Improved cytosine base editing system

Abstract

Provided is an improved cytosine base editing system with reduced genome-wide off-target effects and a narrow editing window, the system comprising a base editing fusion protein comprising an APOBEC3B deaminase or an APOBEC3B deaminase mutant fused to a CRISPR effector protein.

Description

Technical Field

[0001] The present invention belongs to the field of gene editing. Specifically, the present invention relates to an improved cytosine base editing system with significantly reduced genome-wide off-target effects and a narrow editing window. Background of the Invention

[0003] Genome editing technology is a genetic engineering technology based on artificial nucleases to modify the genome in a targeted manner, and it plays an increasingly powerful role in agricultural and medical research. Clustered regularly interspaced short palindromic repeats / CRISPR associated systems (CRISPR) are currently the most widely used genome editing tools. Under the guidance of artificially designed guide RNA, Cas proteins can target any position in the genome. The base editing system is a new gene editing technology developed based on the CRISPR system. It is divided into cytosine base editing system and adenine base editing system. Cytosine deaminase and adenine deaminase are fused with Cas9 single-stranded nickase, respectively. Under the targeting action of guide RNA, Cas9 single-stranded nickase produces a single-stranded DNA region, so the deaminase can efficiently deaminate the C and A nucleotides on the single-stranded DNA at the target position, turning them into U bases and I bases, which are then repaired into T bases and G bases during the cell's own repair process.

[0004] The cytosine base editing system has been found to produce unpredictable off-target phenomena on a genome-wide scale, which is likely due to the overexpression of cytosine deaminase in the genome, resulting in random deamination in highly transcriptionally active regions of the genome. In addition, if there are multiple Cs within the working window of the target site, the existing efficient base editing system often obtains products in which multiple Cs are changed simultaneously, and cannot obtain products with only a single C mutation. The specificity on a genome-wide scale and the accuracy at the target site greatly affect the use of the cytosine base editing system.

[0005] Brief description of the invention

[0006] The specificity and accuracy of the cytosine base editing system may be related to the binding ability of cytosine deaminase to single-stranded DNA. By changing or weakening the binding ability of deaminase to single-stranded DNA without reducing the deamination ability of deaminase, it is possible to obtain a cytosine single-base editing system that is both efficient and has both specificity and accuracy. The inventors optimized Loop1 and Loop7 in the domain of human hA3Bctd (APOBEC3B C-terminal domain) that binds to single-stranded DNA, tested the obtained variants through rice protoplast transformation, detected the efficiency and accuracy of the obtained variants, and tested the specificity of the obtained variants, thereby obtaining a series of efficient, highly specific, and highly accurate base editing systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 . Shows the A3Bctd mutation site selection.

[0009] Figure 2 . Shows the on-target efficiency and off-target efficiency of the base editing system to be tested.

[0010] Figure 3 .Shows the average on-target efficiency and the average off-target efficiency of the base editing systems tested.

[0011] Figure 4 . Combinations of double and triple mutants are shown.

[0012] Figure 5 . Shows the protoplast transformation to verify the targeting and off-target efficiency of double and triple mutants.

[0013] Figure 6 .Shows the average targeting efficiency and average off-target efficiency of the double and triple mutants tested.

[0014] Figure 7 .Shows the working efficiency of different base editing systems at different Cs at four target sites.

[0015] Figure 8 .Shows the average mutation types of editing products of different base editing systems at four targeted sites. DETAILED DESCRIPTION OF THE INVENTION

[0017] I. Definition

[0018] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. For example, the standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following documents: Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook"). At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0019] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and each combination should be considered to have been listed separately 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."

[0020] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of the polypeptide may be retained in certain practical situations (for example, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, a sequence containing the methionine is also covered at this time, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0021] As used herein, the term "CRISPR effector protein" generally refers to nucleases present in naturally occurring CRISPR systems, as well as modified forms thereof, variants thereof, catalytically active fragments thereof, etc. The term encompasses any effector protein based on the CRISPR system that is capable of achieving gene targeting (e.g., gene editing, gene targeting regulation, etc.) in cells.

[0022] Examples of "CRISPR effector proteins" include Cas9 nucleases or variants thereof. The Cas9 nuclease may be a Cas9 nuclease from a different species, such as spCas9 from Streptococcus pyogenes (S. pyogenes) or SaCas9 derived from Staphylococcus aureus (S. aureus). "Cas9 nuclease" and "Cas9" are used interchangeably herein to refer to an RNA-guided nuclease comprising a Cas9 protein or a fragment thereof (e.g., a protein comprising an active DNA cleavage domain of Cas9 and / or a gRNA binding domain of Cas9). Cas9 is a component of the CRISPR / Cas (clustered regularly interspaced short palindromic repeats and related systems) genome editing system that can target and cut a DNA target sequence under the guidance of a guide RNA to form a DNA double-strand break (DSB).

[0023] Examples of "CRISPR effector proteins" may also include Cpf1 nucleases or variants thereof, such as highly specific variants. The Cpf1 nuclease may be a Cpf1 nuclease from a different species, such as a Cpf1 nuclease from Francisella novicida U112, Acidaminococcus sp. BV3L6, and Lachnospiraceae bacterium ND2006.

[0024] "CRISPR effector protein" 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, C2c3 or C2c2 nuclease, for example, including these nucleases or functional variants thereof.

[0025] "Genome" as used herein encompasses not only the chromosomal DNA present in the cell nucleus, but also the organellar DNA present in subcellular components of the cell (eg, mitochondria, plastids).

[0026] As used herein, "organism" includes any organism suitable for genome editing, preferably a eukaryotic organism. 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, such as rice, corn, wheat, sorghum, barley, soybeans, peanuts, Arabidopsis, etc.

[0027] "Genetically modified organism" or "genetically modified cell" means an organism or cell that contains an exogenous polynucleotide or a modified gene or expression control sequence in its genome. For example, the exogenous polynucleotide can be stably integrated into the genome of the organism or cell and inherited for consecutive generations. The exogenous polynucleotide can be integrated into the genome alone or as part of a recombinant DNA construct. The modified gene or expression control sequence is a sequence in the genome of the organism or cell that contains single or multiple deoxynucleotide substitutions, deletions and additions.

[0028] "Exogenous" with respect to a sequence refers to a sequence that is from a foreign species, or, if from the same species, a sequence that has been significantly altered in composition and / or locus from its native form through deliberate human intervention.

[0029] "Polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers that optionally may contain synthetic, non-natural or altered nucleotide bases. Nucleotides are referred to by their single letter names as follows: "A" is adenosine or deoxyadenosine (RNA or DNA, respectively), "C" represents cytidine or deoxycytidine, "G" represents 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.

[0030] "Polypeptide", "peptide", and "protein" are used interchangeably in the present invention to refer to polymers 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 attachment, sulfation, gamma carboxylation, hydroxylation and ADP-ribosylation of glutamic acid residues. In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be performed without changing 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 change biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p.224).

[0031] As used herein, "expression construct" refers to a vector such as a recombinant vector suitable for expressing a nucleotide sequence of interest in an organism. "Expression" refers to the production of a functional product. For example, the expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (such as transcription to generate mRNA or functional RNA) and / or the translation of RNA into a precursor or mature protein.

[0032] The "expression construct" of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, can be a translatable RNA (such as mRNA).

[0033] An "expression construct" of the present invention may comprise regulatory sequences and a nucleotide sequence of interest from different sources, or regulatory sequences and a nucleotide sequence of interest from the same source but arranged in a manner different from that normally found in nature.

[0034] "Regulatory sequence" and "regulatory element" are used interchangeably and refer to nucleotide sequences located upstream (5' non-coding sequence), in the middle or downstream (3' non-coding sequence) of a coding sequence and affecting the transcription, RNA processing or stability or translation of the relevant coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns and polyadenylation recognition sequences.

[0035] "Promoter" refers to a nucleic acid fragment that can control the transcription of another nucleic acid fragment. In some embodiments of the present invention, a promoter is a promoter that can control the transcription of a gene in a cell, whether or not it is derived from the cell. A promoter can be a constitutive promoter or a tissue-specific promoter or a developmentally regulated promoter or an inducible promoter.

[0036] "Constitutive promoter" refers to a promoter that will generally cause a gene to be expressed in most cell types under most circumstances. "Tissue-specific promoter" and "tissue-preferred promoter" are used interchangeably and refer to a promoter that is expressed primarily, but not necessarily exclusively, in one tissue or organ, and may also be expressed in one specific cell or cell type. "Developmentally regulated promoter" refers to a promoter whose activity is determined by developmental events. "Inducible promoter" selectively expresses an operably linked DNA sequence in response to endogenous or exogenous stimuli (environmental, hormonal, chemical signals, etc.).

[0037] As used herein, the term "operably linked" refers to the connection of a regulatory element (e.g., but not limited to, a promoter sequence, a transcription termination sequence, etc.) to a nucleic acid sequence (e.g., a coding sequence or an open reading frame) such that transcription of the nucleotide sequence is controlled and regulated by the transcription regulatory element. Techniques for operably linking a regulatory element region to a nucleic acid molecule are known in the art.

[0038] "Introducing" a nucleic acid molecule (e.g., a plasmid, a linear nucleic acid fragment, RNA, etc.) or a protein into an organism refers to transforming an organism cell 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.

[0039] "Stable transformation" refers to the introduction of an exogenous nucleotide sequence into a genome, resulting in the stable inheritance of the exogenous nucleotide sequence. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any successive generations thereof.

[0040] "Transient transformation" refers to the introduction of a nucleic acid molecule or protein into a cell to perform its function without the stable inheritance of the exogenous nucleotide sequence. In transient transformation, the exogenous nucleic acid sequence is not integrated into the genome.

[0041] "Trait" refers to a physiological, morphological, biochemical, or physical characteristic of a cell or organism.

[0042] "Agronomic traits" specifically refer to measurable indicator parameters of crop plants, including but not limited to: leaf green, grain yield, growth rate, total biomass or accumulation rate, fresh weight at maturity, dry weight at maturity, fruit yield, seed yield, plant total nitrogen content, fruit nitrogen content, seed nitrogen content, plant vegetative tissue nitrogen content, plant total free amino acid content, fruit free amino acid content, seed free amino acid content, plant vegetative tissue free amino acid content, plant total protein content, fruit protein content, seed protein content, plant vegetative tissue protein content, herbicide resistance and drought resistance, nitrogen absorption, root lodging, harvest index, stem lodging, plant height, ear height, ear length, disease resistance, cold resistance, salt resistance and tillering number, etc.

[0043] 2. Improved base editing system

[0044] First, the present invention provides a base editing fusion protein, which comprises an APOBEC3B deaminase or an APOBEC3B deaminase mutant fused to a CRISPR effector protein.

[0045] In the embodiments herein, "base editing fusion protein" and "base editor" are used interchangeably. The base editing fusion protein comprising APOBEC3B deaminase or a mutant thereof of the present invention is capable of efficiently base editing the target sequence, while having significantly reduced genome-wide random off-target effects compared to other base editors. In some embodiments, the base editing fusion protein comprising APOBEC3B deaminase or a mutant thereof of the present invention has a shortened editing window for the target sequence, enabling more precise base editing.

[0046] In some embodiments, the APOBEC3B deaminase mutant is or is derived from human APOBEC3B deaminase. An exemplary wild-type human APOBEC3B deaminase comprises the amino acid sequence shown in SEQ ID NO:19.

[0047] In some embodiments, the APOBEC3B deaminase mutant is or is derived from the C-terminal domain (hA3Bctd, APOBEC3B C-terminal domain) of human APOBEC3B deaminase. An exemplary wild-type hA3Bctd comprises the amino acid sequence of SEQ ID NO:2.

[0048] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises an amino acid substitution at one or more of the following positions relative to wild-type hA3B or hA3Bctd: 210, 211, 214, 230, 240, 281, 308, 311, 313, 314, and 315, wherein the amino acid positions are identified with reference to SEQ ID NO: 19.

[0049] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises an amino acid substitution at one or more of the following positions relative to wild-type hA3B or hA3Bctd: 211, 214, 308, 311, 313, 314, and 315, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0050] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions at positions 211 and 311 relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0051] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions at positions 211 and 313 relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0052] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions at positions 211 and 314 relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0053] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions at positions 311 and 313 relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are identified with reference to SEQ ID NO: 19.

[0054] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions at positions 214 and 314 relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0055] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions at positions 314 and 315 relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0056] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions at positions 211, 311, and 314 relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0057] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions at positions 211, 214, and 313 relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0058] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions at positions 214, 314, and 315 relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0059] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises one or more amino acid substitutions selected from the group consisting of R210A, R210K3, R211K, T214C, T214G, T214S, T214V, L230K, N240A, W281H, F308K, R311K, Y313F, D314R, D314H, Y315M relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are identified with reference to SEQ ID NO: 19.

[0060] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises one or more amino acid substitutions selected from the group consisting of R211K, T214V, F308K, R311K, Y313F, D314R, D314H, and Y315M relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are identified with reference to SEQ ID NO: 19.

[0061] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions R211K and R311K relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0062] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions R211K and Y313F relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0063] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions R211K and D314R relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0064] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions R311K and Y313F relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0065] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions T214V and D314R relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0066] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions D314R and Y315M relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are identified with reference to SEQ ID NO: 19.

[0067] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions R211K, R311K, and D314R relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0068] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions R211K, T214V, and Y313F relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0069] In some embodiments, the APOBEC3B deaminase mutant is derived from human APOBEC3B deaminase (hA3B) or the C-terminal domain of human APOBEC3B deaminase (hA3Bctd) and comprises amino acid substitutions T214V, D314H, and Y315M relative to wild-type hA3B or hA3Bctd, wherein the amino acid positions are determined with reference to SEQ ID NO: 19.

[0070] In some specific embodiments, the APOBEC3B deaminase mutant comprises an amino acid sequence selected from SEQ ID NOs: 3-18, 26-31 and 32-34.

[0071] In some embodiments, the CRISPR effector protein is a "nuclease-inactivated CRISPR effector protein."

[0072] "Nuclease-inactivated CRISPR effector protein" refers to a CRISPR effector protein that lacks double-stranded nucleic acid cleavage activity, but still retains the ability to target DNA guided by gRNA. CRISPR effector proteins that lack double-stranded nucleic acid cleavage activity also encompass nickases, which form nicks in double-stranded nucleic acid molecules but do not completely cut off double-stranded nucleic acids.

[0073] In some preferred embodiments of the present invention, the nuclease-inactivated CRISPR effector protein of the present invention has nickase activity. Without being limited by any theory, it is believed that the mismatch repair of eukaryotic organisms guides the removal and repair of mismatched bases in the DNA chain through a nick on the DNA chain. The U:G mismatch formed by the action of cytidine deaminase may be repaired to C:G. By introducing a nick on a chain containing an unedited G, it will be possible to preferentially repair the U:G mismatch to the desired U:A or T:A.

[0074] In some embodiments, the nuclease-inactivated CRISPR effector protein is a nuclease-inactivated Cas9. The DNA cleavage domain of the Cas9 nuclease is known to contain two subdomains: the HNH nuclease subdomain and the RuvC subdomain. The HNH subdomain cuts the strand complementary to the gRNA, while the RuvC subdomain cuts the non-complementary strand. Mutations in these subdomains can inactivate the nuclease activity of Cas9, forming a "nuclease-inactivated Cas9". The nuclease-inactivated Cas9 still retains the DNA binding ability guided by the gRNA. Therefore, in principle, when fused to another protein, the nuclease-inactivated Cas9 can simply target the other protein to almost any DNA sequence by co-expressing it with a suitable guide RNA.

[0075] The nuclease-inactivated Cas9 of the present invention can be derived from Cas9 of different species, for example, derived from Streptococcus pyogenes (S. pyogenes) Cas9 (SpCas9), or derived from Staphylococcus aureus (S. aureus) Cas9 (SaCas9). Mutating the HNH nuclease subdomain and the RuvC subdomain of Cas9 (for example, comprising mutations D10A and H840A) simultaneously inactivates the nuclease of Cas9 and becomes nuclease-dead Cas9 (dCas9). Mutation-inactivation of one of the subdomains can make Cas9 have nickase activity, i.e., obtain Cas9 nickase (nCas9), for example, nCas9 having only mutation D10A.

[0076] Therefore, in some embodiments of the present invention, the nuclease-inactivated Cas9 of the present invention comprises amino acid substitutions D10A and / or H840A relative to wild-type Cas9.

[0077] In some embodiments of the present invention, the nuclease-inactivated Cas9 may further comprise additional mutations. For example, the nuclease-inactivated SpCas9 may further comprise EQR, VQR or VRER mutations and the SaCas9 may further comprise KKH mutations (Kim et al. Nat. Biotechnol. 35, 371-376.).

[0078] In some specific embodiments of the present invention, the nuclease-inactivated SpCas9 comprises the amino acid sequence shown in SEQ ID NO:35.

[0079] In some embodiments, the nuclease-inactivated CRISPR effector protein is a nuclease-inactivated Cpf1. Cpf1 contains a DNA cleavage domain (RuvC), which can be mutated to inactivate the DNA cleavage activity of Cpf1, forming a "DNA cleavage activity-inactivated Cpf1". The DNA cleavage activity-inactivated Cpf1 still retains the DNA binding ability guided by gRNA. Therefore, in principle, when fused with another protein, the DNA cleavage activity-inactivated Cpf1 can simply target the other protein to almost any DNA sequence by co-expressing it with a suitable guide RNA.

[0080] The DNA cleavage activity-deficient Cpf1 of the present invention can be derived from Cpf1 of different species, for example, Cpf1 proteins derived from Francisella novicida U112, Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006, respectively named FnCpf1, AsCpf1 and LbCpf1.

[0081] In some embodiments, the Cpf1 lacking DNA cleavage activity is FnCpf1 lacking DNA cleavage activity. In some specific embodiments, the FnCpf1 lacking DNA cleavage activity comprises a D917A mutation relative to wild-type FnCpf1.

[0082] In some embodiments, the Cpf1 lacking DNA cleavage activity is AsCpf1 lacking DNA cleavage activity. In some specific embodiments, the AsCpf1 lacking DNA cleavage activity comprises a D908A mutation relative to wild-type AsCpf1.

[0083] In some embodiments, the Cpf1 lacking DNA cleavage activity is LbCpf1 lacking DNA cleavage activity. In some specific embodiments, the LbCpf1 lacking DNA cleavage activity comprises a D832A mutation relative to wild-type LbCpf1.

[0084] In some embodiments of the invention, the APOBEC3B deaminase or APOBEC3B deaminase mutant is fused to the N-terminus of the CRISPR effector protein (e.g., a nuclease-inactive CRISPR effector protein, such as Cas9 or Cpf1).

[0085] In some embodiments of the invention, the APOBEC3B deaminase or APOBEC3B deaminase mutant and the CRISPR effector protein (e.g., a nuclease-inactivated CRISPR effector protein, such as Cas9 or Cpf1) are fused via a linker. The linker can be a non-functional amino acid sequence of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 20-25, 25-50) or more amino acids in length and without secondary or higher structures. For example, the linker can be a flexible linker. Preferably, the linker is 16 or 32 amino acids in length. In some specific embodiments, the linker is an XTEN linker as shown in SEQ ID NO: 36 or 37.

[0086] In cells, uracil DNA glycosylase catalyzes the removal of U from DNA and initiates base excision repair (BER), resulting in the repair of U:G to C:G. Therefore, without being limited by any theory, including a uracil DNA glycosylase inhibitor in the base editing fusion protein of the present invention will be able to increase the efficiency of base editing.

[0087] Therefore, in some embodiments of the present invention, the base editing fusion protein further comprises a uracil DNA glycosylase inhibitor (UGI). In some specific embodiments, the uracil DNA glycosylase inhibitor comprises the amino acid sequence shown in SEQ ID NO:38.

[0088] In some embodiments of the present invention, the base editing fusion protein of the present invention further comprises a nuclear localization sequence (NLS). In general, one or more NLSs in the base editing fusion protein should have sufficient strength to drive the base editing fusion protein to accumulate in the nucleus of the cell in an amount that can achieve its base editing function. In general, the intensity of nuclear localization activity is determined by the number, position, one or more specific NLSs used, or a combination of these factors in the base editing fusion protein.

[0089] In some embodiments of the present invention, the NLS of the base editing fusion protein of the present invention may be located at the N-terminus and / or the C-terminus. In some embodiments of the present invention, the NLS of the base editing fusion protein of the present invention may be located between the APOBEC3B deaminase or APOBEC3B deaminase mutant and the CRISPR effector protein. In some embodiments, the base editing fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs. In some embodiments, the base editing fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the N-terminus. In some embodiments, the base editing fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the C-terminus. In some embodiments, the base editing fusion protein comprises a combination of these, such as one or more NLSs at the N-terminus and one or more NLSs at the C-terminus. When there is more than one NLS, each can be selected to be independent of the other NLSs. In some preferred embodiments of the present invention, the base editing fusion protein comprises at least 2 NLS, for example, the at least 2 NLS are located at the C-terminus. In some embodiments, the NLS is located at the C-terminus of the base editing fusion protein. In some embodiments, the base editing fusion protein comprises at least 3 NLS.

[0090] In general, NLS consists of one or more short sequences of positively charged lysine or arginine amino acids exposed on the surface of the protein, but other types of NLS are also known. Non-limiting examples of NLS include: PKKKRKV or KRPAATKKAGQAKKKK.

[0091] In some embodiments of the invention, the N-terminus of the base editing fusion protein comprises an NLS of the amino acid sequence shown in PKKKRKV. In some embodiments of the invention, the C-terminus of the base editing fusion protein comprises an NLS of the amino acid sequence shown in KRPAATKKAGQAKKKK. In some embodiments of the invention, the C-terminus of the base editing fusion protein comprises an NLS of the amino acid sequence shown in PKKKRKV.

[0092] In addition, depending on the DNA location to be edited, the base editing fusion protein of the present invention may also include other localization sequences, such as cytoplasmic localization sequences, chloroplast localization sequences, mitochondrial localization sequences, etc.

[0093] On the other hand, the present invention also provides the use of the base editing fusion protein of the present invention in base editing a target sequence in a cell genome.

[0094] In another aspect, the present invention also provides a system for base editing a target sequence in a cell genome, comprising at least one of the following i) to v):

[0095] i) the base editing fusion protein of the present invention, and a guide RNA;

[0096] ii) an expression construct comprising a nucleotide sequence encoding the base editing fusion protein of the present invention, and a guide RNA;

[0097] iii) the base editing fusion protein of the present invention, and an expression construct comprising a nucleotide sequence encoding a guide RNA;

[0098] iv) an expression construct comprising a nucleotide sequence encoding the base editing fusion protein of the present invention, and an expression construct comprising a nucleotide sequence encoding a guide RNA;

[0099] v) an expression construct comprising a nucleotide sequence encoding the base editing fusion protein of the present invention and a nucleotide sequence encoding a guide RNA;

[0100] The guide RNA is capable of targeting the base editing fusion protein to a target sequence in the cell genome.

[0101] As used herein, a "base editing system" refers to a combination of components required for base editing a genome in a cell or organism. The individual components of the system, such as a base editing fusion protein, one or more guide RNAs, may exist independently of each other, or may exist in any combination as a composition.

[0102] As used herein, "guide RNA" and "gRNA" are used interchangeably and refer to an RNA molecule that can form a complex with a CRISPR effector protein and can target the complex to a target sequence due to a certain homology with the target sequence. The guide RNA targets the target sequence by base pairing with the complementary strand of the target sequence. For example, the gRNA used by the Cas9 nuclease or its functional variant is generally composed of crRNA and tracrRNA molecules that are partially complementary to form a complex, wherein the crRNA contains a guide sequence (also called a seed sequence) that has sufficient homology to the target sequence so as to hybridize with the complementary strand of the target sequence and guide the CRISPR complex (Cas9+crRNA+tracrRNA) to specifically bind to the target sequence sequence. However, it is known in the art that a single guide RNA (sgRNA) can be designed, which contains the features of both crRNA and tracrRNA. The gRNA used by the Cpf1 nuclease or its functional variant is generally composed of only mature crRNA molecules, which may also be referred to as sgRNA. It is within the capabilities of those skilled in the art to design a suitable gRNA based on the CRISPR effector protein used and the target sequence to be edited.

[0103] In some embodiments, the base editing system of the present invention comprises more than one guide RNA, so that more than one target sequence can be base edited simultaneously.

[0104] In order to obtain effective expression in cells, in some embodiments of the present invention, the nucleotide sequence encoding the base editing fusion protein is codon-optimized for the organism from which the cells to be base edited come.

[0105] Codon optimization refers to a method of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with codons that are more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. Different species exhibit specific preferences for certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) is often correlated with the efficiency of translation of messenger RNA (mRNA), which in turn is believed to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the codons that are most frequently used for peptide synthesis. Therefore, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the Codon Usage Database available at www.kazusa.orjp / codon / , and these tables can be adapted for use in different ways. See, Nakamura et al., 2001. Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

[0106] In some embodiments of the present invention, the guide RNA is a single guide RNA (sgRNA). Methods for constructing suitable sgRNAs according to a given target sequence are known in the art. For example, see the literature: Wang, Y. et al. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat. Biotechnol. 32, 947-951 (2014); Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31, 686-688 (2013); Liang, Z. et al. Targeted mutagenesis in Zea mays using TALENs and the CRISPR / Cas system. J Genet Genomics. 41, 63–68 (2014).

[0107] In some embodiments of the present invention, the nucleotide sequence encoding the base editing fusion protein and / or the nucleotide sequence encoding the guide RNA is operably linked to an expression regulatory element such as a promoter.

[0108] Examples of promoters that can be used in the present invention include, but are not limited to, polymerase (pol) I, pol II or pol III promoters. Examples of pol I promoters include chicken RNA pol I promoters. Examples of pol II promoters include, but are not limited to, cytomegalovirus immediate early (CMV) promoters, Rous sarcoma virus long terminal repeat (RSV-LTR) promoters, and simian virus 40 (SV40) immediate early promoters. Examples of pol III promoters include U6 and H1 promoters. Inducible promoters such as metallothionein promoters can be used. Other examples of promoters include T7 phage promoters, T3 phage promoters, β-galactosidase promoters, and Sp6 phage promoters. When used for plants, the promoter can be a cauliflower mosaic virus 35S promoter, a corn Ubi-1 promoter, a wheat U6 promoter, a rice U3 promoter, a corn U3 promoter, a rice actin promoter.

[0109] Organisms that can be genomic 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 plants are crop plants, including but not limited to wheat, rice, corn, soybeans, sunflowers, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomatoes, tobacco, cassava and potatoes. Preferably, the organism is a plant. More preferably, the organism is rice.

[0110] III. Methods for producing genetically modified organisms

[0111] In another aspect, the present invention provides a method for producing a genetically modified organism, comprising introducing a base editing fusion protein of the present invention, or an expression construct encoding a base editing fusion protein of the present invention, or a system for base editing a target sequence in a cell genome of the present invention into a cell of the organism.

[0112] By introducing the system for base editing a target sequence in a cell genome of the present invention, the guide RNA can target the base editing fusion protein to the target sequence in the cell genome of the organism, resulting in one or more C in the target sequence being replaced by T. In some preferred embodiments, the organism is a plant.

[0113] The selection and design of target sequences that can be recognized and targeted by the CRISPR effector protein and guide RNA complex are within the skills of ordinary technicians in the field.

[0114] In some embodiments of the method of the present invention, it is also included to screen for organisms such as plants with desired nucleotide substitutions. Nucleotide substitutions in organisms such as plants can be detected by T7EI, PCR / RE or sequencing methods, for example, see Shan, Q., Wang, Y., Li, J. & Gao, C. Genome editing in rice and wheat using the CRISPR / Cas system. Nat. Protoc. 9, 2395-2410 (2014).

[0115] In the present invention, the target sequence to be modified can be located anywhere in the genome, for example, within a functional gene such as a protein-coding gene, or, for example, can be located in a gene expression regulatory region such as a promoter region or an enhancer region, thereby achieving modification of the gene function or modification of gene expression.

[0116] C to T base editing in the cell target sequence can be detected by T7EI, PCR / RE or sequencing methods.

[0117] In the method of the present invention, the base editing system can be introduced into cells by various methods well known to those skilled in the art. Methods that can be used to introduce the genome editing system of the present invention into cells include, but are not limited to, calcium phosphate transfection, protoplast fusion, electroporation, liposome transfection, microinjection, viral infection (such as baculovirus, vaccinia virus, adenovirus, adeno-associated virus, lentivirus and other viruses), gene gun method, PEG-mediated protoplast transformation, and Agrobacterium-mediated transformation.

[0118] Cells that can be genome-edited by the methods of the present invention can come from, for example, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, and cats; poultry such as chickens, ducks, and geese; plants, including monocots and dicots, such as rice, corn, wheat, sorghum, barley, soybeans, peanuts, Arabidopsis, and the like.

[0119] The method of the present invention is particularly suitable for producing genetically modified plants, such as crop plants. In the method of producing genetically modified plants of the present invention, the base editing system can be introduced into plants 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, gene gun method, PEG-mediated protoplast transformation, soil Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube channel method, and ovary injection method. Preferably, the base editing system is introduced into the plant by transient transformation.

[0120] 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 stably transform the base editing system into plants. This avoids the potential off-target effects of the stably existing base editing system and also avoids the integration of exogenous nucleotide sequences into the plant genome, thereby having higher biosafety.

[0121] In some preferred embodiments, the introduction is performed in the absence of selection pressure, thereby avoiding integration of the exogenous nucleotide sequence into the plant genome.

[0122] In some embodiments, the introduction includes transforming the base editing system of the present invention into isolated plant cells or tissues, and then regenerating the transformed plant cells or tissues into complete plants. Preferably, the regeneration is carried out in the absence of selection pressure, that is, no selection agent for the selection gene carried on the expression vector is used during tissue culture. Not using a selection agent can improve the regeneration efficiency of the plant and obtain a modified plant that does not contain exogenous nucleotide sequences.

[0123] In other embodiments, the base editing system of the present invention can be transformed into specific parts of the whole plant, such as leaves, stem tips, pollen tubes, panicles or hypocotyls. This is particularly suitable for the transformation of plants that are difficult to regenerate through tissue culture.

[0124] In some embodiments of the present invention, the in vitro expressed protein and / or in vitro transcribed RNA molecule is directly transformed into the plant. The protein and / or RNA molecule can achieve base editing in plant cells and then be degraded by the cells, thereby avoiding the integration of exogenous nucleotide sequences in the plant genome.

[0125] Therefore, in some embodiments, plants without exogenous DNA integration can be obtained by genetically modifying and breeding plants using the methods of the present invention, i.e., non-transgenic (transgene-free) modified plants. In addition, the base editing system of the present invention has high specificity (low off-target rate) when performing base editing in plants, which also improves biosafety.

[0126] Plants that can be base edited by the method of the present invention include monocots and dicots. For example, the plant can be a crop plant, such as wheat, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava or potato.

[0127] In some embodiments of the present invention, the target sequence is related to plant traits such as agronomic traits, whereby the base editing causes the plant to have altered traits relative to wild-type plants. In the present invention, the target sequence to be modified can be located at any position in the genome, for example, in a functional gene such as a protein-coding gene, or, for example, can be located in a gene expression regulatory region such as a promoter region or an enhancer region, thereby achieving modification of the gene function or modification of gene expression. Accordingly, in some embodiments of the present invention, the C to T substitution results in an amino acid substitution in the target protein. In other embodiments of the present invention, the C to T substitution results in changes in the expression of the target gene.

[0128] In some embodiments of the present invention, the method further comprises obtaining progeny of the genetically modified plant.

[0129] In another aspect, the present invention also provides a genetically modified plant or its progeny or part thereof, wherein the plant is obtained by the above method of the present invention. In some embodiments, the genetically modified plant or its progeny or part thereof is non-transgenic.

[0130] In another aspect, the present invention also provides a plant breeding method, comprising hybridizing a first genetically modified plant obtained by the above method of the present invention with a second plant not containing the genetic modification, thereby introducing the genetic modification into the second plant. Example

[0131] In order to facilitate the 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 present invention are provided in the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0132] Example 1: Selection of A3Bctd mutation sites based on protein structure information

[0133] Based on the published structural information of hA3Bctd (PDB: 2NBQ) and the published structural information of full-length hAPOBEC3B (PDB: 5CQD, 5CQH, 5TD5), amino acid point mutations were performed mainly on the key loop regions Loop1 and Loop7 that are closely related to the binding of hA3Bctd to single-stranded DNA, in order to reduce the binding ability to single-stranded DNA. The specific amino acid point mutation positions and types are as follows: Figure 1 shown.

[0134] The candidate single-base editing system was optimized on the A3A-BE3 vector backbone (SEQ ID NO: 1, a base editor containing human APOBEC3A). The artificially synthesized A3Bctd DNA fragment (SEQ ID NO: 2) was used to replace the APOBEC3A sequence in the A3A-BE3 vector using the Gbison method to obtain the A3Bctd-BE3 vector. In the A3Bctd-BE3 vector, the encoded amino acids on A3Bctd were point mutated using fusion PCR and the Gbison method to obtain A3Bctd-R210A-BE3, A3Bctd-R210K-BE3, A3Bctd-R211K-BE3, A3Bctd-T214C-BE3, A3Bctd-T214G-BE3, A3Bctd-T214S-BE3, A3Bctd-T214V-BE3, A3Bctd- Single-base editing vectors with point mutations of td-L230K-BE3, A3Bctd-N240A-BE3, A3Bctd-W281H-BE3, A3Bctd-F308K-BE3, A3Bctd-R311K-BE3, A3Bctd-Y313F-BE3, A3Bctd-D314R-BE3, A3Bctd-D314H-BE3, and A3Bctd-Y315M-BE3 (the deaminase amino acid sequences after point mutations are SEQ ID NOs: 3-18, respectively).

[0135] In addition, the constructed control plasmids include A3A-BE3, YEE-BE3, RK-BE3, eA3A-BE3, A3A-R128A-BE3, A3A-Y130F-BE3, and untruncated APOBEC3B-BE3 (wherein the deaminase sequence is shown in SEQ ID NO: 19-25), wherein YEE and RK are two variants of APOBEC1 deaminase on the BE3 vector, constructed by fusion PCR and Gbison. The A3A deaminase sequence was artificially synthesized, and R128A and Y130F of A3A were constructed by fusion PCR and Gbison.

[0136] Example 2: Verification of editing efficiency and specificity of the A3Bctd-BE3 system carrying a single point mutation in protoplasts

[0137] 2.1. Vector construction

[0138] The guide RNA vectors used in this experiment include pSp-sgRNA and pSa-sgRNA vectors, which are used to construct the eight targets shown in Table 1 below. The target with -T1 is constructed into the pSp-sgRNA vector by enzyme ligation method as a guide RNA vector for detecting targeting efficiency, and the target ending with -SaT1 or -SaT2 is constructed into the pSa-sgRNA vector by enzyme ligation method as a vector for detecting random off-target ability by the TA-AS method.

[0139] The principle of the TA-AS method is to co-transfect with the base editing system to be tested (such as the base editing system based on nSpCas9 in this experiment) other CRISPR systems that are orthogonal to it (i.e., cannot share gRNA) and can produce single-stranded regions, such as the nSaCas9 system, so that the orthogonal other CRISPR system produces a long-term stable single-stranded region at a selected site in the genome. If the base editing system to be tested has a genome-wide random off-target effect, it will deaminize the C base in the single-stranded region and cause undesired editing. The random off-target effect of the single-base editing system can be efficiently and easily detected by high-throughput sequencing of the amplicon of the selected site.

[0140] Table 1.

[0141]

[0142] 2.2. Protoplast transformation

[0143] Conventional BE3, A3A-BE3, YEE-BE3, RK-BE3, eA3A-BE3, A3A-R128A-BE3, A3A-Y130F, untruncated APOBEC3B-BE3 and A3Bctd-BE3 systems were used as controls. Each single-base editing system was co-transformed into rice protoplasts with its own guide RNA vector pSp-sgRNA and pnSaCsa9 and the corresponding pSa-sgRNA in the TA-AS system. After two days of culture, target site amplicon sequencing was performed, and the average values ​​of four targeted sites and four off-target sites were taken to evaluate the targeting efficiency and off-target efficiency. Each target site of each single-base editing system had at least three biological replicates. The results are shown in Figure 2. Figure 2 and Figure 3 As shown in the figure, eight point mutations, R211K, T214V, F308K, R311K, Y313F, D314R, D314H and Y315M, were found to reduce off-target efficiency while maintaining a high mutation efficiency, and seven of them were on Loop1 and Loop7. These seven variants were combined to further improve specificity.

[0144] Example 3: Combination of screened point mutations to further improve specificity

[0145] The seven amino acid mutation sites screened in the previous step were combined to form nine double and triple mutants ( Figure 4 ), the target site mutation efficiency and off-target efficiency of the combined variants were tested at four target sites and four off-target sites as in the above experimental process ( Figure 4 ), the test results showed that the two triple mutants, KKR and VHM, have reduced the off-target efficiency to a level equivalent to the background while maintaining high targeting efficiency ( Figure 5 , Figure 6 ). In particular, the TS-AS system detection results showed that the average off-target efficiency of the four targets detected was only 0.6%, which was 21 times lower than that of the wild-type A3Bctd ( Figure 5 , Figure 6 ).

[0146] Example 4. Analysis of editing characteristics of the new cytosine single-base editing system

[0147] The variant characteristics of all single-base editing systems in four targeted sites, including editing window, preference, and editing product type, were analyzed (the PAM sequence was regarded as positions 21-23). ​​In terms of the editing window, it can be found that the editing efficiency of A3Bctd is comparable to that of A3A-BE3, A3A-R128A, and A3A-Y130F, but its working window is narrower than that of A3A-BE3, A3A-R128A, and A3A-Y130F. Its single amino acid variants A3Bctd-Y313F, A3Bctd-211K, A3Bctd-Y315M, and A3Bctd-T214V can further narrow the working window to 2 to 3bp. Its double or triple mutants can narrow the working window to 1 to 2bp (while slightly sacrificing editing efficiency). Figure 7 ).

[0148] The gene editing products can be divided into three types of mutations according to the number of mutated Cs: single, double, and multiple. Figure 8 The average mutation types of the editing products of all single-base editing systems at the four targeted sites in this experiment are described. Sorting them according to the efficiency of single C mutations shows that although the overall efficiency of the A3A-BE3 series editing systems is relatively high, the proportion of single C mutation products produced is extremely low, and the probability of obtaining single C mutation products is very small. The variant Y313F of A3Bctd has a nearly 10% chance of obtaining an editing product with only one C mutation, and VHM, VR, and KR also show high editing accuracy. It is worth noting that the product accuracy of the VHM and KKR variants is very high, and basically only one or two C mutations will be produced.

Claims

1. A base editing fusion protein comprising an APOBEC3B deaminase mutant fused to a CRISPR effector protein, wherein the amino acid substitution type of the APOBEC3B deaminase mutant is selected from any one of the following (1) to (5): (1) R211K and D314R amino acid substitutions of SEQ ID NO:19, or R22K and D125R amino acid substitutions of SEQ ID NO:2; (2) T214V and D314R amino acid substitutions of SEQ ID NO: 19, or T25V and D125R amino acid substitutions of SEQ ID NO: 2; (3) amino acid substitutions of D314R and Y315M of SEQ ID NO:19, or amino acid substitutions of D125R and Y126M of SEQ ID NO:2; (4) R211K, R311K and D314R amino acid substitutions of SEQ ID NO:19, or R22K, R122K and D125R amino acid substitutions of SEQ ID NO:2; (5) T214V, D314H and Y315M amino acid substitutions of SEQ ID NO:19, or T25V, D125H and Y126M amino acid substitutions of SEQ ID NO:

2.

2. The base editing fusion protein of claim 1, wherein the APOBEC3B deaminase mutant comprises an amino acid sequence selected from SEQ ID NO: 28, 30, 31, 32 and 34.

3. The base editing fusion protein of any one of claims 1-2, wherein the CRISPR effector protein is a CRISPR nickase.

4. The base editing fusion protein of claim 3, wherein the CRISPR effector protein is a nuclease-inactivated Cas9 comprising amino acid substitutions D10A and / or H840A relative to wild-type Cas9, and the nuclease-inactivated Cas9 comprises the amino acid sequence shown in SEQ ID NO:

35.

5. The base editing fusion protein of any one of claims 1-4, wherein the APOBEC3B deaminase mutant is fused to the N-terminus of the CRISPR effector protein.

6. The base editing fusion protein of any one of claims 1-5, wherein the APOBEC3B deaminase mutant and the CRISPR effector protein are fused via a linker, and the linker is a linker shown in SEQ ID NO: 36 or 37.

7. The base editing fusion protein of any one of claims 1 to 6, wherein the base editing fusion protein further comprises a uracil DNA glycosylase inhibitor (UGI), wherein the uracil DNA glycosylase inhibitor comprises the amino acid sequence shown in SEQ ID NO:

38.

8. The base editing fusion protein of any one of claims 1-7, wherein the base editing fusion protein further comprises a nuclear localization sequence (NLS).

9. A system for base editing a target sequence in a cell genome, comprising at least one of the following i) to v): i) the base editing fusion protein of any one of claims 1 to 8, and a guide RNA; ii) an expression construct comprising a nucleotide sequence encoding the base editing fusion protein of any one of claims 1 to 8, and a guide RNA; iii) the base editing fusion protein of any one of claims 1 to 8, and an expression construct comprising a nucleotide sequence encoding a guide RNA; iv) an expression construct comprising a nucleotide sequence encoding the base editing fusion protein of any one of claims 1 to 8, and an expression construct comprising a nucleotide sequence encoding a guide RNA; v) an expression construct comprising a nucleotide sequence encoding the base editing fusion protein of any one of claims 1 to 8 and a nucleotide sequence encoding a guide RNA; The guide RNA is capable of targeting the base editing fusion protein to a target sequence in the cell genome.

10. The system of claim 9, comprising more than one guide RNA or expression construct thereof, so that more than one target sequence can be base edited simultaneously.

11. The system of claim 9 or 10, wherein the nucleotide sequence encoding the base editing fusion protein is codon-optimized for the organism from which the cell to be base edited is derived.

12. The system of any one of claims 9-11, wherein the guide RNA is a single guide RNA.

13. The system of any one of claims 9 to 12, wherein the nucleotide sequence encoding the base editing fusion protein and / or the nucleotide sequence encoding the guide RNA is operably linked to a promoter.

14. A method for producing a genetically modified organism, comprising introducing a base editing fusion protein according to any one of claims 1 to 8, or an expression construct comprising a nucleotide sequence encoding a base editing fusion protein according to any one of claims 1 to 8, or a system for base editing a target sequence in a cell genome according to any one of claims 9 to 13 into an organism cell, wherein the method is not a disease diagnosis or treatment method.

15. The method of claim 14, wherein the organism is a plant.

16. A preparation for producing a genetically modified organism, comprising the base editing fusion protein of any one of claims 1 to 8, or an expression construct comprising a nucleotide sequence encoding the base editing fusion protein of any one of claims 1 to 8, or the system for base editing a target sequence in a cell genome of any one of claims 9 to 13 as an active ingredient of the preparation.

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