Small base editor developed on basis of is200 / is605 transposon family and use thereof

Abstract

A small base editor developed on the basis of the IS200 / IS605 transposon family and a use thereof. Provided is a base editing system, comprising: (1) a mutant of an ISDra2-TnpB protein and / or a nucleic acid encoding the mutant of the ISDra2-TnpB protein; (2) a deaminase and / or a nucleic acid encoding the deaminase; and (3) a guide RNA and / or a nucleic acid encoding the guide RNA, wherein the mutant of the ISDra2-TnpB protein has a genome targeting function but does not have a DNA cleavage function.

Description

A small base editor based on the IS200 / IS605 transposon family and its applications Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a small base editor developed based on the IS200 / IS605 transposon family and its applications. Background Technology

[0002] Base editors are a cutting-edge technology that enables precise and efficient single-base editing at the genome level. They are mainly based on the localization capabilities of the CRISPR / Cas system, combined with the editing functions of base deaminases or other enzymes, and can achieve base conversion or transversion without generating double-strand DNA breaks or relying on homologous recombination repair templates.

[0003] Base editors are mainly divided into cytosine base editors (CBE) and adenine base editors (ABE). The principle of cytosine base editor technology is to fuse cytosine deaminase with dCas or nCas proteins. Utilizing the Cas system's genome targeting and the single-stranded DNA deaminase activity of cytosine deaminase, base editing can be performed independently of DSB (double-strand DNA break), enabling the conversion from cytosine (C) to thymine (T) in cells. Adenine base editing technology is similar, fusing adenine deaminase with the Cas9 protein to achieve the conversion from adenine (A) to guanine (G) in cells. The David Liu laboratory at Harvard University launched cytosine base editors (including BE1, BE2, and BE3) and adenine base editors (including ABE1–ABE7.10) in 2016 and 2017, respectively. BE3 is composed of nCas9 fused with the deaminase rAPOBEC1 and the uracil DNA glycosidase inhibitor UG1. ~37% of its genomic target sequence achieves C-to-T base conversion, while its indels (small insertions or deletions) frequency is low, approximately 1.1%. ABE7.10 is composed of nCas9 fused with two adenine deaminases, achieving A-to-G base conversion in ~50% of its genomic target sequence, while its indels frequency is approximately 0.1%. Subsequently, to further improve editing efficiency and reduce the frequency of indels, BE4 and BE4max were formed by increasing the number of UGI fusions, codon optimization, and adding a nuclear localization sequence (NLS) based on BE3; ABEmax was formed by codon optimization and adding a nuclear localization sequence (NLS) based on ABE7.10. Furthermore, a series of highly efficient deaminases were obtained through phage evolution screening, such as the cytosine base editors evoAPOBEC1-BE4max, evoFERNY-BE4max, and evoCDA1-BE4max, and the adenine base editor ABE8e. Currently, multiple laboratories worldwide have optimized the base editors, resulting in a series of base editors with different editing windows, editing efficiencies, and off-target frequencies.

[0004] However, most existing base editing systems are based on SpCas9. Since the SpCas9 protein contains 1368 amino acids (molecular weight 158.46 kDa), the resulting base editing systems suffer from a large protein size, hindering cell delivery using vectors such as AAV and limiting the application of base editors in gene therapy and other fields. Currently, most base editing systems utilize the CRISPR / Cas system for genome targeting. This system is simple, efficient, and easy to design. However, because the CRISPR / Cas system originates from the bacterial immune system, it involves a Cas protein and gRNA-mediated RNP complex that mediates protein-genome targeting and R-Loop formation. It then uses single-stranded DNA-specific deaminases to edit specific bases within the target editing window. This system exhibits certain off-target effects and immunogenicity. Furthermore, with numerous base editors already built based on the CRISPR / Cas system, its competitive advantage and technological innovation are gradually diminishing.

[0005] Therefore, there is an urgent need to develop a gene editing tool with a small molecular weight that is easy to deliver via vector and for gene therapy. Summary of the Invention

[0006] This invention aims to at least partially address one of the technical problems in related technologies. To this end, one objective of this invention is to provide a base editing system based on the IS200 / IS605 transposon family gene editing tool TnpB, combined with currently discovered highly efficient single-stranded DNA-specific adenine deaminases or cytosine deaminases. It utilizes the TnpB gene-editing active mutants TnpBM1 and TnpBM2 and their reRNAs for genome targeting. By fusing TnpBM1 or TnpBM2 with a deaminase, the deaminase is targeted to the target region to achieve the conversion from cytosine to thymine (C→T) or from adenine to guanine (A→G). This system features a significantly reduced protein size and can be delivered to cells using vectors such as AAV, which is beneficial for downstream applications such as gene therapy.

[0007] Therefore, the present invention provides a base editing system, comprising:

[0008] (1) A mutant of the ISDra2-TnpB protein and / or the nucleic acid encoding the mutant of the ISDra2-TnpB protein;

[0009] (2) Deaminase and / or nucleic acid encoding the deaminase;

[0010] (3) Guide RNA and / or nucleic acid encoding the guide RNA.

[0011] The mutant of the ISDra2-TnpB protein possesses genome targeting function but lacks DNA cleavage function.

[0012] Most mature base editing systems are currently based on SpCas9. However, because the SpCas9 protein contains 1368 amino acids (molecular weight 158.46 kDa), the resulting base editing system molecules are relatively large, hindering cell delivery using vectors such as AAV and limiting its application in gene therapy. This invention is based on the ISDra2TnpB protein, which contains only 408 amino acids (molecular weight 46.41 kDa), significantly smaller than SpCas9. The smaller molecular size of the base editing system based on ISDra2TnpB allows for cell delivery using vectors such as AAV, facilitating downstream applications such as gene therapy.

[0013] According to an embodiment of the present invention, the mutant of the ISDra2-TnpB protein is:

[0014] A mutant with at least 80% sequence homology to the wild-type ISDra2-TnpB protein.

[0015] According to an embodiment of the present invention, the base editing system further includes a nuclear localization signal fragment and / or a nucleic acid encoding the nuclear localization signal fragment.

[0016] According to an embodiment of the present invention, the amino acid sequence of the wild-type ISDra2-TnpB protein is shown in SEQ ID NO:58.

[0017] The amino acid sequence of the wild-type ISDra2-TnpB protein is shown in SEQ ID NO:58 below:

[0018] According to an embodiment of the present invention, the mutant of the ISDra2-TnpB protein is a protein in which the 191st amino acid of the wild-type ISDra2-TnpB protein is mutated from aspartic acid to alanine; and / or

[0019] The mutant of the ISDra2-TnpB protein is a protein in which the 278th amino acid of the wild-type ISDra2-TnpB protein is mutated from glutamic acid to alanine.

[0020] According to an embodiment of the present invention, the base editing system identifies a TAM sequence on a target sequence.

[0021] According to an embodiment of the present invention, the guide RNA comprises:

[0022] 1) Gene targeting regions that can bind complementary to the target sequence;

[0023] 2) A guide segment that binds to the mutant of the ISDra2-TnpB protein.

[0024] According to an embodiment of the present invention, the deaminase includes cytosine deaminase and adenine deaminase.

[0025] According to an embodiment of the present invention, the cytosine deaminase is a DNA or RNA cytosine deaminase; the adenine deaminase is a DNA or RNA adenine deaminase.

[0026] According to an embodiment of the present invention, the adenine deaminase includes at least one selected from TadA, TadA*, and TadA**.

[0027] According to embodiments of the present invention, the cytosine deaminase comprises a subset selected from APOBEC, FERNY deaminase, or variants thereof.

[0028] According to an embodiment of the present invention, when the deaminase includes cytosine deaminase, the base editing system further includes a uracil DNA glycosylase inhibitor (UGI protein).

[0029] According to an embodiment of the present invention, the mutant of the ISDra2-TnpB protein is further linked to at least one UGI protein.

[0030] Another aspect of the present invention provides a recombinant expression vector comprising any two or three of the following:

[0031] (i) The nucleic acid sequence encoding the guide RNA;

[0032] (ii) The nucleic acid sequence of the mutant encoding the SDra2-TnpB protein;

[0033] (iii) The nucleic acid sequence encoding the deaminase.

[0034] The guide RNA, the mutant SDra2-TnpB protein, and the deaminase are as described in the base editing system above.

[0035] This invention utilizes the newly discovered IS200 / IS605 transposon family gene editing tool TnpB, combined with currently discovered highly efficient single-stranded DNA-specific adenine deaminase or cytosine deaminase. TnpB and its reRNA are used for genome targeting. By fusing TnpB with a deaminase, the deaminase is targeted to the target region to achieve the conversion from cytosine to thymine (C→T) or from adenine to guanine (A→G).

[0036] In another aspect, the present invention provides an adeno-associated virus. According to an embodiment of the present invention, the adeno-associated virus is prepared by viral packaging of the aforementioned recombinant expression vector.

[0037] Another aspect of the present invention provides a base editing method, comprising:

[0038] The target gene is brought into contact with the base editing system described above to achieve single-base editing of the target gene.

[0039] In another aspect, the present invention provides a cell obtained by editing the cell using the base editing system or the base editing method described above.

[0040] In another aspect, the present invention provides a kit comprising at least one of the base editing system described above, the recombinant expression vector described above, and the adeno-associated virus described above.

[0041] In another aspect, the present invention provides the use of the aforementioned base editing system, the aforementioned recombinant expression vector, and the aforementioned adeno-associated virus in the preparation of kits for base editing.

[0042] In another aspect, the present invention provides a drug comprising at least one of the base editing system described above, the recombinant expression vector described above, and the adeno-associated virus described above.

[0043] In another aspect, the present invention provides the use of the aforementioned base editing system, the aforementioned recombinant expression vector, and the aforementioned adeno-associated virus in the preparation of drugs for gene therapy.

[0044] In another aspect, the present invention provides the use of the aforementioned base editing system, the aforementioned recombinant expression vector, the aforementioned adeno-associated virus, and the aforementioned drug in gene therapy.

[0045] This invention, based on a small base editor developed from transposon families, is novel and facilitates packaging with vectors such as AAV, enabling precise editing of target gene regions. It can be applied to the precise editing of genomes in human cells, facilitating downstream applications such as scientific research and gene therapy. The development of this small base editor broadens its application in gene therapy and other fields, demonstrating promising market prospects. In another aspect, this invention provides a gene therapy method comprising administering at least one of the following to a subject:

[0046] a. The base editing system described above;

[0047] b. The recombinant expression vectors described above;

[0048] c. The adeno-associated virus mentioned above;

[0049] d. The drugs mentioned above.

[0050] Base editors play a crucial role in gene therapy for monogenic genetic diseases, but most current base editors are based on SpCas9, resulting in excessively large protein sizes that hinder cell delivery for gene therapy applications. The ISDra2TnpB protein, containing only 408 amino acids, is significantly smaller than SpCas9. Therefore, base editing systems based on ISDra2TnpB can be significantly smaller, allowing for cell delivery using vectors such as AAV, thus facilitating downstream gene therapy applications.

[0051] This invention is based on the newly discovered IS200 / IS605 transposon family gene editing tool TnpB, and is a novel base editing system that is not dependent on the CRISPR / Cas system. This base editing system is relatively novel, with few reports to date, and possesses innovation and competitive advantages.

[0052] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0053] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0054] Figure 1 shows a schematic diagram of the in vivo editing vectors PX458-TnpB-NC and PX458-TnpB-HDV-NC in Example 1. In these two vectors, the expression of reRNA-target or reRNA-target-HDV is initiated by the human U6 promoter; the expression of downstream TnpB protein and GFP is initiated by the Pol II promoter.

[0055] Figure 2 shows the in vivo editing activity analysis of TnpB protein in HEK239T cells in Example 1. The in vivo editing activity of TnpB was verified by T7E1 enzyme digestion analysis. The in vivo gene editing activity of TnpB in cells was detected at 12 target sites of four genes: AAVS1, EMX1, AGBL1, and HBG1. SpCas9 was used as a positive control to detect gene editing activity at one target site of the AAVS1 gene.

[0056] Figure 3 shows the gene editing activity analysis of the TnpB mutants TnpB-M1 and TnpB-M2 in HEK239T cells in Example 2. The in vivo editing activity of TnpB, TnpB-M1 and TnpB-M2 was verified by T7E1 restriction enzyme digestion analysis. The in vivo gene editing activity of TnpB, TnpB-M1 and TnpB-M2 was detected at two target sites of the EMX1 and PTEN1 genes.

[0057] Figure 4 shows the CRISPRi inhibitory activity of TnpB-M1 and TnpB-M2 against EMX1 gene expression in HEK239T cells in vivo, analyzed using real-time analysis in Example 2.

[0058] Figure 5 shows the in vivo editing activity analysis of TnpB targeting the A-rich site of the AGBL1 gene using T7E1 enzyme digestion in Example 3;

[0059] Figure 6 shows the in vivo editing activity analysis of TnpB against the A-rich target site of the AGBL1 gene, which was verified by amplicon library construction and sequencing analysis using the target site in Example 3.

[0060] Figure 7 shows the in vivo editing activity of TnpB against the C-rich target site of the AGBL1 gene, verified by T7E1 restriction enzyme digestion analysis in Example 4.

[0061] Figure 8 shows the in vivo editing activity analysis of TnpB against the C-rich target site of the AGBL1 gene, which was verified by amplicon library construction and sequencing analysis using the target site in Example 4.

[0062] Figure 9 shows a schematic diagram of the adenine base editor protein structure designed based on TnpB in Example 5, where TnpBM1 represents the TnpB (D191A) mutant and TnpBM2 represents the TnpB (E278A) mutant;

[0063] Figure 10 shows a schematic diagram of the cytosine base editor protein structure designed based on TnpB in Example 6, where TnpBM1 represents the TnpB (D191A) mutant and TnpBM2 represents the TnpB (E278A) mutant;

[0064] Figure 11 shows the in vivo base editing activity of adenine base editors TnpBM1-ABE1, TnpBM2-ABE1, and TnpBM2-ABE2 evaluated by library construction and sequencing using targeted site amplicon in Example 7.

[0065] Figure 12 shows the in vivo base editing activity of adenine base editors TnpBM1-ABE1, TnpBM1-ABE2, TnpBM1-ABE3 and TnpBM1-ABE4 evaluated by library construction and sequencing using targeted site amplicon in Example 7.

[0066] Figure 13 shows the in vivo base editing activity of cytosine base editors TnpBM1-CBE1, TnpBM2-CBE1, TnpBM2-CBE2, and TnpBM2-CBE3 evaluated by library construction and sequencing using targeted site amplicon in Example 8.

[0067] Figure 14 shows the in vivo base editing activity of cytosine base editors TnpBM1-CBE1, TnpBM1-CBE2, TnpBM1-CBE3, TnpBM1-CBE4, TnpBM1-CBE5 and TnpBM1-CBE6 evaluated by library construction and sequencing using targeted site amplicon in Example 8.

[0068] Detailed description of the invention

[0069] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0070] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0071] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0072] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0073] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0074] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0075] The term "homology" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing corresponding positions in different polypeptide or nucleic acid molecules. When the same position in the sequence of the compared molecules is occupied by the same base or amino acid in different sequences, then the molecules are homologous at that position. The degree of homology between sequences is determined as a function of the number of common matching or homologous positions.

[0076] The term “encoding” refers to a polynucleotide “encoding” a polypeptide, meaning that in its natural state or when manipulated by methods known to those skilled in the art, it can be transcribed and / or translated to produce a target polypeptide and / or fragments thereof, or to produce mRNA capable of encoding the target polypeptide and / or fragments thereof.

[0077] The term "complementary" refers to the fact that nucleic acids (such as RNA) contain nucleotide sequences that enable them to bind nonvalently to another nucleic acid in a sequence-specific, antiparallel manner under appropriate in vitro and / or in vivo temperature and solution ionic strength conditions, i.e., forming Watson-Crick base pairs and / or G / U base pairs, "annealing" or "hybridization".

[0078] It is understood in the art that the sequence of a polynucleotide does not need to be 100% complementary to the sequence of its target nucleic acid, which it can specifically hybridize to. The polynucleotide may hybridize on one or more segments. The polynucleotide may contain at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence complementarity with the target region within the target nucleic acid sequence it is targeting. In the art, the percentage complementarity between specific nucleic acid sequence segments can be routinely determined using known BLAST and PowerBLAST programs.

[0079] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably in this invention and refer to a polymeric form of amino acids of any length, which may include encoded and non-coded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a modified peptide backbone.

[0080] The term "encoding" a specific RNA DNA sequence refers to the DNA nucleic acid sequence transcribed into RNA. DNA polynucleotides can encode RNA (mRNA) that is translated into protein, or they can encode RNA that is not translated into protein (e.g., tRNA, rRNA, or gRNA; also known as "non-coding" RNA or "ncRNA").

[0081] The term "vector" or "expression vector" refers to a replicon, such as a plasmid, bacteriophage, virus, or granule, to which another segment of DNA, or "insertion fragment," can be attached in order to enable the attached segment to replicate within the cell.

[0082] The terms "recombinant expression vector" or "DNA construct" are used interchangeably in this invention to refer to a DNA molecule comprising a vector and at least one insert fragment. Recombinant expression vectors are typically produced for the purpose of expressing and / or amplifying the insert fragment or for constructing other recombinant nucleotide sequences. When exogenous DNA, such as a recombinant expression vector, has been introduced into a cell, the cell has been "genetically modified," "transformed," or "transfected" by said DNA. The presence of exogenous DNA results in permanent or transient genetic alterations. The transformed DNA may or may not integrate into the cell's genome.

[0083] The term "target DNA" refers to a DNA polynucleotide containing a "target site" or "target sequence." The terms "target site" and "target sequence" are used interchangeably in this invention to refer to the nucleic acid sequence present in the target DNA.

[0084] "Cutting" refers to the breaking of the covalent backbone of a DNA molecule.

[0085] The terms "nuclease" and "endonuclease" are used interchangeably to refer to enzymes that have catalytic activity for the degradation of endonucleases for the cleavage of polynucleotides. The "cleavage domain," "active domain," or "nuclease domain" of a nuclease refers to a polypeptide sequence or domain within the nuclease that has catalytic activity for DNA cleavage. The cleavage domain may be contained within a single polypeptide chain, or the cleavage activity may arise from the association of two or more polypeptides.

[0086] The term "treatment" includes preventing the occurrence of disease or symptoms; suppressing disease or symptoms; or alleviating disease.

[0087] The term "subject" refers to any mammalian subject, particularly a human, to whom a diagnosis, treatment, or therapy is intended.

[0088] Base editing system

[0089] Most current base editing systems utilize the CRISPR / Cas system for genome targeting. This invention develops a single-base editing system based on the newly discovered IS200 / IS605 transposon family gene editing tool TnpB. This system mediates genome targeting through transposon proteins and reRNA, representing a novel base editing system independent of the CRISPR / Cas system. This base editing system is novel because its genome targeting via the transposon protein TnpB and reRNA overcomes some inherent limitations of the CRISPR-Cas system, and is expected to have relatively lower off-target effects and immunogenicity. Furthermore, base editing systems independent of the CRISPR / Cas system are rarely reported, giving this system an innovative and competitive advantage.

[0090] Previous base editing systems all targeted SpCas9, whose large protein size limited the application of base editors in gene therapy and other fields. In recent years, the identification of smaller gene editing tools has provided a new foundation for the modification of base editors. This invention utilizes the recently discovered IS200 / IS605 transposon family gene editing tool TnpB as a basis to develop single-base editing systems targeting cytosine and adenine respectively, featuring a significantly reduced protein size, which can be used for subsequent gene therapy and other applications.

[0091] According to a specific embodiment of the present invention, the present invention provides a base editing system, comprising:

[0092] (1) A mutant of the ISDra2-TnpB protein and / or the nucleic acid encoding the mutant of the ISDra2-TnpB protein;

[0093] (2) Deaminase and / or nucleic acid encoding the deaminase;

[0094] (3) Guide RNA and / or nucleic acid encoding the guide RNA.

[0095] The mutant of the ISDra2-TnpB protein possesses genome targeting function but lacks DNA cleavage function.

[0096] According to a specific embodiment of the present invention, when the base editing system provided by the present invention contains mutants of the ISDra2-TnpB protein, all mutant forms that possess genome targeting function but lack DNA cleavage function and can bind to guide RNA are covered within the protection scope of the base editing system provided by the present invention. According to a preferred embodiment of the present invention, the mutant of the ISDra2-TnpB protein is a mutant having at least 80% sequence homology compared to the amino acid sequence of the wild-type ISDra2-TnpB protein (as shown in SEQ ID NO:58). It should be noted that "at least 80% sequence homology" as used in the present invention refers to sequence homology of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher.

[0097] According to a preferred embodiment of the present invention, the mutant of the ISDra2-TnpB protein is a protein in which the 191st amino acid of the wild-type ISDra2-TnpB protein is mutated from aspartic acid to alanine.

[0098] According to another preferred embodiment of the present invention, the mutant of the ISDra2-TnpB protein is a protein in which the 278th amino acid of the wild-type ISDra2-TnpB protein is mutated from glutamic acid to alanine.

[0099] According to a specific embodiment of the present invention, the base editing system further includes a nuclear localization signal fragment and / or a nucleic acid encoding the nuclear localization signal fragment. It should be noted that the base editing system may contain one or more nuclear localization signal fragments.

[0100] According to a specific embodiment of the present invention, the amino acid sequence of the nuclear localization signal fragment in the base editing system provided by the present invention can be KRTADGSEFEPKKKRKV, such as SEQ ID NO:59. It should be noted that, in addition to the amino acid sequence shown in SEQ ID NO:59, other sequences with the same function of nuclear localization signal fragments are also covered within the scope of protection of the present invention.

[0101] Nuclear localization signal fragments (NLS) are short amino acid sequence segments found within nucleophilic proteins, rich in basic amino acid residues such as lysine (Lys) and arginine (Arg). These signal sequences guide protein transport from the cytoplasm to the nucleus. NLS sequences typically contain 4-8 basic amino acid residues, which can be a single cluster (single part, MP NLS) or two clusters (double part, BP NLS). Normally, NLS sequences guide protein entry into the nucleus by binding to importins in the nuclear pore complex. The base editing system provided by this invention allows the coding sequence of the mutant ISDra2-TnpB protein to be linked to an NLS sequence, enabling base editing of the target gene within the cell nucleus using the NLS sequence.

[0102] According to a specific embodiment of the present invention, the base editing system identifies a TAM sequence on a target sequence.

[0103] In this paper, "TAM sequences" (Transposon-Adjacent Motifs) are specific DNA sequences adjacent to transposons that are recognized by transposases during transposon splicing and RNA-guided DNA cleavage. TAM sequences are typically rich in AT bases and play a crucial role in transposon movement and gene editing. TAM sequences have important applications in gene editing technology. For example, the TnpB nuclease can recognize TAM sequences, thereby introducing double-strand breaks at specific locations and achieving gene editing. In this invention, the mutant of the ISDra2-TnpB protein in the base editing system of this invention achieves single-base editing by recognizing TAM sequences on the target sequence.

[0104] According to a specific embodiment of the present invention, the guide RNA comprises:

[0105] 1) Gene targeting regions that can bind complementary to the target sequence;

[0106] 2) A guide segment that binds to the mutant of the ISDra2-TnpB protein.

[0107] The "guide RNA" in this invention is a small non-coding RNA commonly used in gene editing systems. It guides nucleases to specific sites for editing by complementary pairing with the target DNA or RNA sequence. The definition of guide RNA is not limited to gRNA in CRISPR systems, but also includes other RNA molecules with similar functions, such as reRNA.

[0108] "reRNA" is a guide RNA that plays a guiding role in the TnpB nuclease system, helping TnpB recognize and cleave specific DNA sequences. The guide RNA in the base editing system provided by this invention is mainly used to bind to mutants of the ISDra2-TnpB protein and to bind complementary to the target sequence.

[0109] According to a specific embodiment of the present invention, the deaminase includes cytosine deaminase and adenine deaminase. According to a preferred embodiment of the present invention, the cytosine deaminase is a DNA or RNA cytosine deaminase; the adenine deaminase is a DNA or RNA adenine deaminase. According to a further preferred embodiment of the present invention, the adenine deaminase includes at least one selected from TadA, TadA*, and TadA**; the cytosine deaminase includes APOBEC, evoFERNY deaminase, or variants thereof.

[0110] In this article, "TadA" (tRNA-specific adenosine deaminase) is an adenosine deaminase originally used for the adenosine deamination reaction in tRNA. In the field of gene editing, TadA has been engineered for use in adenine base editors (ABE) and cytosine base editors (CBE).

[0111] In this paper, “TadA*” is an engineered adenosine deaminase used in the adenine base editor (ABE). TadA* is modified by single-point mutation or multiple mutations to enhance adenosine deamination activity on DNA, thereby achieving efficient adenine-to-guanine base conversion.

[0112] In this paper, “TadA**” is a further optimized variant of TadA. Through multiple sequence alignment (MSA)-guided protein engineering and TadA homolog screening, a highly efficient cytosine base editor (CBE) was developed, eliminating the bias of specific motifs and the limitation of adenosine deaminase activity.

[0113] In this paper, "APOBEC" (Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like) is a family of proteins with cytosine deaminase activity, capable of catalyzing the deamination of cytosine (C) in single-stranded DNA or RNA into uracil (U). APOBEC-1 is a member of this protein family. The "APOBEC variants" mentioned in this invention can be, for example, evoAPOBEC-1, an evolutionarily optimized cytosine deaminase used to improve the performance of cytosine base editors (CBEs). It was developed using the phage-assisted continuous evolution system (PACE) and exhibits higher editing efficiency and lower off-target effects.

[0114] “evoFERNY” is an evolutionarily optimized cytosine deaminase with high editing efficiency and low off-target effects. It was initially developed by David R. Liu's research team at Harvard University using the phage-assisted continuous evolution system (PACE) to improve the performance of cytosine base editors (CBEs). evoFERNY has demonstrated highly efficient cytosine-to-thymine (C-to-T) editing in various cell lines, particularly in sequence environments where traditional CBEs perform poorly. evoFERNY can recognize a wider range of target selections, including non-traditional PAM sequences such as NG-PAM, thus expanding the application scope of base editing. Compared to traditional cytosine deaminases, evoFERNY excels in reducing off-target editing events, improving the safety of gene editing.

[0115] According to a specific embodiment of the present invention, when the deaminase includes cytosine deaminase, the base editing system further includes a uracil DNA glycosylation inhibitor.

[0116] According to a specific embodiment of the present invention, in the base editing system, the mutant of the ISDra2-TnpB protein is further linked to at least one UGI protein.

[0117] Recombinant expression vectors, adeno-associated viruses, base editing methods

[0118] According to a specific embodiment of the present invention, the present invention provides a recombinant expression vector comprising any two or three of the following:

[0119] (i) The nucleic acid sequence encoding the guide RNA;

[0120] (ii) The nucleic acid sequence of the mutant encoding the SDra2-TnpB protein;

[0121] (iii) The nucleic acid sequence encoding the deaminase.

[0122] The guide RNA, the mutant SDra2-TnpB protein, and the deaminase are as described in the base editing system above.

[0123] According to a specific embodiment of the present invention, the present invention provides an adeno-associated virus, which is formed by viral packaging of the recombinant expression vector described above.

[0124] According to a specific embodiment of the present invention, the present invention provides a base editing method, comprising:

[0125] The target gene is brought into contact with the base editing system described above to achieve single-base editing of the target gene.

[0126] Cells, reagent kits, drugs

[0127] According to a specific embodiment of the present invention, the present invention provides a cell obtained by editing the cell using the base editing system or the base editing method described above.

[0128] According to a specific embodiment of the present invention, the present invention provides a kit comprising at least one of the aforementioned base editing system, the aforementioned recombinant expression vector, and the aforementioned adeno-associated virus.

[0129] According to a specific embodiment of the present invention, the present invention provides a drug comprising at least one of the aforementioned base editing system, the aforementioned recombinant expression vector, and the aforementioned adeno-associated virus.

[0130] The use of drugs in gene therapy and gene therapy methods

[0131] According to a specific embodiment of the present invention, the present invention provides the use of the aforementioned base editing system, the aforementioned recombinant expression vector, the aforementioned adeno-associated virus, and the aforementioned drug in gene therapy.

[0132] According to one specific embodiment of the present invention, the present invention provides a gene therapy method comprising administering to a subject at least one of the following:

[0133] a. The base editing system described above;

[0134] b. The recombinant expression vectors described above;

[0135] c. The adeno-associated virus mentioned above;

[0136] d. The drugs mentioned above.

[0137] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0138] Example 1: In vivo editing activity analysis of the transposon family gene editing tool TnpB

[0139] To clarify the targeted in vivo editing activity of TnpB, we first constructed two TnpB-based in vivo editing vectors, PX458-TnpB-NC and PX458-TnpB-HDV-NC, using the PX458 vector (Figure 1 and Table 1; both achieve the objectives of this invention, but PX458-TnpB-HDV-NC offers more precise targeting and better results; unless otherwise specified, PX458-TnpB-HDV-NC will be used subsequently). Then, 12 target sites were selected for the AAVS1, AGBL1, EMX1, and HBG1 / 2 genes (Table 2). These target sites were inserted into the in vivo editing vector PX458-TnpB-NC using Golden Gate cloning technology to obtain the targeted editing plasmid. Next, the obtained plasmid was transfected into HEK239T cells for in vivo editing experiments. Finally, the genome of the edited cells was extracted and the in vivo editing activity of TnpB was verified by PCR amplification and T7E1 restriction enzyme digestion analysis. The results are shown in Figure 2. Specific-sized cleavage bands could be detected for AAVS1-g1 (weak signal), AAVS1-g2, AAVS1-g3, EMX1-g1, AGBL1-g1, AGBL1-g2, and HBG1-g3, indicating that TnpB has in vivo editing activity in human cells.

[0140] Table 1. PX458-TnpB-NC and PX458-TnpB-HDV-NC vector sequence information

[0141] Table 2 Target site sequence information

[0142] Example 2: Obtaining TnpB gene-editing active mutants

[0143] To obtain TnpB gene-editing mutants for subsequent base editor construction, we mutated key amino acids in the RuvC domain of TnpB, including TnpM1 (D191A) and TnpM2 (E278A), and then evaluated their in vivo editing activity and in vivo gene expression inhibition (CRISPRi) effect. First, we constructed targeting plasmids for the PTEN1 and EMX1 genes using TnpB, TnpBM1 (also referred to as TnpB-M1 in Figure 3), and TnpBM2 (also referred to as TnpB-M2 in Figure 3). Then, we analyzed their intracellular gene-editing activity through cell transfection and T7E1 restriction enzyme digestion (Tables 3 and 4). The results, as shown in Figure 3, indicate that, unlike TnpB, no intracellular gene-editing activity was detected in the mutants TnpM1 (D191A) and TnpM2 (E278A). Furthermore, we analyzed the effects of TnpBM1 and TnpBM2 on the expression of the target gene EMX1 using real-time PCR (Table 4). The results are shown in Figure 4. The mutants TnpBM1 and TnpBM2 still exhibited an inhibitory effect on EMX1 gene expression, demonstrating a CRISPRi effect. These results indicate that the TnpBM1 and TnpBM2 mutations do not affect the targeting function of TnpB and can be used for the next step of base editor construction.

[0144] Table 3 Target Site Sequence Information

[0145] Table 4 Primer sequence information for T7E1 and Real-time PCR detection

[0146] Example 3: Design and Screening of Targets for Adenine Base Editor Activity Assessment

[0147] To evaluate the activity and characteristics of the adenine base editor, we first selected a set of adenine (A)-rich target sites (AT1–AT9) for the AGBL1 gene (Table 5). Then, we constructed target editing plasmids for AT1–AT9 using the TnpB gene editing vector PX458-TnpB-HDV-NC. We screened active targets by analyzing the TnpB target editing activity for subsequent base editing activity and characteristic evaluation. The results are shown in Figure 5. In vivo, all nine target sites (AT1–AT9) designed for ABE were effective TnpB editing sites, with AT8 showing weaker activity. Significant non-gene editing mutations were observed at AT1, AT2, AT6, and AT8. Further, we selected AT2–AT5, AT7, and AT9 sites, and analyzed the gene editing activity of each site using amplicon sequencing. The results showed that gene editing occurred at all detected sites, with editing efficiencies ranging from 10% to 50% (before deducting transfection efficiency) (Figure 6).

[0148] Table 5. Sequence information of the adenine base editor target site.

[0149] Example 4: Design and Screening of Targets for Cytosine Base Editor Activity Assessment

[0150] To evaluate the activity and characteristics of the cytosine base editor, we selected a set of cytosine (C)-rich target sites (CT1–CT7) for the AGBL1 gene (Table 6). Then, we constructed target editing plasmids for CT1–CT7 using the in vivo TnpB editing vector PX458-TnpB-HDV-NC. We screened active targets by analyzing the TnpB targeting activity for subsequent evaluation of base editing activity and characteristics. The results are shown in Figure 7. In vivo, 6 out of the 7 target sites (CT2–CT7) designed for CBE were effective TnpB editing sites, with CT6 showing relatively weak activity. Furthermore, we selected CT2–CT7 sites and analyzed the gene editing activity of each site using amplicon sequencing. The results showed that gene editing occurred at all detected sites, with editing efficiencies ranging from 5% to 30% (before deducting transfection efficiency) (Figure 8).

[0151] Table 6. Cytosine base editor target site sequence information

[0152] Example 5: Design of an adenine base editor

[0153] Based on previous research results, we selected TnpBM1 and TnpBM2 to construct adenine base editing vectors. Current research indicates that the ABE7.10 and ABE8e base editors constructed based on SpCas9 have high intracellular gene editing efficiency. In this study, six adenine base editing vectors, PX458-TnpBM1-ABE1-NC, PX458-TnpBM2-ABE1-NC, PX458-TnpBM1-ABE2-NC, PX458-TnpBM2-ABE2-NC, PX458-TnpBM1-ABE3-NC and PX458-TnpBM1-ABE4-NC (Table 7), were constructed by fusing the deaminase sequences in the ABE7.10 and ABE8e base editors with the TnpBM1 or TnpBM2 sequences. The corresponding base editors were named TnpBM1-ABE1, TnpBM2-ABE1, TnpBM1-ABE2, TnpBM2-ABE2, TnpBM1-ABE3 and TnpBM1-ABE4 (Figure 9).

[0154] Table 7 Sequence information of adenine base editing vector

[0155] Example 6: Design of a cytosine base editor

[0156] Based on previous research results, we selected TnpBM1 and TnpBM2 for constructing cytosine base editing vectors. Current research indicates that three base editors based on SpCas9—BE4max, evoAPOBEC1-BE4max, and evoFERNY-BE4max—have high intracellular gene editing efficiency. Based on these highly efficient cytosine base editors, we constructed nine cytosine base editors using TnpB, with the base editing vectors being PX458-TnpBM1-CBE1-NC, PX458-TnpBM2-CBE1-NC, PX458-TnpBM1-CBE2-NC, PX458-TnpBM2-CBE2-NC, PX458-TnpBM1-CBE3-NC, PX458-TnpBM2-CBE3-NC, and PX458-TnpB. M1-CBE4-NC, PX458-TnpBM1-CBE5-NC, and PX458-TnpBM1-CBE6-NC (Table 8) are named TnpBM1-CBE1, TnpBM2-CBE1, TnpBM1-CBE2, TnpBM2-CBE2, TnpBM1-CBE3, TnpBM2-CBE3, TnpBM1-CBE4, TnpBM1-CBE5, and TnpBM1-CBE6 respectively (Figure 10).

[0157] Table 8. Cytosine base editing vector sequence information

[0158] Example 7: Evaluation of the activity and properties of the adenine base editor

[0159] To evaluate the activity and characteristics of the adenine base editor, based on the previous screening results of effective editing targets for TnpB in vivo, we selected six targets: AT2–AT4, AT5, AT7, and AT9 (Table 5). We first designed three adenine base editing vectors, PX458-TnpBM1-ABE1-NC, PX458-TnpBM2-ABE1-NC, and PX458-TnpBM2-ABE2-NC (Table 7), to construct targeted editing plasmids. Then, we performed cell transfection and used targeted amplicon libraries for library construction and sequencing to evaluate the gene editing activity and characteristics of the three adenine base editors, TnpBM1-ABE1, TnpBM2-ABE1, and TnpBM2-ABE2 (Figure 9). Analysis revealed that the adenine base editors TnpBM1-ABE1 and TnpBM2-ABE1 exhibited low-level (less than 0.5% base editing activity) base editing activity (adenine A to guanine G) only at the AT3 site, with TnpBM1-ABE1 showing higher base editing activity than TnpBM2-ABE1. TnpBM2-ABE2 showed base editing activity at the AT3, AT5, AT7, and AT9 sites, with editing activities ranging from 0.2% to 2.5% for different positions. The base editing activity of TnpBM2-ABE2 at the AT3 site was significantly higher than that of TnpBM2-ABE1 (Figure 11). These results indicate that TnpBM2-ABE2 has higher base editing activity than TnpBM2-ABE1, and TnpBM1-ABE1 has higher base editor activity than TnpBM2-ABE1.

[0160] Furthermore, we designed three other base editing vectors using TnpBM1: PX458-TnpBM1-ABE2-NC, PX458-TnpBM1-ABE3-NC, and PX458-TnpBM1-ABE4-NC (Table 7), and constructed targeting plasmids for four target sites: AT3, AT5, AT7, and AT9. Then, we performed a new round of cell transfection and targeted amplicon library construction and sequencing to evaluate the gene editing activity and characteristics of the four adenine base editors obtained: TnpBM1-ABE1, TnpBM1-ABE2, TnpBM1-ABE3, and TnpBM1-ABE4 (Figure 9). Analysis revealed that three adenine base editors, TnpBM1-ABE1, TnpBM1-ABE2, and TnpBM1-ABE4, exhibited gene editing activity at the AT3 site, with their base editing activities decreasing in the order of TnpBM1-ABE4, TnpBM1-ABE2, and TnpBM1-ABE1. At the AT5, AT7, and AT9 sites, only TnpBM1-ABE4 showed base editing activity (Figure 12). These results indicate that TnpBM1-ABE4 exhibits the highest base editor activity.

[0161] Based on the above results, we obtained an adenine base editor constructed based on the small transposon protein TnpB. We found that the base editing activity of the base editor constructed by fusing TnpBM1 (D191A) mutant with TadA8e was high, and the base editing activity of TnpBM1-ABE4 constructed by fusing TadA8e with the carboxyl terminus of TnpBM1 was the highest.

[0162] Example 8: Evaluation of the activity and properties of the cytosine base editor

[0163] To evaluate the activity and characteristics of cytosine base editors, based on the previous screening results of effective editing targets for TnpB in vivo, we selected six targets, C2 to C7. First, using TnpBM1 and TnpBM2, we designed four cytosine base editing vectors: PX458-TnpBM1-CBE1-NC, PX458-TnpBM2-CBE1-NC, PX458-TnpBM2-CBE2-NC, and PX458-TnpBM2-CBE3-NC (Table 8) to construct targeted editing plasmids. Then, we performed cell transfection and used targeted amplicon libraries for library construction and sequencing to evaluate the gene editing activity and characteristics of the four cytosine base editors: TnpBM1-CBE1, TnpBM2-CBE1, TnpBM2-CBE2, and TnpBM2-CBE3 (Figure 9). Analysis revealed that the base editing activities of the base editors TnpBM1-CBE1, TnpBM2-CBE1, TnpBM2-CBE2, and TnpBM2-CBE3 decreased sequentially at the CT3 and CT5 sites. However, at the CT6 site, TnpBM1-CBE1, TnpBM2-CBE2, and TnpBM2-CBE3 exhibited base editing activity, with TnpBM2-CBE2 showing higher activity than TnpBM1-CBE1 and TnpBM2-CBE3. No significant base editing activity was detected at the CT7 site (Figure 13). These results indicate that the fusion of the TnpB(D191A) mutant TnpBM1 with a deaminase can achieve high base editing activity.

[0164] To further clarify the base editing activity of the TnpB cytosine base editor, we designed five other base editing vectors using TnpBM1: PX458-TnpBM1-CBE2-NC, PX458-TnpBM1-CBE3-NC, PX458-TnpBM1-CBE4-NC, PX458-TnpBM1-CBE5-NC, and PX458-TnpBM1-CBE6-NC (Table 7), and constructed targeting plasmids for four target sites: CT3, CT5, CT6, and CT7. Then, a new round of cell transfection and targeted amplicon library construction and sequencing were performed to evaluate the gene editing activity and characteristics of the six cytosine base editors obtained: TnpBM1-CBE1, TnpBM1-CBE2, TnpBM1-CBE3, TnpBM1-CBE4, TnpBM1-CBE5 and TnpBM1-CBE6 (Figure 10). Analysis revealed that base editing activities were detected in base editors TnpBM1-CBE1, TnpBM1-CBE2, TnpBM1-CBE3, TnpBM1-CBE4, and TnpBM1-CBE5 at the CT3 and CT5 sites, with TnpBM1-CBE1, TnpBM1-CBE2, and TnpBM1-CBE3 exhibiting relatively high base editing activity. At the CT6 site, base editing activities were detected in base editors TnpBM1-CBE1, TnpBM1-CBE2, and TnpBM1-CBE3, with TnpBM1-CBE3 showing relatively high activity. At the CT7 site, only TnpBM1-CBE1 and TnpBM1-CBE2 showed relatively low base editing activity (Figure 14). The above results indicate that the three base editors, TnpBM1-CBE1, TnpBM1-CBE2, and TnpBM1-CBE3, have relatively high base editing activity.

[0165] Based on the above results, we obtained a cytosine base editor constructed based on the small transposon protein TnpB. We found that the base editing activity of the base editor constructed by fusing the TnpB(D191A) mutant TnpBM1 with the detected cytosine deaminase was high, and the gene editing activity of the base editor constructed by fusing the deaminase with the amino terminus of TnpBM1 was relatively high.

[0166] First, this invention experimentally verified the in vivo targeted gene editing activity of TnpB, a novel transposon family of small gene editing tools (Example 1), demonstrating that TnpB possesses in vivo targeted gene editing activity in human cells. Second, we performed point mutations on key amino acids related to TnpB's gene editing activity and evaluated the in vivo editing activity and in vivo gene expression suppression (CRISPRi) effect of the mutants (Example 2), obtaining TnpB mutants TnpBM1 (D191A) and TnpM2 (E278A) that possess only genome targeting function but lack dsDNA cleavage function. Then, we selected a set of adenine (A)-rich target sites (A1-A9) and a set of cytosine (C)-rich target sites (C1-C7) for the AGBL1 gene. We constructed corresponding target editing plasmids using the in vivo editing vector PX458-TnpB-HDV-NC (Example 1) to analyze gene editing activity (Examples 3 and 4). We screened and obtained TnpB gene editing active sites for the activity evaluation of TnpB adenine and cytosine base editors. Simultaneously, we designed and constructed an adenine base editor using the obtained TnpB mutants TnpBM1 (D191A) and TnpM2 (E278A) combined with currently discovered highly efficient single-stranded DNA-specific adenine deaminases (Example 5); and designed and constructed a cytosine base editor using the TnpB mutants TnpBM1 (D191A) and TnpM2 (E278A) combined with currently discovered highly efficient single-stranded DNA-specific cytosine deaminases and uracil DNA glycosylase inhibitors (Example 6). Finally, we constructed corresponding cytosine base editors and adenine base editor targeting plasmids using the screened TnpB gene editing active sites. We evaluated their in vivo gene editing activity in human cells through cell transfection, obtaining a series of adenine and cytosine base editing systems developed based on the transposon family gene editing tool TnpB, and evaluated their base editing activity (Examples 7 and 8).

[0167] This invention utilizes the newly discovered IS200 / IS605 transposon family gene editing tool TnpB, combined with currently discovered highly efficient single-stranded DNA-specific adenine deaminase or cytosine deaminase. It uses the TnpB gene editing active mutants TnpBM1 and TnpBM2 and their reRNA for genome targeting. By using the TnpBM1 or TnpBM2 fusion deaminase, the deaminase is targeted to the target region to achieve the conversion from cytosine to thymine (C→T) or from adenine to guanine (A→G).

[0168] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0169] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A base editing system, wherein, include: (1) A mutant of the ISDra2-TnpB protein and / or the nucleic acid encoding the mutant of the ISDra2-TnpB protein; (2) Deaminase and / or nucleic acid encoding the deaminase; (3) Guide RNA and / or nucleic acid encoding the guide RNA. The mutant of the ISDra2-TnpB protein possesses genome targeting function but lacks DNA cleavage function.

2. The base editing system of claim 1, wherein, The mutant of the ISDra2-TnpB protein is: A mutant with at least 80% sequence homology to the wild-type ISDra2-TnpB protein.

3. The base editing system of claim 1, wherein, The base editing system further includes a nuclear localization signal fragment and / or a nucleic acid encoding the nuclear localization signal fragment.

4. The base editing system of claim 2, wherein, The amino acid sequence of the wild-type ISDra2-TnpB protein is shown in SEQ ID NO:

58.

5. The base editing system of claim 4, wherein, The mutant of the ISDra2-TnpB protein is a protein in which the 191st amino acid of the wild-type ISDra2-TnpB protein is mutated from aspartic acid to alanine; and / or The mutant of the ISDra2-TnpB protein is a protein in which the 278th amino acid of the wild-type ISDra2-TnpB protein is mutated from glutamic acid to alanine.

6. The base editing system of claim 1, wherein, The base editing system identifies the TAM sequence on the target sequence.

7. The base editing system according to claim 1, wherein, The guide RNA includes: 1) Gene targeting regions that can bind complementary to the target sequence; 2) A guide segment that binds to the mutant of the ISDra2-TnpB protein.

8. The base editing system of claim 1, wherein, The deaminases include cytosine deaminase and adenine deaminase.

9. The base editing system according to claim 8, wherein, The cytosine deaminase is a DNA or RNA cytosine deaminase; the adenine deaminase is a DNA or RNA adenine deaminase.

10. The base editing system of claim 8, wherein, The adenine deaminase includes at least one selected from TadA, TadA*, and TadA**; The cytosine deaminases include those selected from APOBEC, FERNY deaminases, or variants thereof.

11. The base editing system according to claim 1, wherein, When the deaminase includes cytosine deaminase, the base editing system further includes a uracil DNA glycosylation inhibitor.

12. The base editing system according to claim 11, wherein, The mutant of the ISDra2-TnpB protein is further linked to at least one UGI protein.

13. A recombinant expression vector, wherein, Includes any two or three of the following: (i) The nucleic acid sequence encoding the guide RNA; (ii) The nucleic acid sequence of the mutant encoding the SDra2-TnpB protein; (iii) The nucleic acid sequence encoding the deaminase. The guide RNA, the mutant SDra2-TnpB protein, and the deaminase are as described in any one of claims 1-12 in the base editing system.

14. An adeno-associated virus, wherein, The adeno-associated virus is prepared by viral packaging of the recombinant expression vector of claim 13.

15. A base editing method, wherein, include: The target gene is contacted with the base editing system of any one of claims 1-12 to achieve single base editing on the target gene.

16. A type of cell, in which, The cells were obtained by editing using the base editing system of any one of claims 1-12 or the base editing method of claim 15.

17. A kit, wherein, It includes at least one of the base editing system according to any one of claims 1-12, the recombinant expression vector according to claim 13, and the adeno-associated virus according to claim 14.

18. Use of the base editing system of any one of claims 1-12, the recombinant expression vector of claim 13, and the adeno-associated virus of claim 14 in the preparation of a kit for base editing.

19. A drug, wherein, It includes at least one of the base editing system according to any one of claims 1-12, the recombinant expression vector according to claim 13, and the adeno-associated virus according to claim 14.

20. Use of the base editing system of any one of claims 1-12, the recombinant expression vector of claim 13, and the adeno-associated virus of claim 14 in the preparation of a medicament for gene therapy.

21. Use of the base editing system of any one of claims 1-12, the recombinant expression vector of claim 13, the adeno-associated virus of claim 14, and the drug of claim 19 in gene therapy.

22. A method of gene therapy wherein, This includes administering at least one of the following to the subject: a. The base editing system according to any one of claims 1-12; b. The recombinant expression vector according to claim 13; c. The adeno-associated virus of claim 14; d. The medicament according to claim 19.

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