exist independently, or can exist in any combination as a composition.

As used herein, "guide RNA" and "gRNA" are used interchangeably and refer to an RNA that is capable of forming a complex with a CRISPR effector protein and is capable of targeting the complex to a target sequence due to certain identity with the target sequence. molecular. Guide RNA targets a target sequence by base pairing with its complementary strand. For example, the gRNA used by Cas9 nuclease or its functional variants is usually composed of crRNA and tracrRNA molecules that are partially complementary to form a complex, where the crRNA contains sufficient identity with the target sequence to hybridize to the complementary strand of the target sequence and guide The guide sequence (also called the seed sequence) that the CRISPR complex (Cas9+crRNA+tracrRNA) specifically binds to the target sequence. However, it is known in the art that single guide RNAs (sgRNAs) can be designed that contain characteristics of both crRNA and tracrRNA. The gRNA used by Cpf1 nuclease or its functional variants is usually composed only of mature crRNA molecules, which can also be called sgRNA. It is within the capabilities of those skilled in the art to design appropriate gRNA based on the CRISPR nuclease used and the target sequence to be edited.

Those skilled in the art will know that if the base editing fusion protein is not based on a CRISPR effector protein, the system may not require a guide RNA or an expression construct encoding it.

In some embodiments, after the base editing system of the present invention is introduced into the cell, the base editing fusion protein and the guide RNA can form a complex, and the complex specifically targets the target under the guidance of the guide RNA. Target the target sequence and cause one or more A's to be replaced by G's in the target sequence.

In some embodiments, the at least one guide RNA can be directed to a target sequence located on the sense strand (eg, the protein coding strand) and/or the antisense strand within the target nucleic acid region of the genome. When the guide RNA targets the sense strand (eg, protein-coding strand), the base editing composition of the present invention can cause one or more A's in the target sequence on the sense strand (eg, protein-coding strand) to be replaced by G. When the guide RNA targets the antisense strand, the base editing composition of the present invention can cause one or more Ts within the target sequence on the sense strand (eg, protein coding strand) to be replaced with Cs.

In order to obtain efficient expression in cells, in some embodiments of the invention, the base editing fusion encoding The nucleotide sequence of the protein is codon-optimized for the organism whose genome is to be modified.

Codon optimization refers to replacing at least one codon of the native sequence (e.g., about or more than about 1, 2, 3, 4, 5, 10) with a codon that is more frequently or most frequently used in the host cell's genes. , 15, 20, 25, 50 or more codons while maintaining the native amino acid sequence and modifying the nucleic acid sequence to enhance expression in the host cell of interest. Different species display certain codons for specific amino acids specific preferences. Codon bias (differences in codon usage between organisms) is often related to the efficiency of messenger RNA (mRNA) translation, which is thought to depend on the nature of the codons being translated and Availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs within a cell generally reflects the codons most frequently used for peptide synthesis. Thus, genes can be tailored to be most efficient in a given organism based on codon optimization. Optimal gene expression. Codon utilization tables are readily available, for example in the Codon Usage Database available at www.kazusa.orjp/codon/ , and these tables can be adjusted in different ways Applicable. See, Nakamura Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

Organisms whose genomes can be modified by the base editing system of the present invention include any organisms suitable for base editing, preferably eukaryotic organisms. Examples of organisms include, but are not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, cats; poultry such as chickens, ducks, geese; plants, including monocots and dicots For example, the plant is a crop plant, including but not limited to wheat, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugar cane, tomato, tobacco, cassava and potato.

4. Methods of producing genetically modified cells

In another aspect, the invention also provides a method for producing at least one genetically modified cell, comprising introducing the base editing system of the invention into at least one of the cells, thereby causing a target nucleic acid region in the at least one cell One or more nucleotide substitutions within. In some embodiments, the one or more nucleotide substitutions are A to G substitutions.

In some embodiments, the method further includes the step of screening said at least one cell for cells having the desired one or more nucleotide substitutions.

In some embodiments, the methods of the invention are performed in vitro. For example, the cells are isolated cells, or cells in an isolated tissue or organ.

In another aspect, the invention also provides genetically modified organisms comprising genetically modified cells or progeny cells thereof produced by the methods of the invention. Preferably, the genetically modified cell or progeny thereof has the desired one or more nucleotide substitutions.

In the present invention, the target nucleic acid region to be modified can be located anywhere in the genome, such as within a functional gene such as a protein-coding gene, or can be located in a gene expression regulatory region such as a promoter region or enhancer region, thereby achieving the desired modification. Modification of gene function or modification of gene expression. In some embodiments, the desired nucleotide substitution results in a desired modification of gene function or gene expression.

In some embodiments, the target nucleic acid region is associated with a trait of the cell or organism. In some implementations In some embodiments, mutations in the target nucleic acid region result in changes in the characteristics of the cell or organism. In some embodiments, the target nucleic acid region is located in the coding region of the protein. In some embodiments, the target nucleic acid region encodes a functionally relevant motif or domain of a protein. In some preferred embodiments, one or more nucleotide substitutions in the target nucleic acid region results in amino acid substitutions in the amino acid sequence of the protein. In some embodiments, the one or more nucleotide substitutions result in changes in the function of the protein.

In the method of the present invention, the base editing system can be introduced into cells through various methods well known to those skilled in the art.

Methods that can be used to introduce the base editing system of the present invention into cells include, but are not limited to: calcium phosphate transfection, protoplast fusion, electroporation, lipofection, microinjection, viral infection (such as baculovirus, vaccinia virus, Adenovirus, adeno-associated virus, lentivirus and other viruses), biolistic method, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation.

Cells that can be base edited by the method of the present invention can be from, for example, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, cats; poultry such as chickens, ducks, geese; plants, including Monocots and dicots, preferably crop plants, include but are not limited to wheat, rice, corn, soybean, sunflower, sorghum, rape, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava and potato.

5. Application in plants

The base editing fusion proteins, base editing systems and methods of producing genetically modified cells of the present invention are particularly suitable for genetic modification of plants. Preferably, the plant is a crop plant, including but not limited to wheat, rice, corn, soybean, sunflower, sorghum, rape, alfalfa, cotton, barley, millet, sugar cane, tomato, tobacco, cassava and potato. More preferably, the plant is rice.

In another aspect, the invention provides a method of producing a genetically modified plant, comprising introducing a base editing system of the invention into at least one said plant, thereby resulting in a target nucleic acid region in the genome of said at least one plant One or more nucleotide substitutions within.

In some embodiments, the method further includes screening the at least one plant for plants having the desired one or more nucleotide substitutions.

In the method of the present invention, the base editing composition can be introduced into the plant by various methods well known to those skilled in the art. Methods that can be used to introduce the base editing system of the present invention into plants include, but are not limited to: biolistic method, PEG-mediated protoplast transformation, soil Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube channel method, and Intraventricular injection method. Preferably, the base editing composition is introduced into the plant by transient transformation.

In the method of the present invention, the modification of the target sequence can be achieved by simply introducing or producing the base editing fusion protein and guide RNA in plant cells, and the modification can be stably inherited without the need to encode the base. Plants are stably transformed with exogenous polynucleotides that are components of the editing system. This avoids the potential off-target effects of the stably existing (continuously produced) base editing composition, and also avoids the integration of exogenous nucleotide sequences in the plant genome, thereby achieving higher biological safety.

In some preferred embodiments, the introduction is performed in the absence of selection pressure, thereby avoiding exogenous nucleotides Integration of sequences into plant genomes.

In some embodiments, the introduction includes transforming the base editing system of the invention into isolated plant cells or tissues, and then regenerating the transformed plant cells or tissues into intact plants. Preferably, the regeneration is performed in the absence of selection pressure, that is, without the use of any selection agent against the selection gene carried on the expression vector during tissue culture. Not using a selection agent can increase the regeneration efficiency of plants and obtain modified plants that do not contain foreign nucleotide sequences.

In other embodiments, the base editing system of the present invention can be transformed into specific parts of an intact plant, such as leaves, shoot tips, pollen tubes, young ears or hypocotyls. This is particularly suitable for the transformation of plants that are difficult to regenerate in tissue culture.

In some embodiments of the invention, in vitro expressed proteins and/or in vitro transcribed RNA molecules (eg, the expression construct is an in vitro transcribed RNA molecule) are directly transformed into the plant. The protein and/or RNA molecules can achieve base editing in plant cells and are subsequently degraded by the cells, avoiding the integration of exogenous nucleotide sequences in the plant genome.

Therefore, in some embodiments, genetic modification and breeding of plants using the methods of the present invention can result in plants whose genomes are free of exogenous polynucleotide integration, that is, non-transgene-free modified plants.

In some embodiments of the invention, wherein the modified target nucleic acid region is associated with a plant trait, such as an agronomic trait, whereby the one or more nucleotide substitutions result in the plant having altered properties relative to a wild-type plant. (Preferably improved) traits, such as agronomic traits.

In some embodiments, the method further includes the step of screening plants with the desired one or more nucleotide substitutions and/or a desired trait, such as an agronomic trait.

In some embodiments of the invention, the method further includes obtaining progeny of the genetically modified plant. Preferably, the genetically modified plant or progeny thereof has the desired nucleotide substitution(s) and/or a desired trait such as an agronomic trait.

In another aspect, the present invention also provides a genetically modified plant or a progeny thereof or a part thereof, wherein said plant is obtained by the above-mentioned method of the present invention. In some embodiments, the genetically modified plant or progeny thereof or parts thereof are non-transgenic. Preferably, the genetically modified plant or its progeny has the desired genetic modification and/or the desired traits such as agronomic traits.

In another aspect, the present invention also provides a plant breeding method, comprising combining a genetically modified first plant containing one or more nucleotide substitutions in the target nucleic acid region obtained by the above-mentioned method of the present invention with a plant that does not contain The one or more nucleotide substitutions are crossed into a second plant, thereby introducing the one or more nucleotide substitutions into the second plant. Preferably, the genetically modified first plant has desirable traits such as agronomic traits.

6. Therapeutic Application

The invention also covers the use of the base editing system of the invention in disease treatment.

By modifying disease-related genes through the base editing system of the present invention, the up-regulation, down-regulation, inactivation, activation or mutation correction of disease-related genes can be achieved, thereby achieving prevention and/or treatment of diseases. For example, the present invention The target nucleic acid region may be located within the protein coding region of a disease-related gene, or may be located in a gene expression regulatory region such as a promoter region or enhancer region, thereby enabling functional modification of the disease-related gene or modification of the disease-related gene. Modification of expression. Therefore, modification of disease-related genes described herein includes modifications to the disease-related genes themselves (such as protein coding regions), as well as modifications to their expression regulatory regions (such as promoters, enhancers, introns, etc.).

A "disease-associated" gene refers to any gene that produces a transcription or translation product at abnormal levels or in an abnormal form in cells derived from disease-affected tissue as compared to non-disease control tissues or cells. Where altered expression is associated with the emergence and/or progression of a disease, it may be a gene that is expressed at an abnormally high level; it may be a gene that is expressed at an abnormally low level. Disease-associated genes also refer to genes that have one or more mutations or genetic variants that are directly responsible for or in linkage disequilibrium with one or more genes responsible for the etiology of the disease. The mutation or genetic variation is, for example, a single nucleotide variation (SNV). The products of transcription or translation may be known or unknown, and may be at normal or abnormal levels.

Accordingly, the invention also provides methods of treating a disease in a subject in need thereof, comprising delivering to said subject an effective amount of a base editing system of the invention to modify a gene associated with said disease.

The present invention also provides the use of the base editing system of the invention for preparing a pharmaceutical composition for treating a disease in a subject in need thereof, wherein the base editing system is used to modify a gene associated with the disease.

The present invention also provides pharmaceutical compositions for treating diseases in a subject in need thereof, which comprise the base editing system of the present invention, and optionally a pharmaceutically acceptable carrier, wherein the base editing system is used to modify and genes related to the disease.

In some embodiments, the subject is a mammal, such as a human.

Examples of such diseases include, but are not limited to, tumors, inflammation, Parkinson's disease, cardiovascular disease, Alzheimer's disease, autism, drug addiction, age-related macular degeneration, schizophrenia, genetic disorders, and the like.

7. Test kit

The invention also includes a kit for use in the method of the invention, the kit comprising the base editing fusion protein of the invention and/or an expression construct containing a nucleotide sequence encoding the base editing fusion protein, or comprising Base editing system of the present invention. Kits generally include labels indicating the intended use and/or method of use of the contents of the kit. The term label includes any written or recorded material on or provided with the kit or otherwise provided with the kit. The kit of the present invention may also contain suitable materials for constructing the expression vector in the base editing system of the present invention. The kit of the present invention may also include reagents suitable for transforming the base editing fusion protein or base editing system of the present invention into cells.

8. Method for preparing adenine deaminase for base editing

In another aspect, the present invention also provides a method for obtaining/preparing adenine deaminase for base editing, comprising

1) Identification of an adenine deaminase containing the characteristic sequence motif VX _n NX ₁₀ HAEX _n PCXMC; and

2) The adenine deaminase containing the characteristic sequence motif VX _n NX ₁₀ HAEX _n PCXMC corresponds to The amino acid at amino acid position 108 of SEQ ID NO:14 is mutated to N.

In some embodiments, the method includes converting the adenine deaminase comprising the characteristic sequence motif VX _n NX ₁₀ HAEX _n PCXMC at amino acid positions corresponding to positions 106-109 of SEQ ID NO: 14 The amino acid mutations are VRNS, ALNK, ALNS, ARNK, ARNS, VLNK, VLNS, or VRNK.

Example

In order to facilitate understanding of the present invention, the present invention will be described more fully below with reference to relevant specific embodiments and accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough understanding of the present disclosure will be provided.

Materials and Methods

1. Carrier construction

The excavated deaminase sequences were constructed, and the sequences were double-codon optimized for rice and wheat by Genscript. The sequence was constructed into the PABE-7 vector backbone (addgene #115628). The plasmid of the reporter system used in the examples was constructed in advance by the inventors (Li, C., Zong, Y., Wang, Y., Jin, S., Zhang, D., Song, Q., Zhang, R .,&Gao,C.(2018).Expanded base editing in rice and wheat using a Cas9-adenosine deaminase fusion.Genome biology,19(1),59.).

2. Protoplast isolation and transformation

The protoplasts used in the present invention come from rice variety Zhonghua 11.

2.1 Rice seedling culture

Rice seeds were first rinsed with 75% ethanol for 1 minute, then treated with 4% sodium hypochlorite for 30 minutes, and washed more than 5 times with sterile water. Cultivate on M6 medium for 3-4 weeks at 26°C, protected from light.

2.2 Protoplast isolation

(1) Cut off the rice stalk, cut the middle part into 0.5-1mm silk with a blade, put it into 0.6M Mannitol solution to protect from light for 10 minutes, then filter it with a filter, and put it into 50mL enzymatic hydrolysis solution (0.45 μm membrane filtration), vacuum (pressure about 15Kpa) for 30 minutes, take out and place on a shaker (10 rpm) for enzymatic hydrolysis at room temperature for 5 hours;

(2) Add 30-50mL W5 to dilute the enzymatic hydrolyzate, filter the enzymatic hydrolyzate with a 75μm nylon filter and place it in a round-bottom centrifuge tube (50mL);

(3) 23℃, 250g (rcf), rise 3 and drop 3, centrifuge for 3 minutes, discard the supernatant;

(4) Gently suspend the cells with 20mL W5 and repeat step (3)

(5) Add appropriate amount of MMG to suspend until transformation.

2.3 Rice protoplast transformation

(1) Add 10 μg of each required transformation vector to a 2mL centrifuge tube, mix well, and use a sharpened pipette tip to pipette 200 μL protoplasts, flick to mix, add 220 μL PEG4000 solution, flick to mix, induce transformation at room temperature in the dark for 20-30 minutes;

(2) Add 880μL W5 and mix gently by inverting, 250g (rcf), rise 3 and drop 3, centrifuge for 3 minutes, discard the supernatant;

(3) Add 1mL of WI solution, mix gently by inverting, transfer gently to a flow tube, and incubate in the dark at room temperature for 48 hours.

3. Observe cell fluorescence with flow cytometry

Protoplast GFP-negative and positive populations were analyzed by flow cytometry using a FACSAria III (BD Biosciences) instrument.

Example 1. Sequence search for candidate adenine deaminase that can be used for base editing

TadA is an adenine deaminase that acts on tRNA. The deaminase in the adenine base editing system currently used is a TadA variant of Escherichia coli. The branch it belongs to is the Tad1/ADAR branch. Rubio et al. have studied and summarized that TadA deaminase has the characteristics of an amino acid sequence containing H(C)xE and PCxxC (where x represents an arbitrary amino acid) (Rubio, M.A., Pastar, I., Gaston, K.W., Ragone, F.L.,Janzen,C.J.,Cross,G.A.,Papavasiliou,F.N.,&Alfonzo,J.D.(2007).An adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U deamination of DNA.Proceedings of the National Academy of Sciences of the United States of America,104(19),7821-7826.). In order to find a new type of TadA deaminase, further annotation was carried out based on proteins that matched the characteristic sequence in the Uniprot sprot (https://www.uniprot.org/uniprot/) database, and the characteristic sequence was further analyzed and Revise. The inventor found that when the characteristic sequence is VnxN10xHAEnxPCxMC (nx represents any number of any amino acids, 10x represents 10 any number of amino acids), most of the results found in the Uniprot sprot database (Table 1) and Uniprot tremble database (Table 2) For the protein sequence annotated as TadA, it was proved that this characteristic sequence has high confidence for searching for novel adenine deaminase.

Table 1. Protein functions and proportions found in the Uniprot sprot database using modified characteristic sequences

Table 2. Protein functions and proportions found in the Uniprot tremble database using modified characteristic sequences

Example 2. Transformation of No. 135 potential new TadA deaminase

The inventor found that Iyer et al. (Iyer, L.M., Zhang, D., Rogozin, I.B., & Aravind, L. (2011). Evolution of the deaminase fold and multiple origins of eukaryotic editing and mutagenic nucleic acid deaminases from bacterial toxin systems. Nucleic acids research, 39(22), 9473-9497. The potential deaminase listed as number 135 has this characteristic sequence. And the sequence has a very low similarity with E. coli TadA deaminase, only 41.89% (Figure 1 ). In order to make it act on DNA, the inventors referred to the ABE8e variant sequence (Richter, M.F., Zhao, K.T., Eton, E., Lapinaite, A., Newby, G.A., Thuronyi, B.W., Wilson, C., Koblan,L.W.,Zeng,J.,Bauer,D.E.,Doudna,J.A.,&Liu,D.R.(2020).Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.Nature biotechnology,38(7),883 -891.), mutating the 101-104 amino acids (corresponding to the 106-109 amino acids of ABE8e). The inventor found that when the D at position 103 is changed to N, the No. 135 deaminase can be The function of deamination of adenine at the target site on DNA (Figure 2 and Table 3). Therefore, the protein numbered 135 has the function of deamination of adenine on single-stranded DNA. Based on this protein, a new type of adenine can be established Base editing system.

Table 3. No.135 potential deaminase 101-104 amino acid mutation information and reporting system luminescence status

Example 3. Modification of proteins in the guanine deaminase branch into novel adenine deaminase

Through the VnxN10xHAEnxPCxMC feature sequence search results, it was found that in addition to the sequences annotated as TadA in the database, there are also some proteins annotated as other functions, such as guanine deaminase, tRNA isoleucine synthase, HAD hydrolase, etc. and some proteins whose functions have not yet been elucidated. The inventors selected some of these proteins and found that they were highly similar to TadA in structure, but had extremely low sequence similarity to TadA (Figure 4). Therefore, based on the guanine deaminase branch listed by Iyer et al., proteins numbered 1299 and 1417 were selected for comparison with ecTadA, and it was found that their similarity to ecTadA was only 47.24% and 42.66% (Figure 3). According to the comparison results, the four key amino acids of 1299 and 1417 were modified (replaced with VRNS). The results of protoplast experiments showed that the modified proteins can make the reporter system glow (Figures 5 and 6), that is, they can achieve the target site gland Deamination of purines.

sequence list

Claims (22)

Use of a cytosine deaminase for gene editing, such as base editing, in an organism or a cell of an organism, wherein the adenine deaminase 1) Contains the characteristic sequence motif VX _n NX ₁₀ HAEX _n PCXMC; and/or 2) Contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, An amino acid sequence with 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity at the amino acid position corresponding to position 108 of SEQ ID NO: 14 The amino acid is N. A base editing fusion protein comprising a nucleic acid targeting domain and an adenine deamination domain, wherein the adenine deamination domain comprises at least one (eg, one or two) adenine deaminase polypeptides, adenine deaminase 1) Contains the characteristic sequence motif VX _n NX ₁₀ HAEX _n PCXMC; and/or 2) Contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, An amino acid sequence with 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity at the amino acid position corresponding to position 108 of SEQ ID NO: 14 The amino acid is N. The use of claim 1 or the base editing fusion protein of claim 2, wherein the adenine deaminase i) The amino acid at the amino acid position corresponding to amino acid position 106 of SEQ ID NO: 14 is A or V; ii) the amino acid at amino acid position corresponding to amino acid position 107 of SEQ ID NO: 14 is L or R; and/or iii) The amino acid at the amino acid position corresponding to amino acid position 109 of SEQ ID NO: 14 is K or S. The use or base editing fusion protein of claim 3, wherein the amino acids of the adenine deaminase at the amino acid positions corresponding to positions 106-109 of SEQ ID NO: 14 are VRNS, ALNK, ALNS, ARNK, ARNS , VLNK, VLNS, or VRNK. The use or base editing fusion protein of claim 4, wherein the adenine deaminase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-9, 11 and 13. The base editing fusion protein of any one of claims 2-5, wherein the nucleic acid targeting domain comprises at least one CRISPR effector polypeptide. The base editing fusion protein of claim 6, wherein the CRISPR effector protein is nuclease-inactivated Cas9, for example, the nuclease-inactivated Cas9 includes the amino acid sequence shown in SEQ ID NO: 16. The base editing fusion protein of any one of claims 2-7, wherein the nucleic acid targeting domain and the adenine deamination domain are fused through a linker. The base editing fusion protein of any one of claims 2 to 8, wherein the base editing fusion protein includes in the following order from the N-terminus to the C-terminus: an adenine deamination domain and a nucleic acid targeting domain. The base editing fusion protein of any one of claims 2-9, wherein the base editing fusion protein further comprises one or more nuclear localization sequences (NLS). A base editing system for modifying target nucleic acid regions in the genome of an organism or organism cell, which includes: i) The base editing fusion protein of one of claims 2-10 and/or an expression construct containing a nucleotide sequence encoding the base editing fusion protein; and/or ii) at least one guide RNA and/or at least one expression construct comprising a nucleotide sequence encoding said at least one guide RNA, wherein said at least one guide RNA is directed to at least one target sequence within said target nucleic acid region. The base editing system of claim 11, wherein the nucleotide sequence encoding the base editing fusion protein is codon-optimized for the organism whose genome is to be modified. The base editing system of claim 11 or 12, wherein the organism is a eukaryotic organism, including mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, cats; poultry such as chickens, Ducks and geese; plants, including monocots and dicots, for example, the plants are crop plants, including but not limited to wheat, rice, corn, soybean, sunflower, sorghum, rape, alfalfa, cotton, barley, millet, Sugar cane, tomato, tobacco, cassava and potato. A method of producing at least one genetically modified cell, comprising introducing the base editing system of any one of claims 11-13 into at least one of said cells, thereby resulting in said at least one cell within a target nucleic acid region. One or more nucleotides, for example, the one or more nucleotide substitutions are A to G substitutions. 14. The method of claim 14, further comprising the step of screening said at least one cell for cells having the desired one or more nucleotide substitutions. The method of claim 14 or 15, wherein the base editing system is introduced into the cell by a method selected from the group consisting of: calcium phosphate transfection, protoplast fusion, electroporation, lipofection, microinjection, viral infection (such as rod-shaped viruses, vaccinia virus, adenovirus, adeno-associated virus, lentivirus and other viruses), biolistic method, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation. The method of any one of claims 14-16, wherein the cells are from mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, cats; poultry such as chickens, ducks, geese; plants, Included are monocots and dicots, preferably crop plants such as wheat, rice, corn, soybean, sunflower, sorghum, rape, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava and potato. A pharmaceutical composition for treating a disease in a subject in need thereof, comprising the base editing system of any one of claims 11-13, and optionally a pharmaceutically acceptable carrier, wherein the base editing system The system is used to modify genes associated with the disease. The pharmaceutical composition of claim 18, wherein said subject is a mammal, such as a human. The pharmaceutical composition of claim 18 or 19, wherein the disease is selected from the group consisting of tumors, inflammation, Parkinson's disease, cardiovascular disease, Alzheimer's disease, autism, drug addiction, age-related macular degeneration, and schizophrenia. , hereditary diseases, etc. A method of obtaining/preparing adenine deaminase for base editing, comprising 1) Identification of an adenine deaminase containing the characteristic sequence motif VX _n NX ₁₀ HAEX _n PCXMC; and 2) Mutation of the amino acid at the amino acid position corresponding to position 108 of SEQ ID NO: 14 in the adenine deaminase containing the characteristic sequence motif VX _n NX ₁₀ HAEX _n PCXMC to N. The method of claim 21 , wherein the method comprises converting the adenine deaminase containing the characteristic sequence motif VX _n NX ₁₀ HAEX _n PCXMC at the amino acid positions corresponding to positions 106-109 of SEQ ID NO: 14 The amino acid mutation is VRNS, ALNK, ALNS, ARNK, ARNS, VLNK, VLNS, or VRNK.