WO2021233442A1 - 一种核酸表达的方法 - Google Patents

一种核酸表达的方法 Download PDF

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WO2021233442A1
WO2021233442A1 PCT/CN2021/095310 CN2021095310W WO2021233442A1 WO 2021233442 A1 WO2021233442 A1 WO 2021233442A1 CN 2021095310 W CN2021095310 W CN 2021095310W WO 2021233442 A1 WO2021233442 A1 WO 2021233442A1
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nucleic acid
promoter
acid construct
gene
coding sequence
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PCT/CN2021/095310
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French (fr)
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谢洪涛
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山东舜丰生物科技有限公司
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Publication of WO2021233442A1 publication Critical patent/WO2021233442A1/zh

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04001—Cytosine deaminase (3.5.4.1)
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    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04002—Adenine deaminase (3.5.4.2)

Definitions

  • the present invention relates to the field of biotechnology, in particular, to a method of nucleic acid expression.
  • the purpose of the present invention is to provide a method for improving the single-base editing efficiency in plants.
  • the first aspect of the present invention provides a nucleic acid construct having a 5'-3' (5' to 3') formula I structure:
  • P1, S1, L1, S2, and S3 are the elements used to form the construct, respectively;
  • P1 is the first promoter sequence, and the first promoter includes the promoter of elongation factor
  • S1 and S2 are each independently one or more (a) coding sequence of gene editing enzyme, (b) coding sequence of adenine deaminase and/or coding sequence of cytosine deaminase;
  • L1 is the coding sequence without or connecting peptide
  • S3 is the coding sequence of no or uracil glycosidase inhibitor UGI;
  • each "-" is independently a bond or a nucleotide linking sequence.
  • said S1 is the coding sequence of adenine deaminase and/or the coding sequence of cytosine deaminase
  • said S2 is the coding sequence of gene editing enzyme.
  • S1 is the coding sequence of adenine deaminase
  • S3 is none.
  • S1 is the coding sequence of cytosine deaminase
  • S3 is the coding sequence of the uracil glycosidase inhibitor UGI.
  • the elongation factor includes eukaryotic elongation factor or prokaryotic elongation factor.
  • the eukaryotic elongation factor includes EF1 ⁇ , EF1 ⁇ , and EF2.
  • the prokaryotic elongation factor includes EF-Tu, EF-Ts, and EF-G; preferably, includes EF1 ⁇ ; preferably, includes EF1 ⁇ in plants.
  • the plant is selected from the group consisting of corn, rice, soybean, Arabidopsis, tobacco, tomato, or a combination thereof.
  • the first promoter is derived from one or more plants selected from the group consisting of corn, rice, soybean, Arabidopsis, tobacco, and tomato.
  • the first promoter is tomato EF1a promoter.
  • sequence of the first promoter is shown in SEQ ID NO.:1.
  • the length of the L1 nucleotide sequence is independently 3-120 nt, preferably 3-96 nt, and preferably a multiple of 3.
  • the length of the amino acid sequence encoded by L1 is independently 3-40aa, preferably 6-32aa, preferably 18-32aa, and preferably 24-32aa.
  • the length of the nucleotide linking sequence is 1-300 nt, preferably 1-100 nt.
  • nucleotide linking sequence does not affect the normal transcription and translation of each element.
  • the gene editing enzyme is an enzyme selected from the group of editing tools: CRISPR enzyme, TALEN enzyme, ZFN enzyme, or a combination thereof.
  • the gene editing enzyme is derived from microorganisms; preferably from bacteria.
  • the source of the gene editing enzyme is selected from the group consisting of Streptococcus pyogenes, Staphylococcus aureus, Streptococcus canis, or a combination thereof.
  • the gene editing enzyme has double-stranded or single-stranded DNA cleavage activity, or no cleavage activity.
  • the gene editing enzyme is a CRISPR enzyme with single-stranded DNA cutting activity.
  • the gene editing enzymes include wild-type or mutant gene editing enzymes.
  • the identity of the gene editing enzyme and the mutant gene editing enzyme is ⁇ 80%, preferably ⁇ 90%; more preferably ⁇ 95%, more preferably, ⁇ 98% Or 99%.
  • the mutant gene editing enzyme is passed through one or more of the wild-type gene editing enzyme, preferably 1-15, preferably 1-10, preferably 1 -7, more preferably 2-5, amino acid substitutions, deletions; and/or 1-5, preferably 1-4, more preferably 1-3, most preferably 1-2 amino acids Added to form.
  • the gene editing enzyme is selected from the group consisting of Cas9, Cas12, Cas13, Cms1, MAD7, or a combination thereof.
  • the gene editing enzyme is selected from the group consisting of nCas9, dCas9, nCas9NG, nCas9X, nCas12, nCas13, or a combination thereof.
  • amino acid sequence of the gene editing enzyme is shown in SEQ ID NO.: 2.
  • the coding sequence of the gene editing enzyme is selected from the following group:
  • the coding sequence of the gene editing enzyme is shown in SEQ ID NO.:3.
  • the adenine deaminase includes wild type and mutant type.
  • the adenine deaminase includes wild-type and/or mutant TadA.
  • the adenine deaminase includes TadA.
  • the mutant type of adenine deaminase includes TadA7-10.
  • the adenine deaminase is a fusion protein formed by TadA and TadA7-10.
  • the coding sequence of the adenine deaminase is selected from the following group:
  • the homology between the nucleotide sequence and the sequence shown in SEQ ID NO.: 5 or 19 is ⁇ 75% (preferably ⁇ 85%, more preferably ⁇ 90% or ⁇ 95% or ⁇ 98% or ⁇ 99%) polynucleotides;
  • the coding sequence of the adenine deaminase is shown in SEQ ID NO. 5 or 19.
  • amino acid sequence of the adenine deaminase is shown in SEQ ID NO.:4.
  • the cytosine deaminase includes wild type and mutant type.
  • the cytosine deaminase includes APOBEC.
  • the APOBEC is selected from the following group: APOBEC1 (A1), APOBEC2 (A2), APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3H, APOBEC4 (A4), activation-induced deaminase ( activation induced cytidinedeaminase, AID), or a combination thereof.
  • the mutants of the cytosine deaminase include CBE2.0, CBE2.1, CBE2.2, CBE2.3, and CBE2.4.
  • amino acid sequence of the cytosine deaminase is shown in any one of SEQ ID NO.: 6, 8-11.
  • nucleic acid construct can also be operably linked to one or more positioning signal sequences.
  • the localization signal is selected from the group consisting of nuclear localization signal, chloroplast localization signal, mitochondrial localization signal, or a combination thereof.
  • the location signal includes a nuclear location signal, preferably, it includes 1-2 nuclear location signals.
  • the nuclear localization signal includes bpNLS and SV40.
  • nucleotide sequence of the nuclear localization signal is shown in any one of SEQ ID NO.: 12-14.
  • amino acid sequence of the nuclear localization signal is shown in SEQ ID NO.: 15.
  • nucleotide sequence of the S3 element is shown in SEQ ID NO.: 16.
  • nucleic acid construct is further operably connected to one or more second nucleic acid constructs represented by formula II:
  • P2 is the second promoter sequence
  • Y1 is the coding sequence of gRNA
  • each "-" is independently a bond or a nucleotide linking sequence.
  • gRNA sequences when at least two nucleic acid constructs of formula II are contained, their gRNA sequences may be different from each other.
  • nucleic acid construct of formula II is located at the 5'end or 3'end of the nucleic acid construct of formula I or distributed on both ends thereof.
  • the gRNA includes crRNA, tracrRNA, and sgRNA.
  • the second promoter is derived from one or more plants selected from the group consisting of rice, corn, soybean, Arabidopsis, tobacco or tomato.
  • the second promoter includes an RNA polymerase III-dependent promoter.
  • the second promoter is an RNA polymerase III-dependent promoter.
  • the second promoter is selected from the group consisting of U6, U3, U6a, U6b, U6c, U6-1, U3b, U3d, U6-26, U6-29, H1, or a combination thereof.
  • the second promoter includes U6 promoter.
  • nucleotide elements of the present invention are connected in-frame to express a fusion protein with correct amino acid sequence.
  • nucleic acid construct of Formula I and the nucleic acid construct of Formula II each independently have a terminator.
  • nucleic acid construct of Formula I and the nucleic acid construct of Formula II share the same terminator.
  • the terminator includes a terminator suitable for plant gene editing.
  • the terminator is selected from the group consisting of NOS, Poly A, T-UBQ, rbcS, or a combination thereof.
  • the construct has a structure of formula IIIa or formula IIIb:
  • nucleic acid construct can also be operably connected to the first integration element (I1) and the second integration element (I2).
  • the first integration element includes a 5'homology arm sequence.
  • the second integration element includes a 3'homology arm sequence.
  • one or more additional expression cassettes are additionally inserted between the I1 and I2 elements.
  • the additional expression cassette is independent of the expression cassette containing the nucleic acid construct of Formula I and the expression cassette containing the nucleic acid construct of Formula II.
  • the additional expression cassette expresses a substance selected from the group consisting of marker genes.
  • the marker gene includes a resistance gene (such as a hygromycin resistance gene, a herbicide resistance gene), a fluorescent gene, or a combination thereof.
  • the second aspect of the present invention provides a vector containing the nucleic acid construct according to the first aspect of the present invention.
  • the vector is a plant expression vector.
  • the vector is an expression vector that can be transfected or transformed into plant cells.
  • the carrier is an Agrobacterium Ti carrier.
  • the construct is integrated into the T-DNA region of the vector.
  • the carrier is cyclic or linear.
  • the third aspect of the present invention provides a host cell containing the nucleic acid construct according to the first aspect of the present invention, or its genome integrates one or more nucleic acid constructs according to the first aspect of the present invention.
  • the cell is a plant cell.
  • the plant is selected from the group consisting of monocots, dicots, gymnosperms, or combinations thereof.
  • the plant is selected from the group consisting of gramineous plants, legumes, cruciferous plants, Solanaceae, Umbelliferae, or a combination thereof.
  • the plant is selected from the group consisting of Arabidopsis, wheat, barley, oats, corn, rice, sorghum, millet, soybean, peanut, tobacco, tomato, cabbage, rape, spinach, lettuce, Cucumber, chrysanthemum, water spinach, celery, lettuce, or a combination thereof.
  • the host cell is used to introduce the nucleic acid construct of claim 1 into the cell by a method selected from the group consisting of: Agrobacterium transformation method, gene gun method, microinjection method, electric shock method, Ultrasonic method and polyethylene glycol (PEG) mediated method.
  • a method selected from the group consisting of: Agrobacterium transformation method, gene gun method, microinjection method, electric shock method, Ultrasonic method and polyethylene glycol (PEG) mediated method.
  • the fourth aspect of the present invention provides a reagent combination, including:
  • P1 is the first promoter sequence, and the first promoter includes the promoter of elongation factor
  • S1 and S2 are each independently one or more (a) coding sequence of gene editing enzyme, (b) coding sequence of adenine deaminase and/or coding sequence of cytosine deaminase;
  • L1 is the coding sequence without or connecting peptide
  • S3 is the coding sequence of no or uracil glycosidase inhibitor UGI;
  • P2 is the second promoter
  • Y1 is the coding sequence of gRNA
  • the first carrier and the second carrier are different carriers.
  • first nucleic acid construct and the second nucleic acid construct are located on different vectors.
  • the first carrier and the second carrier are the same carrier.
  • first nucleic acid construct and the second nucleic acid construct are located on the same vector.
  • the fifth aspect of the present invention provides a kit containing the reagent combination according to the fourth aspect of the present invention.
  • the kit further contains a label or instructions.
  • the sixth aspect of the present invention provides a method for gene editing of plants, including the steps:
  • nucleic acid construct according to the first aspect of the present invention, the vector according to the second aspect of the present invention, or the reagent combination according to the fourth aspect of the present invention is introduced into the plant cell of the plant to be edited, so that the Gene editing in plant cells.
  • the introduction is by Agrobacterium.
  • the introduction is by gene gun.
  • the gene editing is site-directed base substitution (or mutation).
  • the site-directed substitution includes mutating A to G.
  • the site-directed substitution includes mutating C to T.
  • the plants include any higher plant types that can be transformed, including monocotyledonous plants, dicotyledonous plants and gymnosperms.
  • the plant is a dicotyledonous plant.
  • the plant is selected from the group consisting of gramineous plants, legumes, cruciferous plants, Solanaceae, Umbelliferae, or a combination thereof.
  • the plant is selected from the group consisting of Arabidopsis, wheat, barley, oats, corn, rice, sorghum, millet, soybean, peanut, tobacco, tomato, cabbage, rape, spinach, lettuce, Cucumber, chrysanthemum, water spinach, celery, lettuce, or a combination thereof.
  • the seventh aspect of the present invention provides a method for preparing gene-edited plant cells, including the steps:
  • nucleic acid construct according to the first aspect of the present invention, the vector according to the second aspect of the present invention, or the reagent combination according to the fourth aspect of the present invention are introduced (or transfected) into a plant cell so that the chromosomes in the plant cell Site-specific replacement (or mutation) occurs, thereby producing the gene-edited plant cell.
  • the transfection adopts the Agrobacterium transformation method or the gene gun bombardment method.
  • the eighth aspect of the present invention provides a nucleic acid construct according to the first aspect of the present invention, a vector according to the second aspect of the present invention, a host cell according to the third aspect of the present invention, and a nucleic acid construct according to the fourth aspect of the present invention.
  • the reagent combination and the use of the kit according to the fifth aspect of the present invention are used for gene editing of plants; or, used to improve the efficiency of base editing, preferably, to improve the efficiency of single base editing in plants.
  • the ninth aspect of the present invention provides a method for preparing a gene-edited plant, including the steps:
  • the gene-edited plant cell prepared by the method of the seventh aspect of the present invention is regenerated into a plant body, thereby obtaining the gene-edited plant.
  • the tenth aspect of the present invention provides a gene-edited plant prepared by the method described in the ninth aspect of the present invention.
  • the eleventh aspect of the present invention provides a method for improving the efficiency of base editing in plants, the method comprising using the nucleic acid construct according to the first aspect of the present invention, the vector according to the second aspect of the present invention, and the The host cell according to the third aspect, the reagent combination according to the fourth aspect of the present invention, or the kit according to the fifth aspect of the present invention is a step of performing base editing on plants.
  • Figure 1 shows the structure of the ABE single-base editor containing slEF1a.
  • Figure 2 shows the efficiency of different promoters in single-base editing in tomato.
  • FIG. 3 shows the single-base editing efficiency in soybeans using different promoters and different base editors.
  • an efficient EF promoter (such as tomato EF promoter), which was constructed in the single-base editing system of ABE and CBE to drive ( a) Expression of a fusion protein composed of a gene editing enzyme and (b) adenine deaminase and/or cytosine deaminase.
  • the promoter significantly improves the editing efficiency in plants.
  • the inventor completed the present invention.
  • homologous arm refers to the flanking sequences that are identical to the genomic sequence on both sides of the foreign sequence to be inserted on the targeting vector, and are used to identify and recombine regions.
  • plant promoter refers to a nucleic acid sequence capable of initiating transcription of a nucleic acid in a plant cell.
  • the plant promoter can be derived from plants, microorganisms (such as bacteria, viruses), animals, etc., or synthetic or engineered promoters.
  • the term "gene editing” or “base mutation” or “base editing” refers to a substitution, insertion and/or deletion of a base at a certain position in a nucleotide sequence. ).
  • the "edit” or “mutation” in the present invention is preferably a single-base mutation.
  • base substitution refers to the mutation of a base at a certain position in the nucleotide sequence to another different base, such as the mutation of A to G.
  • A.T to G.C refers to the mutation or replacement of an A-T base pair at a certain position with a G-C base pair in a double-stranded nucleic acid sequence (especially a genomic sequence).
  • C.G to T.A refers to the mutation or replacement of a C-G base pair at a certain position with a T-A base pair in a double-stranded nucleic acid sequence (especially a genomic sequence).
  • the term "gene editing enzyme” refers to CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), TALEN (transcription activator-like effector nuclease technology Tanscription Activator-like (TAL) effector nucleases), ZFN (zinc finger nuclease) and other editing tools nucleases.
  • the gene editing enzyme is a CRISPR enzyme, also known as Cas protein, and its types include but are not limited to: Cas9 protein, Cas12 protein, Cas13 protein, Cas14 protein, Csm1 protein, and FDK1 protein.
  • the Cas protein refers to a family of proteins, which can have different structures according to different sources, such as SpCas9 derived from Streptococcus pyogenes, and SaCas9 derived from Staphylococcus (Staphylococcus aureus); it can also be based on structure Features (such as structural domains) are classified in the lower position.
  • the Cas12 family includes Cas12a (also known as Cpf1), Cas12b, Cas12c, and Cas12i.
  • the Cas protein may have double-stranded or single-stranded or no cleavage activity.
  • the Cas protein of the present invention may be wild-type or its mutant.
  • the mutation type of the mutant includes amino acid substitution, substitution or deletion.
  • the mutant may or may not change the enzyme cleavage activity of the Cas protein.
  • the Cas protein of the present invention has only single-strand cleavage activity or no cleavage activity, and it is a mutant of the wild-type Cas protein.
  • the Cas protein of the present invention is Cas9, Cas12, Cas13 or Cas14 with single-strand cleavage activity.
  • the Cas9 protein of the present invention includes SpCas9n (D10A), nSpCas9NG, SaCas9n, ScCas9n, XCas9n, where "n” represents nick, that is, a Cas protein with only single-strand cleavage activity.
  • the term "coding sequence of Cas protein” refers to a nucleotide sequence encoding Cas protein.
  • the skilled person will realize that because of the codon degeneracy, a large number of polynucleotide sequences can encode the same polypeptide .
  • technicians will also realize that different species have certain preferences for codons, and may optimize the codons of Cas protein according to the needs of expression in different species. These variants are all referred to by the term "Cas protein.
  • Encoding sequence specifically covers.
  • the term specifically includes a full-length sequence that is substantially the same as the Cas gene sequence, and a sequence that encodes a protein that retains the function of the Cas protein.
  • gRNA is also called guide RNA or guide RNA, and has the meaning commonly understood by those skilled in the art.
  • guide RNAs can include direct repeats and guide sequences, or consist essentially of direct repeats and guide sequences (also called spacers in the context of endogenous CRISPR systems). (spacer)) composition.
  • gRNA can include crRNA and tracrRNA, or only crRNA, depending on the Cas protein it depends on.
  • crRNA and tracrRNA can be artificially modified and fused to form single guide RNA (sgRNA).
  • the gRNA of the present invention may be natural, or artificially modified or designed and synthesized.
  • the targeting sequence is any polynucleotide sequence that has sufficient complementarity with the target sequence to hybridize with the target sequence and guide the specific binding of the CRISPR/Cas complex to the target sequence, usually having 17- Sequence length of 23nt.
  • the degree of complementarity between the targeting sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, Or at least 99%. Determining the best alignment is within the abilities of those of ordinary skill in the art. For example, there are published and commercially available alignment algorithms and programs, such as but not limited to ClustalW, Smith-Waterman algorithm in matlab, Bowtie, Geneious, Biopython, and SeqMan.
  • plant includes whole plants, plant organs (such as leaves, stems, roots, etc.), seeds and plant cells, and their progeny.
  • the types of plants that can be used in the method of the present invention are not particularly limited, and generally include any plant types that can be subjected to gene editing technology, including monocots, dicots, gymnosperms, and angiosperms, and mainly woody plants.
  • expression cassette refers to a polynucleotide sequence containing sequence components for the gene to be expressed and the elements required for expression.
  • the components required for expression include a promoter and a polyadenylation signal sequence.
  • the expression cassette of the present invention optionally contains other sequences, including (but not limited to): enhancers, secretion signal peptide sequences and the like.
  • nucleotide sequence is from 5'to 3', unless otherwise specified.
  • uracil DNA glycosylase inhibitor can inhibit the uracil DNA glycosylase in the cell from catalyzing U back to C.
  • the EF promoter refers to the promoter of the elongation factor
  • the elongation factor (EF) refers to the protein factor that promotes the extension of the polypeptide chain during mRNA translation.
  • Elongation factors in eukaryotes include: EF1 ⁇ , EF1 ⁇ and EF2.
  • Elongation factors in prokaryotes include EF-Tu, EF-Ts and EF-G.
  • EF1a is eukaryotic elongation factor 1 ⁇ , which is an important part of protein biosynthesis.
  • EF1A catalyzes the binding of aminoacyl tRNA to the ribosomal A site through a GTP-dependent mechanism.
  • EF1A accounts for 3-10% of the total soluble protein and is considered to be one of the most abundant soluble proteins in the cytoplasm.
  • the EF promoter includes, but is not limited to: EF1a promoter, EF1 ⁇ promoter, EF2 promoter, EF-Tu, EF-Ts, EF-G.
  • the promoter of the present invention refers to an EF1a promoter element derived from a Solanaceae plant (preferably, from tomato or similar plants).
  • a typical sequence of the promoter of the present invention is shown in SEQ ID NO.:1.
  • this term also includes promoters from other different Solanaceae plants that are homologous to the promoter shown in SEQ ID NO.:1.
  • the term also includes the derived promoters or active fragments of the promoter shown in SEQ ID NO.:1 or its homologous promoters, mainly these derived promoters or active fragments retain the function of high gene editing efficiency, such as retention At least 50% of the specific promoter function of the promoter shown in SEQ ID NO.:1 (expressed in terms of the expression level of the foreign gene that can be activated).
  • Solanaceae includes tomatoes, potatoes, eggplants, peppers, wolfberries, and tobacco.
  • promoter or “promoter region (domain) refers to a nucleic acid sequence that accurately and effectively initiates the transcription function of a gene, and guides the transcription of the gene nucleic acid sequence into mRNA, which usually exists in the coding sequence of the target gene. Upstream (5' end), generally, a promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription.
  • the promoter or promoter region (domain) includes promoter variants, which can be obtained by inserting or deleting regulatory regions, performing random or site-directed mutations, and the like.
  • the present invention also includes the preferred promoter sequence of the present invention (SEQ ID NO.: 1) that has 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more , Most preferably 98% or more, such as 99%) homologous nucleic acid, which also has the function of specifically improving the efficiency of plant gene editing.
  • “Homology” refers to the level of similarity (ie sequence similarity or identity) between two or more nucleic acids according to the percentage of positional identity.
  • the example of the present invention provides a promoter EF1a derived from the Solanaceae family, such as tomato, it is derived from other similar plants (especially belonging to the same family as tomato) and has certain homology with the promoter of the present invention.
  • sexual (conservative) promoters are also included in the scope of the present invention, as long as those skilled in the art can easily isolate the promoter from other plants based on the information provided in this application after reading this application.
  • exogenous or heterologous refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, if the combination of the promoter and the target gene sequence is not normally naturally occurring, the promoter is foreign to the target gene. A specific sequence is “foreign” to the cell or organism into which it is inserted.
  • trans-regulatory element refers to a conservative base sequence that regulates the transcription initiation and transcription efficiency of a gene.
  • the promoter of the present invention may be operably linked to a foreign gene, and the foreign gene may be foreign (heterologous) relative to the promoter.
  • the foreign gene (also called target gene) of the present invention is not particularly limited, and can be a gene encoding a protein with specific functions, such as (a) gene editing enzyme and (b) adenine deaminase and/or cell Pyrimidine deaminase.
  • exogenous genes include (but are not limited to): resistance genes, selectable marker genes, epitope tags, reporter gene sequences, nuclear localization signal sequences, transcription activation domains (e.g., transcription activation domains (e.g., , VP64), transcription repression domain (for example, KRAB domain or SID domain), nuclease domain (for example, Fok1), viral capsid protein gene, antibody gene; and domains having activities selected from the following: Nucleotide deaminase, methylase activity, demethylase, transcription activation activity, transcription inhibition activity, transcription release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA Cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity and nucleic acid binding activity.
  • transcription activation domains e.g., transcription activation domains (e.g., , VP
  • the resistance genes are selected from the following group: herbicide resistance genes, antiviral genes, cold tolerance genes, high temperature tolerance genes, drought tolerance genes, flood tolerance genes, or insect resistance genes.
  • the selection marker gene is selected from the following group: gus ( ⁇ -glucuronidase) gene, hyg (hygromycin) gene, neo (neomycin) gene, or gfp (green fluorescent protein) gene.
  • the present invention also provides a gene expression cassette, which has the following elements in order from 5'to 3': a promoter, a gene ORF sequence, and a terminator.
  • a promoter sequence is shown in SEQ ID NO.:1 or the homology with the sequence shown in SEQ ID NO.:1 is ⁇ 90%, preferably ⁇ 95%, more preferably ⁇ 98%.
  • the present invention also provides a recombinant vector comprising the promoter and/or gene expression cassette of the present invention.
  • the downstream of the promoter of the recombinant vector contains a multiple cloning site or at least one restriction site.
  • the target gene is ligated into a suitable multiple cloning site or restriction site, so that the target gene and the promoter are operably linked.
  • the recombinant vector includes (from 5'to 3'direction): a promoter, a target gene, and a terminator.
  • the recombinant vector may also include elements selected from the following group: 3'polynucleotideization signal; untranslated nucleic acid sequence; transport and targeting nucleic acid sequence; resistance selection marker (dihydrofolate reductase, Neomycin resistance, hygromycin resistance and green fluorescent protein, etc.); enhancer; or operator.
  • Those of ordinary skill in the art can use well-known methods to construct an expression vector containing the promoter and/or target gene sequence of the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the promoter, expression cassette or vector of the present invention can be used to transform an appropriate host cell to allow the host to express protein.
  • the host cell can be a prokaryotic cell, such as Escherichia coli, Streptomyces, Agrobacterium; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell.
  • a prokaryotic cell such as Escherichia coli, Streptomyces, Agrobacterium
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a plant cell.
  • the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.).
  • Agrobacterium transformation or gene gun transformation can also be used to transform plants, such as leaf disc method, immature embryo transformation method, flower bud soaking method, etc.
  • the transformed plant cells, tissues or organs can be regenerated by conventional methods to obtain transgenic plants.
  • the method for preparing transgenic plants is: transfer the vector carrying the promoter and the target gene (the two are operably linked) into Agrobacterium, and then Agrobacterium transfers the vector containing the promoter and the target gene.
  • the fragment is integrated into the chromosome of the plant.
  • the transgenic recipient plants involved are, for example, Arabidopsis, wheat, barley, oats, maize, rice, sorghum, millet, soybean, peanut, tobacco, tomato, cabbage, rape, spinach, lettuce, cucumber, chrysanthemum, water spinach, celery, Lettuce and so on.
  • the recombinant vector is pCAMBIA1300 vector, and the promoter of the present invention is constructed into this vector to transform plants.
  • the present invention clones the EF promoter (such as tomato SlEF1a promoter), and uses the promoter to drive the expression of the fusion protein coding sequence of Cas enzyme and deaminase, and finally obtains a pair of dicotyledon High-efficiency single-base substitution and gene knock-out system in plants.
  • EF promoter such as tomato SlEF1a promoter
  • adenine deaminase is an enzyme that catalyzes the hydrolysis and deamination of adenine to produce hypoxanthine and ammonia.
  • Adenine A is converted to hypoxanthine I.
  • Hypoxanthine I can be paired with cytosine, and it is read and copied as guanine (G) at the DNA level, resulting in the conversion of A ⁇ T pairing to G ⁇ C pairing.
  • G guanine
  • TadA adenine deaminase derived from Escherichia coli, has been artificially modified to obtain ecTadA mutants. The dimer of TadA and ecTadA is currently commonly used adenine deaminase.
  • the applicable TadA includes both the wild-type form and the specific mutant form TadA7-10, or a combination of the wild-type form and the mutant form.
  • TadA7-10 can perform deamination reaction with DNA as a substrate.
  • the coding sequence of adenine deaminase in the nucleic acid construct can be codon optimized in a host-preferred manner according to the applicable host.
  • cytosine deaminase is an enzyme that can catalyze the deamination of cytosine in cells to form uracil, which converts cytosine C to uracil U, and damages DNA by polymerase in the process of re-replication. Function, uracil will be recognized as T during DNA replication, causing C ⁇ G pairing to convert to T ⁇ A pairing.
  • APOBEC1 A1
  • APOBEC2 A2
  • APOBEC3A ⁇ H 3A, 3B, 3C, 3D, 3E, 3F, 3H
  • APOBEC4 A4
  • activation-induced deaminase activation induced cytidinedeaminase
  • the applicable cytosine deaminase includes both wild-type and specific mutant forms (such as CBE2.0, CBE2.1, CBE2.2, CBE2.3, CBE2.4), or Contains a combination of wild-type and mutant forms.
  • the mutant form of cytosine deaminase can perform deamination reactions using DNA as a substrate.
  • the cytosine deaminase coding sequence in the nucleic acid construct can be codon optimized in a host-preferred manner according to the applicable host.
  • the preferred cytosine deaminase is CBE2.0, CBE2.1, CBE2.2, CBE2.3, CBE2.4.
  • amino acid sequence of CBE2.0 is shown in SEQ ID NO.: 6, and its nucleotide sequence is shown in SEQ ID NO.: 7.
  • amino acid sequence of CBE2.1 is shown in SEQ ID NO.: 8.
  • amino acid sequence of CBE2.2 is shown in SEQ ID NO.:9.
  • amino acid sequence of CBE2.3 is shown in SEQ ID NO.: 10.
  • the amino acid sequence of CBE2.4 is shown in SEQ ID NO.: 11.
  • the present invention provides a nucleic acid construct for gene editing of plants, the nucleic acid construct has a 5'-3' formula I structure:
  • P1, S1, L1, S2, S3 are the elements used to form the construct, respectively
  • each "-" is a bond or a nucleotide linking sequence.
  • nucleic acid construct is further operably connected to one or more second nucleic acid constructs represented by formula II:
  • P2 and Y1 are defined as described in the first aspect of the present invention.
  • the nucleic acid construct can also be operably connected to the first integration element (I1) and the second integration element (I2).
  • the I1 element (or the integration element on the left) and the I2 element (or the integration element on the right) can cooperate to integrate the elements between them (ie the nucleotide sequence from P1 to Y1) into the genome of the plant cell .
  • I1 and I2 are Ti elements from Agrobacterium. Of course, other elements that can play a similar integration role can also be used in the present invention.
  • the various elements used in the constructs of the present invention are either known in the art or can be prepared by methods known to those skilled in the art.
  • the corresponding elements can be obtained by conventional methods, such as PCR method, fully artificial chemical synthesis method, and enzyme digestion method, and then connected together by well-known DNA ligation technology to form the construct of the present invention.
  • Inserting the construct of the present invention into an exogenous vector constitutes the vector of the present invention.
  • the vector of the present invention is transformed into plant cells so as to mediate the integration of the vector of the present invention into the chromosome of the plant cell and express it in the plant body to prepare a gene-edited plant cell.
  • the gene-edited plant cell of the present invention is regenerated into a plant body, thereby obtaining a gene-edited plant.
  • nucleic acid construct constructed by the present invention can be introduced into plant cells through conventional plant recombination technology (for example, Agrobacterium transfer technology), thereby obtaining the nucleic acid construct (or a vector carrying the nucleic acid construct) Or obtain a plant cell in which the nucleic acid construct is integrated in the genome.
  • plant recombination technology for example, Agrobacterium transfer technology
  • the plant individual integrated with the nucleic acid construct can be isolated or removed in its progeny by conventional screening or other means known in the art, thereby obtaining a gene-edited plant that does not contain the nucleic acid construct body.
  • the present invention is to use a specific EF promoter, such as tomato EF1a, to drive the expression of the coding sequence of a gene editing enzyme (such as Cas9) and a deaminase fusion protein, thereby improving gene editing efficiency.
  • a gene editing enzyme such as Cas9
  • a deaminase fusion protein thereby improving gene editing efficiency.
  • the main feature of the vector is to link a specific EF promoter (such as tomato EF1a), deaminase and Cas fusion protein coding sequence, optionally also including nuclear localization signal, UGI coding sequence, to form the present invention Specific nucleic acid constructs.
  • a specific EF promoter such as tomato EF1a
  • deaminase and Cas fusion protein coding sequence optionally also including nuclear localization signal, UGI coding sequence
  • the Cas protein is a mutant Cas protein that has no cleavage activity or a single-stranded cleavage activity.
  • the Cas protein of the present invention may be nCas9, and its amino acid sequence is shown in SEQ ID NO.:2.
  • the proteins are usually connected by some flexible short peptides, namely Linker (connecting peptide sequence).
  • Linker can use XTEN, and its coding sequence is shown in SEQ ID NO.: 17, and its amino acid sequence is shown in SEQ ID NO.: 18.
  • the vector can be, for example, a plasmid, virus, cosmid, phage, etc., which are well known to those skilled in the art and have been described in many ways in the art.
  • the expression vector in the present invention is a plasmid.
  • the expression vector may include a promoter, a ribosome binding site for translation initiation, a polyadenylation site, a transcription terminator, an enhancer, and the like.
  • the expression vector may also contain one or more selectable marker genes for selection of host cells containing the vector. Such selectable markers include genes encoding dihydrofolate reductase, genes conferring resistance to neomycin, genes conferring resistance to tetracycline or ampicillin, and the like.
  • nucleic acid construct of the present invention can be inserted into the vector by various methods, for example, by digesting the insert and the vector with an appropriate restriction endonuclease and then ligating.
  • a variety of cloning techniques are known in the art, and these are all within the knowledge of those skilled in the art.
  • the vectors applicable in the present invention include plasmids that can be obtained from commercial channels, such as but not limited to: pBR322 (ATCC37017), pCAMBIA1300, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison, WI, USA) )pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia), pKK232-8, pCM7, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmaci
  • the method for introducing the construct of formula I of the present invention into cells or integrating into the genome there is no particular limitation on the method for introducing the construct of formula I of the present invention into cells or integrating into the genome. It can be carried out by a conventional method, for example, the construct of formula I or the corresponding vector is introduced into the plant cell by a suitable method.
  • Representative introduction methods include but are not limited to: Agrobacterium transfection method, gene gun method, microinjection method, electric shock method, ultrasonic method, and polyethylene glycol (PEG)-mediated method.
  • the recipient plants which include various crop plants (such as grasses), forestry plants, horticultural plants (such as floral plants) and the like.
  • crop plants such as grasses
  • forestry plants such as floral plants
  • horticultural plants such as floral plants
  • Representative examples include, but are not limited to: rice, soybeans, tomatoes, corn, tobacco, wheat, sorghum, potatoes and the like.
  • the DNA in the transformed plant cell is allowed to express the fusion protein and gRNA.
  • the gene editing enzyme such as Cas9 nuclease fused with adenine deaminase and/or cytosine deaminase, under the guidance of the corresponding gRNA, mutates the A at the target position to G (and thus makes the T of the complementary strand mutate to C ) Or mutate the C of the target position to T (and then mutate the G of the complementary strand to A).
  • the invention can be used in the field of plant genetic engineering, for plant research and breeding, especially for genetic improvement of agricultural crops, forestry crops or horticultural plants with economic value.
  • the present invention combines a specific promoter (such as Ef1a promoter) with a gene editing enzyme (such as Cas9 nuclease), adenine deaminase and/or cytosine deaminase for the first time, and optionally also includes a nuclear localization signal
  • a specific promoter such as Ef1a promoter
  • a gene editing enzyme such as Cas9 nuclease
  • adenine deaminase and/or cytosine deaminase for the first time
  • the coding sequences of UGI are linked together to form the specific nucleic acid construct of the present invention.
  • the nucleic acid construct of the present invention successfully realizes gRNA-guided base-directed mutation (such as A mutation to G) in plants, and the mutation efficiency Very high (can be as high as ⁇ 70% or higher).
  • the specific nucleic acid construct of the present invention can edit some gene sites where other promoters do not work, and break the barrier of gene editing restricted by genotype.
  • the specific nucleic acid construct of the present invention can edit some plants where other promoters do not work, such as soybeans, which effectively expands the application range of the gene editing system and breaks the species barrier.
  • Solyc05g012020 which affects fruit development in tomato, is selected as the target gene, and 6 target sites are selected to design sgRNA.
  • the sequences of the 6 sgRNAs are as follows:
  • sgRNA1 TACTGGAGTTGTACCTGGA (SEQ ID NO.: 20),
  • sgRNA2 GGAACAGCTGAACGTCAAT (SEQ ID NO.: 21),
  • sgRNA3 GAACAGCCTTCTCATCATGA (SEQ ID NO.: 22),
  • sgRNA4 GGTGAGGATTTGGGACAATT (SEQ ID NO.: 23),
  • sgRNA5 CTGTGAATCTGATGAAGTTT (SEQ ID NO.: 24),
  • sgRNA6 GAAAAGTAATAACAAAGGGC (SEQ ID NO.: 25).
  • the expression cassette of the ABE single-base editor was obtained by homologous recombination technology (see Figure 1).
  • the nucleotide sequence of the adenine deaminase ABE7.10 is shown in SEQ ID NO.: 5 or 19.
  • the nucleotide sequence of the SlEF1a promoter is shown in SEQ ID NO.:1, and the specific operations are as follows:
  • PCR reaction conditions are: 95°C pre-denaturation for 5 minutes, 98°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 45s, 35 cycles, 72°C extension for 5 minutes
  • the marker gene is detected (taking GUS as an example), a few pieces of cotyledons after sterilization for 7 days are taken for GUS staining, and the infection time is adjusted according to the size of the staining area. (It is not necessary to do it for each batch, it is done regularly to check the activity of the bacteria).
  • the slEF1a promoter achieves up to 70% editing efficiency in single-base editing, which is 2-20 times higher than other promoters (see Figure 2).
  • the slEF1a promoter can efficiently drive the expression of the fusion protein of deaminase and Cas9, effectively expand the scope of application of single-base editing tools, and is of great significance to the improvement of plant traits and variety cultivation.
  • Example 1 investigate the SlEF1a promoter (pS1EF1a), the CaMV35S promoter (35S) and the AA6 promoter (pAA6, reference: CN101370939A) and ABE7.10 (SEQ ID NO.: 5 or 19) And the editing efficiency when Cas9 is used in conjunction; as shown in Figure 3, the "A to G gRNA1" is the result of using different promoters in combination with the above adenine deaminase. In soybeans, the SlEF1a promoter is used The resulting editing efficiency is much higher than CaMV35S promoter and AA6 promoter.
  • adenine deaminase is replaced with cytosine deaminase.
  • the amino acid sequence of the cytosine deaminase is shown in SEQ ID NO.: 6, 8-11. In this embodiment, SEQ is preferred.
  • ID NO.: 6 shows the cytosine deaminase, to investigate the editing efficiency of different promoters when used in conjunction with cytosine deaminase and Cas9; as shown in Figure 3, the "C to T gRNA2" is In order to use different promoters in combination with the above-mentioned cytosine deaminase, the editing efficiency of using the SlEF1a promoter in soybeans is much higher than that of the CaMV35S promoter and the AA6 promoter.

Abstract

提供了一种核酸表达的方法及核酸构建物，采用特定启动子驱动的核酸构建物，在植物中实现了gRNA引导的高效的碱基定点突变。

Description

一种核酸表达的方法 技术领域
本发明涉及生物技术领域，具体地，涉及一种核酸表达的方法。
背景技术
目前在双子叶植物中单碱基编辑效率低，大部分双子叶植物如大豆目前还不能进行单碱基编辑，而拟南芥/番茄等植物，单碱基编辑效率很低，严重影响了生物技术育种在农业生产上的应用。因此，提高在双子叶植物中的单碱基编辑编辑效率在农业生产中具有极大的商业价值。
因此，本领域迫切需要开发一种提高在植物中的单碱基编辑效率的方法。
发明内容
本发明的目的在于提供一种提高在植物中的单碱基编辑效率的方法。
本发明第一方面提供了一种核酸构建物，所述核酸构建物具有5’-3’(5’至3’)的式I结构：
P1-S1-L1-S2-S3  (I)；
式中，
P1、S1、L1、S2、S3分别为用于构成所述构建物的元件；
P1为第一启动子序列，所述第一启动子包括延伸因子的启动子；
S1、S2各自独立地为一个或多个(a)基因编辑酶的编码序列、(b)腺嘌呤脱氨酶的编码序列和/或胞嘧啶脱氨酶的编码序列；
L1为无或连接肽的编码序列；
S3为无或尿嘧啶糖苷酶抑制剂UGI的编码序列；
并且，各“-”独立地为键或核苷酸连接序列。
在另一优选例中，所述的S1为腺嘌呤脱氨酶的编码序列和/或胞嘧啶脱氨酶的编码序列，所述S2为基因编辑酶的编码序列。
在另一优选例中，当S1为腺嘌呤脱氨酶的编码序列，S3为无。
在另一优选例中，当S1为胞嘧啶脱氨酶的编码序列，S3为尿嘧啶糖苷酶抑制 剂UGI的编码序列。
在另一优选例中，所述延伸因子包括真核延伸因子或原核延伸因子。
在另一优选例中，所述真核延伸因子包括EF1α、EF1β、EF2。
在另一优选例中，所述原核延伸因子包括EF-Tu、EF-Ts、EF-G；优选地，包括EF1α；优选地，包括植物中的EF1α。
在另一优选例中，所述植物选自下组：玉米、水稻、大豆、拟南芥、烟草、番茄、或其组合。
在另一优选例中，所述第一启动子来源于选自下组的一种或多种植物：玉米、水稻、大豆、拟南芥、烟草、番茄。
在另一优选例中，所述第一启动子为番茄EF1a启动子。
在另一优选例中，所述第一启动子的序列如SEQ ID NO.:1所示。
在另一优选例中，所述的L1核苷酸序列长度各自独立地为3-120nt,较佳的为3-96nt，并且优选为3的倍数。
在另一优选例中，所述的L1编码的氨基酸序列长度各自独立的为3-40aa，较佳的为6-32aa，较佳的为18-32aa，较佳的为24-32aa。
在另一优选例中，所述的核苷酸连接序列长度为1-300nt，较佳地1-100nt。
在另一优选例中，所述核苷酸连接序列不影响各元件的正常转录和翻译。
在另一优选例中，所述基因编辑酶为选自下组的编辑工具的酶：CRISPR酶、TALEN酶、ZFN酶、或其组合。
在另一优选例中，所述基因编辑酶来源于微生物；优选地来源于细菌。
在另一优选例中，所述基因编辑酶的来源选自下组：酿脓链球菌(Streptococcus pyogenes)、葡萄球菌(Staphylococcus aureus)、犬链球菌(Streptococcus canis)、或其组合。
在另一优选例中，所述基因编辑酶具有双链或单链DNA切割活性、或无切割活性。
在另一优选例中，所述基因编辑酶为具有单链DNA切割活性的CRISPR酶。
在另一优选例中，所述基因编辑酶包括野生型或突变型的基因编辑酶。
在另一优选例中，所述的基因编辑酶与所述的突变的基因编辑酶的同一性≥80％，较佳地≥90％；更佳地≥95％，更佳地，≥98％或99％。
在另一优选例中，所述突变的基因编辑酶由所述的野生型的基因编辑酶经过一个或多个，较佳地1-15个，较佳地1-10个，较佳的1-7个，更佳地2-5个， 氨基酸取代、缺失；和/或经过1-5，较佳地1-4个，更佳地1-3个，最佳地1-2个氨基酸的添加形成的。
在另一优选例中，所述基因编辑酶选自下组：Cas9、Cas12、Cas13、Cms1、MAD7、或其组合。
在另一优选例中，所述基因编辑酶选自下组：nCas9、dCas9、nCas9NG、nCas9X、nCas12、nCas13、或其组合。
在另一优选例中，所述基因编辑酶的氨基酸序列如SEQ ID NO.:2所示。
在另一优选例中，所述基因编辑酶的编码序列选自下组：
(i)序列如SEQ ID NO.:3所示的多核苷酸；
(ii)核苷酸序列与SEQ ID NO.:3所示序列的同源性≥75％(较佳地≥85％，更佳地≥90％或≥95％或≥98％或≥99％)的多核苷酸；
(iii)在SEQ ID NO.:3所示多核苷酸的5'端和/或3'端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸；
(iv)与(i)-(iii)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述基因编辑酶的编码序列如SEQ ID NO.:3所示。
在另一优选例中，所述腺嘌呤脱氨酶包括野生型和突变型。
在另一优选例中，所述腺嘌呤脱氨酶包括野生型和/或突变型的TadA。
在另一优选例中，所述腺嘌呤脱氨酶包括TadA。
在另一优选例中，所述腺嘌呤脱氨酶的突变型包括TadA7-10。
在另一优选例中，所述腺嘌呤脱氨酶为TadA与TadA7-10形成的融合蛋白。
在另一优选例中，所述腺嘌呤脱氨酶的编码序列选自下组：
(i)序列如SEQ ID NO.:5或19所示的多核苷酸；
(ii)核苷酸序列与SEQ ID NO.:5或19所示序列的同源性≥75％(较佳地≥85％，更佳地≥90％或≥95％或≥98％或≥99％)的多核苷酸；
(iii)在SEQ ID NO.:5或19所示多核苷酸的5'端和/或3'端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸；
(iv)与(i)-(iii)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述腺嘌呤脱氨酶的编码序列如SEQ ID NO.5或19所示。
在另一优选例中，所述腺嘌呤脱氨酶的氨基酸序列如SEQ ID NO.:4所示。
在另一优选例中，所述胞嘧啶脱氨酶包括野生型和突变型。
在另一优选例中，所述胞嘧啶脱氨酶包括APOBEC。
在另一优选例中，所述APOBEC选自下组：APOBEC1(A1)、APOBEC2(A2)、APOBEC3A、APOBEC3B、APOBEC3C、APOBEC3D、APOBEC3E、APOBEC3F、APOBEC3H、APOBEC4(A4)、活化诱导脱氨酶(activation induced cytidine deaminase,AID)、或其组合。
在另一优选例中，所述胞嘧啶脱氨酶的突变型包括CBE2.0、CBE2.1、CBE2.2、CBE2.3、CBE2.4。
在另一优选例中，所述胞嘧啶脱氨酶的氨基酸序列如SEQ ID NO.:6、8-11中任一所示。
在另一优选例中，所述核酸构建物还可与一个或多个定位信号序列可操作的连接。
在另一优选例中，所述的定位信号选自下组：核定位信号、叶绿体定位信号、线粒体定位信号、或其组合。
在另一优选例中，所述的定位信号包括核定位信号，优选地，包括1-2个核定位信号。
在另一优选例中，所述核定位信号包括bpNLS、SV40。
在另一优选例中，所述核定位信号的核苷酸序列如SEQ ID NO.:12-14中任一所示。
在另一优选例中，所述核定位信号的氨基酸序列如SEQ ID NO.:15所示。
在另一优选例中，所述S3元件的核苷酸序列如SEQ ID NO.:16所示。
在另一优选例中，所述的核酸构建物还进一步可操作地与一个或多个式II所示的第二核酸构建物相连：
P2-Y1(II)
式中，
P2为第二启动子序列；
Y1为gRNA的编码序列；
并且，各“-”独立地为键或核苷酸连接序列。
在另一优选例中，当含有至少两个式II核酸构建物时，其gRNA序列可以互不相同。
在另一优选例中，所述式II核酸构建物位于式I核酸构建物的5’端或3’端或 分布于其两端。
在另一优选例中，所述gRNA包括crRNA、tracrRNA、sgRNA。
在另一优选例中，所述第二启动子来源于选自下组的一种或多种植物：水稻、玉米、大豆、拟南芥、烟草或番茄。
在另一优选例中，所述第二启动子包括RNA聚合酶III依赖的启动子。
在另一优选例中，所述第二启动子为RNA聚合酶III依赖的启动子。
在另一优选例中，所述第二启动子选自下组：U6、U3、U6a、U6b、U6c、U6-1、U3b、U3d、U6-26、U6-29、H1、或其组合。
在另一优选例中，所述第二启动子包括U6启动子。
在另一优选例中，本发明的上述核苷酸元件是按阅读框(in-frame)连接的，从而表达氨基酸序列正确的融合蛋白。
在另一优选例中，所述的式I核酸构建物和式II核酸构建物还各自独立地具有终止子。
在另一优选例中，所述的式I核酸构建物和式II核酸构建物共用相同的终止子。
在另一优选例中，所述终止子包括适用于植物基因编辑的终止子。
在另一优选例中，所述终止子选自下组：NOS、Poly A、T-UBQ、rbcS、或其组合。
在另一优选例中，所述的构建物具有式IIIa或式IIIb结构：
P1-S1-L1-S2-S3-P2-Y1  (IIIa)；
P2-Y1-P1-S1-L1-S2-S3  (IIIb)；
式中，各元件的定义如上所述。
在另一优选例中，所述的核酸构建物还可操作地连接第一整合元件(I1)和第二整合元件(I2)。
在另一优选例中，所述第一整合元件包括5’同源臂序列。在另一优选例中，所述第二整合元件包括3’同源臂序列。
在另一优选例中，在所述的I1和I2元件之间，还含有额外插入的一个或多个额外的表达盒。
在另一优选例中，所述的额外表达盒是独立于含有式I核酸构建物的表达盒和含有式II核酸构建物的表达盒的。
在另一优选例中，所述的额外表达盒表达选自下组的物质：标记基因。
在另一优选例中，所述标记基因包括抗性基因(如潮霉素抗性基因、除草剂抗 性基因)、荧光基因、或其组合。
本发明第二方面提供了一种载体，所述载体含有本发明第一方面所述的核酸构建物。
在另一优选例中，所述载体为植物表达载体。
在另一优选例中，所述的载体为可转染或转化植物细胞的表达载体。
在另一优选例中，所述的载体为农杆菌Ti载体。
在另一优选例中，所述的构建物整合到所述载体的T-DNA区。
在另一优选例中，所述载体是环状的或线性的。
本发明第三方面提供了一种宿主细胞，所述细胞含有本发明第一方面所述的核酸构建物，或其基因组整合有一个或多个本发明第一方面所述的核酸构建物。
在另一优选例中，所述的细胞为植物细胞。
在另一优选例中，所述的植物选自下组：单子叶植物、双子叶植物、裸子植物、或其组合。
在另一优选例中，所述的植物选自下组：禾本科植物、豆科植物、十字花科植物、茄科、伞形科、或其组合。
在另一优选例中，所述的植物选自下组：拟南芥、小麦、大麦、燕麦、玉米、水稻、高粱、粟、大豆、花生、烟草、番茄、白菜、油菜、菠菜、生菜、黄瓜、茼蒿、空心菜、芹菜、油麦菜、或其组合。
在另一优选例中，所述的宿主细胞是用选自下组的方法将权利要求1所述的核酸构建物导入细胞的：农杆菌转化法、基因枪法、显微注射法、电击法、超声波法和聚乙二醇(PEG)介导法。
本发明第四方面提供了一种试剂组合，包括：
(i)第一核酸构建物，或含有所述第一核酸构建物的第一载体，所述第一核酸构建物具有从5’-3’的式I结构：
P1-S1-L1-S2-S3     (I)
其中，
P1为第一启动子序列，所述第一启动子包括延伸因子的启动子；
S1、S2各自独立地为一个或多个(a)基因编辑酶的编码序列、(b)腺嘌呤脱氨酶的编码序列和/或胞嘧啶脱氨酶的编码序列；
L1为无或连接肽的编码序列；
S3为无或尿嘧啶糖苷酶抑制剂UGI的编码序列；
并且，“-”为键或核苷酸连接序列；
(ii)第二核酸构建物，或含有所述第二核酸构建物的第二载体，所述第二核酸构建物具有从5’-3’的式(II)所示的结构：
P2-Y1  (II)；
其中，P2为第二启动子；
Y1为gRNA的编码序列；
并且，“-”为键或核苷酸连接序列。
在另一优选例中，所述第一载体和所述第二载体为不同的载体。
在另一优选例中，所述第一核酸构建物和所述第二核酸构建物位于不同的载体上。
在另一优选例中，所述第一载体和所述第二载体为同一载体。
在另一优选例中，所述第一核酸构建物和所述第二核酸构建物位于同一载体上。
本发明第五方面提供了一种试剂盒，所述试剂盒含有本发明第四方面所述的试剂组合。
在另一优选例中，所述试剂盒还含有标签或说明书。
本发明第六方面提供了一种对植物进行基因编辑的方法，包括步骤：
(i)提供待编辑植物；和
(ii)将本发明第一方面所述的核酸构建物、本发明第二方面所述的载体或本发明第四方面所述的试剂组合导入所述待编辑植物的植物细胞，从而在所述植物细胞内进行基因编辑。
在另一优选例中，所述导入为通过农杆菌导入。
在另一优选例中，所述导入为通过基因枪导入。
在另一优选例中，所述的基因编辑为定点碱基替换(或突变)。
在另一优选例中，所述定点替换(或突变)包括将A突变为G。
在另一优选例中，所述定点替换(或突变)包括将C突变为T。
在另一优选例中，所述的植物包括任何可进行转化技术的高等植物类型，包括单子叶植物、双子叶植物和裸子植物。
在另一优选例中，所述的植物为双子叶植物。
在另一优选例中，所述的植物选自下组：禾本科植物、豆科植物、十字花科 植物、茄科、伞形科、或其组合。
在另一优选例中，所述的植物选自下组：拟南芥、小麦、大麦、燕麦、玉米、水稻、高粱、粟、大豆、花生、烟草、番茄、白菜、油菜、菠菜、生菜、黄瓜、茼蒿、空心菜、芹菜、油麦菜、或其组合。
本发明第七方面提供了一种制备经基因编辑的植物细胞的方法，包括步骤：
将本发明第一方面所述的核酸构建物、本发明第二方面所述的载体或本发明第四方面所述的试剂组合导入(或转染)植物细胞，使得所述植物细胞中的染色体发生定点替换(或突变)，从而制得所述经基因编辑的植物细胞。
在另一优选例中，所述的转染采用农杆菌转化法或基因枪轰击法。
本发明第八方面提供了一种本发明第一方面所述的核酸构建物、本发明第二方面所述的载体、本发明第三方面所述的宿主细胞、本发明第四方面所述的试剂组合、本发明第五方面所述的试剂盒的用途，用于对植物进行基因编辑；或者，用于提高碱基编辑效率，优选，提高植物中的单碱基编辑效率。
本发明第九方面提供了一种制备经基因编辑的植物的方法，包括步骤：
将本发明第七方面所述方法制备的所述经基因编辑的植物细胞再生为植物体，从而获得所述经基因编辑的植物。
本发明第十方面提供了一种经基因编辑的植物，所述的植物是用本发明第九方面所述的方法制备的。
本发明第十一方面提供了一种提高植物中的碱基编辑效率的方法，所述方法包括利用本发明第一方面所述的核酸构建物、本发明第二方面所述的载体、本发明第三方面所述的宿主细胞、本发明第四方面所述的试剂组合，或本发明第五方面所述的试剂盒对植物进行碱基编辑的步骤。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了含有slEF1a的ABE单碱基编辑器的结构。
图2显示了不同启动子在番茄中单碱基编辑中的效率。
图3显示了利用不同启动子和不同的碱基编辑器在大豆中的单碱基编辑效 率。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地发现一种高效的EF启动子(如番茄EF启动子)，将该启动子构建于ABE和CBE的单碱基编辑系统中，用以驱动(a)基因编辑酶与(b)腺嘌呤脱氨酶和/或胞嘧啶脱氨酶共同构成的融合蛋白的表达，该启动子在植物中显著提高了编辑效率。在此基础上，本发明人完成了本发明。
术语
如本文所用，术语“同源臂”指打靶载体上待插入的外源序列两侧的与基因组序列完全一致的侧翼序列，用于识别并发生重组的区域。
如本文所用，术语“植物启动子”指能够在植物细胞中启动核酸转录的核酸序列。该植物启动子可以是来源于植物、微生物(如细菌、病毒)或动物等，或者是人工合成或改造过的启动子。
如本文所用，术语“基因编辑”或“碱基突变”或“碱基编辑”指核苷酸序列的某一位置处发生碱基的替换(substitution)、插入(insertion)和/或缺失(deletion)。本发明中所述“编辑”或“突变”优选为单碱基突变。
如本文所用，术语“碱基替换”指核苷酸序列的某一位置处的碱基突变为另一不同的碱基，比如A突变为G。
如本文所用，术语“A.T到G.C”指在双链核酸序列(尤其是基因组序列)中，某一位置上的A-T碱基对突变为或替换为G-C碱基对。
如本文所用，术语“C.G到T.A”指在双链核酸序列(尤其是基因组序列)中，某一位置上的C-G碱基对突变为或替换为T-A碱基对。
如本文所用，术语“基因编辑酶”指适用于CRISPR(规律成簇间隔短回文重复序列Clustered Regularly Interspaced Short Palindromic Repeats)、TALEN(转录激活因子样效应物核酸酶技术Tanscription Activator-like(TAL)effector nucleases)、ZFN(锌指核酸技术，Zinc finger nuclease)等编辑工具的核酸酶。优选地，所述基因编辑酶为CRISPR酶，又名Cas蛋白，其种类包括但并不限于：Cas9蛋白、Cas12蛋白、Cas13蛋白、Cas14蛋白、Csm1蛋白、FDK1蛋白。所述的Cas蛋白是指蛋白家族，可以根据其来源不同而具有不同的结构，如来源于酿 脓链球菌(Streptococcus pyogenes)的SpCas9、来源于葡萄球菌(Staphylococcus aureus)的SaCas9；还可以根据结构特征(如结构域)进行下位分类，如Cas12家族包括Cas12a(又名Cpf1)、Cas12b、Cas12c、Cas12i等。所述的Cas蛋白可以具有双链或单链或无切割活性。本发明所述的Cas蛋白可以是野生型或其突变体，所述的突变体的突变类型包括氨基酸的替换、取代或缺失，所述的突变体可以改变也可以不改变Cas蛋白的酶切活性。优选地，本发明所述的Cas蛋白只具有单链切割活性或无切割活性，其为野生型Cas蛋白的一种突变体。优选地，本发明Cas蛋白为具有单链切割活性的Cas9、Cas12、Cas13或Cas14。在一优选实施方式中，本发明的Cas9蛋白包括SpCas9n(D10A)、nSpCas9NG、SaCas9n、ScCas9n、XCas9n，其中“n”表示nick，即只具有单链切割活性的Cas蛋白。突变已知Cas蛋白获得具有单链或无切割活性的Cas蛋白为本领域的常规技术手段。本领域技术人员所知，现有技术中已报到的多种具有核酸切割活性的Cas蛋白，该公知蛋白或其改造后的变体均可以实现本发明的功能，本文通过引用方式将其纳入保护范围。
如本文所用，术语“Cas蛋白的编码序列”指编码Cas蛋白的核苷酸序列。在插入的多聚核苷酸序列被转录和翻译从而产生功能性Cas蛋白的情况下，技术人员会认识到，因为密码子的简并性，有大量多聚核苷酸序列可以编码相同的多肽。另外，技术人员也会认识到不同物种对于密码子具有一定的偏好性，可能会根据在不同物种中表达的需要，会对Cas蛋白的密码子进行优化，这些变异体都被术语“Cas蛋白的编码序列”所具体涵盖。此外，术语特定地包括了全长的、与Cas基因序列基本相同的序列，以及编码出保留Cas蛋白功能的蛋白质的序列。
如本文所用，所述的“gRNA”又称为guide RNA或导向RNA，并且具有本领域技术人员通常理解的含义。一般而言，导向RNA可以包含同向(direct)重复序列和导向序列(guide sequence)，或者基本上由或由同向重复序列和导向序列(在内源性CRISPR系统背景下也称为间隔序列(spacer))组成。gRNA在不同的CRISPR系统中，依据其所依赖的Cas蛋白的不同，可以包括crRNA和tracrRNA，也可以只含有crRNA。crRNA和tracrRNA可以经过人工改造融合形成single guide RNA(sgRNA)。本发明所述的gRNA可以是天然的，也可以是经过人工改造或设计合成的。在某些情况下，导向序列是与靶序列具有足够互补性从而与所述靶序列杂交并引导CRISPR/Cas复合物与所述靶序列的特异性结合的任何多核苷酸序列，通 常具有17-23nt的序列长度。在某些实施方案中，当最佳比对时，导向序列与其相应靶序列之间的互补程度为至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、或至少99％。确定最佳比对在本领域的普通技术人员的能力范围内。例如，存在公开和可商购的比对算法和程序，诸如但不限于ClustalW、matlab中的史密斯-沃特曼算法(Smith-Waterman)、Bowtie、Geneious、Biopython以及SeqMan。
如本文所用，术语“植物”包括全植株、植物器官(如叶、茎、根等)、种子和植物细胞以及它们的子代。可用于本发明方法的植物的种类没有特别限制，一般包括任何可进行基因编辑技术的植物类型，包括单子叶、双子叶植物和裸子植物、被子植物，主要包括木本植物。
如本文所用，术语“表达盒”是指含有待表达基因以及表达所需元件的序列组件的一段多聚核苷酸序列。表达所需的组件包括启动子和聚腺苷酸化信号序列。此外，本发明的表达盒还任选地含有其他序列，包括(但并不限于)：增强子、分泌信号肽序列等。
在本发明中，核苷酸序列的描述是从5’至3’方向，除非特别注明。
如本文所用，“尿嘧啶糖苷酶抑制剂(uracil DNA glycosylase inhibitor，UGI)”能够抑制胞内的尿嘧啶DNA糖苷酶将U再催化回C。
EF启动子
EF启动子是指延伸因子的启动子，延伸因子(elongation factors，EF)是指在mRNA翻译时促进多肽链延伸的蛋白质因子。真核生物中延伸因子包括：EF1α、EF1β和EF2。原核生物中延伸因子包括EF-Tu、EF-Ts以及EF-G。EF1a是真核延伸因子1α，它是蛋白质生物合成的重要组成部分。EF1A通过GTP依赖性机制催化氨酰基tRNA与核糖体A位点的结合。EF1A占可溶性蛋白总量的3-10％，被认为是细胞质中最丰富的可溶性蛋白之一。
在一优选实施方式中，EF启动子包括，但并不限于：EF1a启动子、EF1β启动子、EF2启动子、EF-Tu、EF-Ts、EF-G。
在一优选实施方式中，本发明的启动子指来源于茄科植物(较佳地，来自番茄或类似植物)的EF1a启动子元件。
一种典型的本发明的启动子的序列如SEQ ID NO.:1所示。
应理解，该术语还包括来自其他不同茄科植物的与SEQ ID NO.:1所示启动子同源的启动子。此外，该术语还包括SEQ ID NO.:1所示启动子或其同源启 动子的衍生启动子或活性片段，主要这些衍生启动子或活性片段保留了高效的基因编辑效率的功能，例如保留至少50％SEQ ID NO.:1所示启动子的特异启动功能(以可以被启动的外源基因的表达量进行表示)。
如本文所用，术语“茄科植物”包括番茄、马铃薯、茄子、辣椒、枸杞、烟草。
如本文所用，术语“启动子”或“启动子区(域)”是指一种准确有效起始基因转录功能的核酸序列，引导基因核酸序列转录为mRNA，其通常存在于目的基因编码序列的上游(5’端)，一般地，启动子或启动子区域提供RNA聚合酶和正确起始转录所必需的其它因子的识别位点。
在本文中，所述启动子或启动子区(域)包括启动子的变体，启动子变体可以通过插入或删除调控区域，进行随机或定点突变等来获得。
本发明还包括与本发明的优选启动子序列(SEQ ID NO.:1)具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％)同源性的核酸，所述核酸也具有特异性提高植物基因编辑效率的功能。“同源性”是指按照位置相同的百分比，两条或多条核酸之间的相似水平(即序列相似性或同一性)。
应理解，尽管本发明的实例中提供了来源于茄科，比如番茄的启动子EF1a，但是来源于其它类似的植物(尤其是与番茄属于同一科)的、与本发明启动子具有一定同源性(保守性)的启动子，也包括在本发明的范围内，只要本领域技术人员在阅读了本申请后根据本申请提供的信息可以方便地从其它植物中分离得到该启动子。
如本文所用，“外源的”或“异源的”是指不同来源的两条或多条核酸或蛋白质序列之间的关系。例如，如果启动子与目的基因序列的组合通常不是天然存在的，则启动子对于该目的基因来说是外源的。特定序列对于其所插入的细胞或生物体来说是“外源的”。
如本文所用，“顺式调控元件”是指对基因的转录起始和转录效率起调节作用的保守性碱基序列。
本发明的启动子可以被可操作地与外源基因连接，该外源基因相对于启动子而言可以是外源(异源)的。本发明所述的外源基因(也称为目的基因)没有特别的限制，可以为编码具有特定功能蛋白的基因，比如(a)基因编辑酶和(b)腺嘌呤脱氨酶和/或胞嘧啶脱氨酶。
所述外源基因的代表性例子包括(但不限于)：抗性基因、筛选标记基因、表位标签、报告基因序列、核定位信号序列、转录激活结构域(例如，转录激活结构域(例如，VP64)、转录抑制结构域(例如，KRAB结构域或SID结构域)、核酸酶结构域(例如，Fok1)，病毒衣壳蛋白基因，抗体基因；以及具有选自下列的活性的结构域：核苷酸脱氨酶，甲基化酶活性,去甲基化酶,转录激活活性,转录抑制活性,转录释放因子活性,组蛋白修饰活性,核酸酶活性,单链RNA切割活性,双链RNA切割活性,单链DNA切割活性,双链DNA切割活性和核酸结合活性。
所述的抗性基因选自下组：抗除草剂基因、抗病毒基因、耐寒基因、耐高温基因、抗旱基因、抗涝基因、或抗虫基因。所述的筛选标记基因选自下组：gus(β-葡萄糖苷酸酶)基因、hyg(潮霉素)基因、neo(新霉素)基因、或gfp(绿色荧光蛋白)基因。
本发明还提供了一种基因表达盒，所述表达盒从5’-3’依次具有下列元件：启动子、基因ORF序列、和终止子。优选地，所述启动子序列如SEQ ID NO.:1所示或与SEQ ID NO.:1所示序列的同源性≥90％，较佳地≥95％，更佳地≥98％。
本发明还提供了一种包括本发明的启动子和/或基因表达盒的重组载体。作为一种优选的方式，重组载体的启动子下游包含多克隆位点或至少一个酶切位点。当需要表达目的基因时，将目的基因连接入适合的多克隆位点或酶切位点内，从而将目的基因与启动子可操作地连接。作为另一种优选方式，所述的重组载体包括(从5’到3’方向)：启动子、目的基因、和终止子。如果需要，所述的重组载体还可以包括选自下组的元件：3’多聚核苷酸化信号；非翻译核酸序列；转运和靶向核酸序列；抗性选择标记(二氢叶酸还原酶、新霉素抗性、潮霉素抗性以及绿色荧光蛋白等)；增强子；或操作子。
本领域普通技术人员可以使用熟知的方法构建含有本发明所述的启动子和/或目的基因序列的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。
本发明的启动子、表达盒或载体，可以用于转化适当的宿主细胞，以使宿主表达蛋白质。宿主细胞可以是原核细胞，如大肠杆菌，链霉菌属、农杆菌：或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物细胞。本领域一般技术人员都清楚如何选择适当的载体和宿主细胞。用重组DNA转化宿主细胞 可用本领域技术人员熟知的常规技术进行。当宿主为原核生物(如大肠杆菌)时，可以用CaCl 2法处理，也可用电穿孔法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法(如显微注射、电穿孔、脂质体包装等)。转化植物也可使用农杆菌转化或基因枪转化等方法，例如叶盘法、幼胚转化法、花芽浸泡法等。对于转化的植物细胞、组织或器官可以用常规方法再生成植株，从而获得转基因的植物。
作为本发明的一种优选方式，制备转基因植物的方法是：将携带启动子和目的基因(两者可操作地连接)的载体转入农杆菌，农杆菌再将含启动子和目的基因的载体片段整合到植物的染色体上。涉及的转基因受体植物例如是拟南芥、小麦、大麦、燕麦、玉米、水稻、高粱、粟、大豆、花生、烟草、番茄、白菜、油菜、菠菜、生菜、黄瓜、茼蒿、空心菜、芹菜、油麦菜等。在本发明的实例中，所述的重组载体是pCAMBIA1300载体，将本发明的启动子构建到该载体中，转化植株。
在一优选实施方式中，本发明克隆了EF启动子(如番茄SlEF1a启动子)，并使用该启动子驱动Cas酶与脱氨酶的融合蛋白编码序列的表达，最终获得了一种对双子叶植物高效率单碱基替换和基因敲除的系统。
腺嘌呤脱氨酶
如本文所用，术语“腺嘌呤脱氨酶”为催化腺嘌呤水解脱氨基生成次黄嘌呤和氨的酶。将腺嘌呤A转变为次黄嘌呤I，次黄嘌呤I可与胞嘧啶配对，在DNA水平被当成鸟嘌呤(G)进行读码与复制，导致A·T配对转换为G·C配对。TadA腺嘌呤脱氨酶，来源于大肠杆菌，经过人工改造目前已获的ecTadA突变体。TadA与ecTadA的二聚体为目前常用的腺嘌呤脱氨酶。
在本发明中，适用的TadA既包含野生型的形式也包含其特定的突变形式TadA7-10，也可包含野生型的形式和突变形式的组合。TadA7-10能够以DNA作为底物进行脱氨反应。
在本发明中，核酸构建物中腺嘌呤脱氨酶编码序列可以根据适用宿主的不同，而采用宿主偏好的方式进行密码子优化。
胞嘧啶脱氨酶
如本文所用，术语“胞嘧啶脱氨酶(APOBEC)”为能够催化细胞内胞嘧啶脱氨形 成尿嘧啶的酶,将胞嘧啶C转变为尿嘧啶U，损伤DNA在重新复制过程中被聚合酶作用，尿嘧啶在DNA复制过程中会被识别成T，导致C·G配对转换为T·A配对。已发现的APOBECs家族成员有11个，包括APOBEC1(A1)、APOBEC2(A2)、APOBEC3A～H(3A、3B、3C、3D、3E、3F、3H)、APOBEC4(A4)以及活化诱导脱氨酶(activation induced cytidine deaminase,AID)。
在本发明中，适用的胞嘧啶脱氨酶既包含野生型的形式也包含其特定的突变形式(如CBE2.0、CBE2.1、CBE2.2、CBE2.3、CBE2.4)，也可包含野生型的形式和突变形式的组合。突变形式的胞嘧啶脱氨酶能够以DNA作为底物进行脱氨反应。
在本发明中，核酸构建物中胞嘧啶脱氨酶编码序列可以根据适用宿主的不同，而采用宿主偏好的方式进行密码子优化。
在本发明的一个优选的实施方式中，优选的胞嘧啶脱氨酶为CBE2.0、CBE2.1、CBE2.2、CBE2.3、CBE2.4。
CBE2.0的氨基酸序列如SEQ ID NO.:6所示，其核苷酸序列如SEQ ID NO.:7所示。
CBE2.1的氨基酸序列如SEQ ID NO.:8所示。
CBE2.2的氨基酸序列如SEQ ID NO.:9所示。
CBE2.3的氨基酸序列如SEQ ID NO.:10所示。
CBE2.4的氨基酸序列如SEQ ID NO.:11所示。
本发明的构建物
本发明提供了一种核酸构建物，用于对植物进行基因编辑，所述的核酸构建物具有5’-3’的式I结构：
P1-S1-L1-S2-S3  (I)；
式中，
P1、S1、L1、S2、S3分别为用于构成所述构建物的元件
，其定义如本发明第一方面所述；
并且，各“-”为键或核苷酸连接序列。
在一优选实施方式中，所述的核酸构建物还进一步可操作地与一个或多个式II所示的第二核酸构建物相连：
P2-Y1  (II)；
式中，P2、Y1的定义如本发明第一方面所述。
在一优选实施方式中，所述的核酸构建物还可操作地连接第一整合元件(I1)和第二整合元件(I2)。
其中，I1元件(或左侧整合元件)和I2元件(或右侧整合元件)可协同作用，从而将位于其间的元件(即从P1至Y1的核苷酸序列)整合到植物细胞的基因组中。
代表性的I1和I2是来自于农杆菌的Ti元件。当然，其他可起到类似整合作用的元件也可用于本发明。
本发明的构建物中所用的各种元件或者是本领域中已知的，或者可用本领域技术人员已知的方法制备。例如，可通过常规方法，如PCR方法、全人工化学合成法、酶切方法获得相应的元件，然后通过熟知的DNA连接技术连接在一起，就形成了本发明的构建物。
将本发明的构建物插入外源载体(尤其是适合转基因植物操作的载体)，就构成了本发明的载体。
将本发明的载体转化植物细胞从而介导本发明的载体对植物细胞染色体进行整合，并在植物体内表达，制得经基因编辑的植物细胞。
将本发明的经基因编辑的植物细胞再生为植物体，从而获得经基因编辑的植物。
将本发明构建好的上述核酸构建物，通过常规的植物重组技术(例如农杆菌转让技术)，可以导入植物细胞，从而获得携带所述核酸构建物(或带有所述核酸构建物的载体)的植物细胞，或获得基因组中整合有所述核酸构建物的植物细胞。
本发明中整合有所述核酸构建物的植物个体，在其子代可通过常规筛选或采用本领域已知的其他手段进行分离或去除，从而制得经基因编辑且不含有核酸构建物的植物体。
具体地，本发明是将一种特定的EF启动子，如番茄EF1a驱动基因编辑酶(如Cas9)与脱氨酶融合蛋白编码序列的表达，从而提高基因编辑效率。
载体构建
该载体的主要特征是将特定的EF启动子(如番茄EF1a)、脱氨酶和Cas融合蛋白的编码序列，任选地还包括核定位信号、UGI编码序列连接在一起，从而形成本发明的特定的核酸构建物。当该核酸构建物在细胞质中表达后，该核酸构建物所编码的融合蛋白可以非常高效地被转移至细胞核内，并由式II构 建物所编码的guide RNA引导至基因组中的靶点位置，从而在靶点位置进行A.T到G.C或C.G到T.A的碱基替换，并基本上避免或消除了发生插入/缺失的风险，并且可显著提高基因编辑的效率。
由于腺嘌呤脱氨基酶将A突变为G，胞嘧啶脱氨基酶将C突变为T并不需要Cas蛋白的DNA双链切割活性。因此，在本发明中Cas蛋白是无切割活性或具有单链切割活性的突变的Cas蛋白。在一优选实施方式中，本发明的Cas蛋白可以是nCas9，其氨基酸序列如SEQ ID NO.:2所示。一般的，为了增加融合蛋白的活性，蛋白间一般通过一些柔性短肽连接，即Linker(连接肽序列)。优选的，该Linker可以选用XTEN，其编码序列如SEQ ID NO.:17所示，其氨基酸序列如SEQ ID NO.:18所示。
选择适用于植物细胞的guide RNA的表达框，并将其与上述融合蛋白的开放表达框(ORF)构建在同一载体。
本发明中，载体可以是例如质粒、病毒、粘粒、噬菌体等类型，它们是本领域技术人员所熟知的，在本领域中众多描述。优选地，本发明中的表达载体是质粒。表达载体可包含启动子、翻译起始的核糖体结合位点、聚腺苷酸化位点、转录终止子、增强子等。表达载体中也可以含有一个或多个可选择标记基因以便用于选择包含载体的宿主细胞。这种可选择的标记包括编码二氢叶酸还原酶的基因，或赋予新霉素耐受性的基因，赋予对四环素或氨苄青霉素耐受性的基因等。
本发明的核酸构建物可通过多种方法插入载体中，例如通过用适当的限制性核酸内切酶消化插入物和载体后进行连接。多种克隆技术在本领域中是已知的，这些均在本领域技术人员的知识范围内。
本发明中适用的载体包括可从商业渠道获得的质粒，例如但不限于：pBR322(ATCC37017)，pCAMBIA1300，pKK223-3(Pharmacia Fine Chemicals，Uppsala，Sweden)，GEM1(Promega Biotec，Madison，WI，USA)pQE70，pQE60，pQE-9(Qiagen)，pD10，psiX174pBluescript II KS，pNH8A，pNH16a，pNH18A，pNH46A(Stratagene)，ptrc99a，pKK223-3，pKK233-3，pDR540，pRIT5(Pharmacia)，pKK232-8，pCM7，pSV2CAT，pOG44，pXT1，pSG(Stratagene)，pSVK3，pBPV，pMSG，和pSVL(Pharmacia)等。
遗传转化
在本发明中，对于将本发明的式I构建物导入细胞或整合到基因组的方法，没有特别限制。可以用常规的方法进行，例如将式I构建物或相应的载体通过合适的方法导入到植物细胞中。代表性的导入方法包括但并不限于：农杆菌转染法、基因枪法、显微注射法、电击法、超声波法、和聚乙二醇(PEG)介导法等。
在本发明中，对于受体植物没有特别限制，其中包括各种不同的农作物植物(如禾本科植物)、林业植物、园艺植物(如花卉植物)等。代表性的例子包括但不限于：水稻、大豆、番茄、玉米、烟草、小麦、高粱、马铃薯等。
上述DNA载体或片段导入植物细胞后，使转化的植物细胞中的DNA表达该融合蛋白和gRNA。融合腺嘌呤脱氨基酶和/或胞嘧啶脱氨酶的基因编辑酶(如Cas9核酸酶)在相应gRNA的引导下，将靶点位置的A突变为G(进而使得互补链的T突变为C)或将靶点位置的C突变为T(进而使得互补链的G突变为A)。
对于用本发明方法进行植物基因组定点替换后的植物细胞或组织或器官，可以用常规方法再生获得相应的经基因编辑的植株。例如，通过组织培养，再生获得碱基替换后的植株。
应用
本发明可以用于植物基因工程领域，用于植物研究和育种，尤其是具有经济价值的农作物、林业作物或园艺植物的遗传改良。
本发明的主要优点包括：
(1)本发明首次将特定的启动子(如Ef1a启动子)与基因编辑酶(如Cas9核酸酶)、腺嘌呤脱氨酶和/或胞嘧啶脱氨酶，任选地还包括核定位信号、UGI的编码序列连接在一起，从而形成本发明的特定的核酸构建物，本发明的核酸构建物在植物中成功实现了gRNA引导的碱基定点突变(如A突变为G)，并且突变效率非常高(可高达≥70％或更高)。
(2)本发明的特定的核酸构建物可以编辑一些其他启动子不起作用的基因位点，破除基因编辑受基因型限制的障碍。
(3)本发明的特定的核酸构建物可编辑一些其他启动子不起作用的植物，如大豆，有效扩大了基因编辑系统的使用范围，破除物种障碍。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。本发明中所涉及的实验材料和试剂如无特殊说明均可从市售渠道获得。
实施例1 不同启动子在番茄中的单碱基编辑效率
1、靶标选择
选择番茄中影响果实发育的Solyc05g012020作为靶标基因，选择6个靶位点设计sgRNA，所设计的6个sgRNA的序列如下：
sgRNA1：TACTGGAGTTGTACCTGGA(SEQ ID NO.:20)，
sgRNA2：GGAACAGCTTGAACGTCAAT(SEQ ID NO.:21)，
sgRNA3：GAACAGCCTTCTCATCATGA(SEQ ID NO.:22)，
sgRNA4：GGTGAGGATTTGGGACAATT(SEQ ID NO.:23)，
sgRNA5：CTGTGAATCTGATGAAGTTT(SEQ ID NO.:24)，
sgRNA6：GAAAAGTAATAACAAAGGGC(SEQ ID NO.:25)。
2、载体构建
通过同源重组技术获得ABE单碱基编辑器的表达盒(参见图1)，所述腺嘌呤脱氨酶ABE7.10的核苷酸序列如SEQ ID NO.:5或19所示，所述SlEF1a启动子的核苷酸序列如SEQ ID NO.:1所示，具体操作如下：
A)以番茄基因组DNA为模版，用正/反向引物pSlEF1a-F/pSlEF1a-R对目标片段进行扩增，获得PCR产物(长度约1583bp,引物退火温度为58)。
Figure PCTCN2021095310-appb-000001
PCR反应条件为：95℃预变性5分钟，98℃变性30秒，58℃退火30秒，72℃延伸45s，35个循环，72℃后延伸5分钟
B)用限制内切酶Sbf1和SalI酶切回收载体骨架
proAtU6-gRNA-pro35S-ABE7.10-nspCas9
C)通过同源重组将A获得PCR产物连入B获得的骨架载体中，获得单碱基编辑载体proAtU6-gRNA-proSlEF1a-ABE7.10-nspCas9
Figure PCTCN2021095310-appb-000002
PCR反应条件为：50℃30min
D)转化大肠杆菌，挑单克隆测序验证片段成功连入载体。
以同样方法构建含有35S、UBI、AtRPS5A、SlRPS5A1、SlRPS5A2、SlTCTP启动子的单碱基编辑载体。
3、遗传转化
(A)上述构建质粒直接转化农杆菌EHA105：
(1)农杆菌感受态细胞中加入质粒DNA，之后冰浴30min,放入液氮中5min，然后立即放入37℃水浴锅中水浴5min，冰上放置5min
(2)取出离心管，加入700ul YEP培养基，振荡培养2～4hr。
(3)取出菌液与含相应抗生素的YEP培养基平板上涂板，在培养箱中倒置培养,2天左右菌落可见。
(B)番茄转基因
(1)取7-10d苗龄的番茄无菌幼苗(子叶完全展开，第一真叶微露)，将子叶剪成5mm见方的叶片(切去叶片尖端和少部分基部，留取中间部分)，正面朝上放置在预培养培养基中，25℃暗培养2d。
(2)将-80℃保存的菌液在固体YEB培养基上划线，28℃暗培养2d。挑取单菌落加入5ml液体YEB培养基，28℃，200rpm，培养1d。取2ml菌液加入50ml新鲜的YEB培养基，28℃，200rpm。4℃，5000rpm离心10min，用侵染缓冲液重悬菌体，将OD600调至0.6-0.8左右。
(3)将预培养2d的子叶在菌液中侵染5-10min，在滤纸皿上吸干多余菌液后，正面朝上放置于共培养培养基(也可用不带菌的侵染液浸湿的滤纸)上， 25℃暗培养2d。
(4)将共培养2d的子叶转移至除菌培养基中，25℃培养7d，前2-3d暗培养，后4-5d光照培养。共培养7d后，将子叶转移至筛选培养基中，培养30-45d。每15d继代一次。
(5)在除菌结束后进行标记基因的检测(以GUS为例)，取数片除菌7d后的子叶进行GUS染色，根据染色面积大小调整侵染时间。(不用每批都进行，定期进行以检查菌的活性)。
(6)检测农杆菌对子叶的伤害，取除菌7d后的子叶若干，使其继续在除菌培养基中生长30d左右，每15天继代一次，观察子叶分化率，判断菌液对子叶的伤害程度，调整侵染时间。
(7)待分化出的幼菌长至约2cm时，将幼苗切下，转移至生根培养基中，培养至根长出。
(8)将分化出的健壮幼苗转移至含抗生素的生根培养基中生根培养一周，室温练苗2-3天后，温室基质栽培。
(9)基因编辑检测。取每株植物的叶片，提取基因组DNA，在gRNA的靶向位点两侧设计引物。扩增得到的片段进行Sanger测序，确定每株植物的基因型。
4、实验结果
slEF1a启动子在单碱基编辑中最高达到70％编辑效率，相比其他启动子提高2-20倍(参见图2)。
5、实验结论
slEF1a启动子可以高效驱动脱氨酶与Cas9的融合蛋白的表达，有效扩大单碱基编辑工具的适用范围，对植物性状改良、品种培育具有重要意义。
实施例2 不同启动子在大豆中的单碱基编辑效率
选择大豆中的GmELF3a和GmALS1基因，选择不同的启动子以及不同的碱基编辑器考察不同启动子在大豆中的单碱基编辑效率，所使用的gRNA如下表所示：
Figure PCTCN2021095310-appb-000003
Figure PCTCN2021095310-appb-000004
首选，按照实施例1的方式，考察SlEF1a启动子(pSlEF1a)、CaMV35S启动子(35S)以及AA6启动子(pAA6，参考文献：CN101370939A)在和ABE7.10(SEQ ID NO.:5或19)以及Cas9配合使用时的编辑效率；如图3所示，所述“A to G gRNA1”即为利用不同启动子与上述腺嘌呤脱氨酶配合使用时的结果，在大豆中，利用SlEF1a启动子所产生的编辑效率要远高于CaMV35S启动子以及AA6启动子。
另外，采用上述方式，将上述腺嘌呤脱氨酶替换为胞嘧啶脱氨酶，所述胞嘧啶脱氨酶的氨基酸序列如SEQ ID NO.:6、8-11所示，本实施例优选SEQ ID NO.:6所示的胞嘧啶脱氨酶，考察不同的启动子在与胞嘧啶脱氨酶和Cas9配合使用时的编辑效率；如图3所示，所述“C to T gRNA2”即为利用不同启动子与上述胞嘧啶脱氨酶配合使用时的结果，在大豆中，利用SlEF1a启动子所产生的编辑效率要远高于CaMV35S启动子以及AA6启动子。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (13)

  1. 一种核酸构建物，其特征在于，所述核酸构建物具有5’-3’(5’至3’)的式I结构：
    P1-S1-L1-S2-S3 (I)；
    式I中：
    P1、S1、L1、S2、S3分别为用于构成所述构建物的元件；
    P1为第一启动子序列，所述第一启动子为延伸因子启动子；
    S1、S2各自独立地为一个或多个(a)基因编辑酶的编码序列、(b)腺嘌呤脱氨酶的编码序列和/或胞嘧啶脱氨酶的编码序列；
    L1为无或连接肽的编码序列；
    S3为无或尿嘧啶糖苷酶抑制剂UGI的编码序列；
    并且，各“-”独立地为键或核苷酸连接序列。
  2. 如权利要求1所述的核酸构建物，其特征在于，所述第一启动子为EF1α启动子，优选，番茄EF1α启动子。
  3. 如权利要求1或2所述的核酸构建物，其特征在于，所述的S1为腺嘌呤脱氨酶的编码序列和/或胞嘧啶脱氨酶的编码序列，所述S2为基因编辑酶的编码序列。
  4. 一种载体，其特征在于，所述载体含有权利要求1-3任一所述的核酸构建物。
  5. 一种宿主细胞，其特征在于，所述细胞含有权利要求1-3任一所述的核酸构建物，或其基因组整合有一个或多个权利要求1-3任一所述的核酸构建物。
  6. 一种试剂组合，其特征在于，包括：
    (i)第一核酸构建物，或含有所述第一核酸构建物的第一载体，所述第一核酸构建物具有从5’-3’的式I结构：
    P1-S1-L1-S2-S3  (I)
    其中，
    P1为第一启动子序列，所述第一启动子为延伸因子启动子；
    S1、S2各自独立地为一个或多个(a)基因编辑酶的编码序列、(b)腺嘌呤脱氨酶的编码序列和/或胞嘧啶脱氨酶的编码序列；
    L1为无或连接肽的编码序列；
    S3为无或尿嘧啶糖苷酶抑制剂UGI的编码序列；
    并且，“-”为键或核苷酸连接序列；
    (ii)第二核酸构建物，或含有所述第二核酸构建物的第二载体，所述第二核酸构建物具有从5’-3’的式(II)所示的结构：
    P2-Y1  (II)；
    其中，P2为第二启动子；
    Y1为gRNA的编码序列；
    并且，“-”为键或核苷酸连接序列。
  7. 如权利要求6所述的试剂组合，其特征在于，所述第一启动子为EF1α启动子，优选，番茄EF1α启动子。
  8. 一种试剂盒，其特征在于，所述试剂盒含有权利要求6-7任一所述的试剂组合。
  9. 一种对植物进行基因编辑的方法，其特征在于，包括步骤：
    (i)提供待编辑植物；和
    (ii)将权利要求1-3任一所述的核酸构建物、权利要求4所述的载体或权利要求6或7所述的试剂组合导入所述待编辑植物的植物细胞，从而在所述植物细胞内进行基因编辑。
  10. 一种制备经基因编辑的植物细胞的方法，其特征在于，包括步骤：
    将权利要求1-3任一所述的核酸构建物、权利要求4所述的载体或权利要求6或7所述的试剂组合导入植物细胞，使得所述植物细胞中的染色体发生定点替换(或突变)，从而制得所述经基因编辑的植物细胞。
  11. 权利要求1-3任一所述的核酸构建物、权利要求4所述的载体、权利要求5所述的宿主细胞、权利要求6或7所述的试剂组合、权利要求8所述的试剂盒的用途，其特征在于，
    用于对植物进行基因编辑，
    或者，用于提高碱基编辑效率，优选，提高植物中的单碱基编辑效率。
  12. 一种制备经基因编辑的植物的方法，其特征在于，包括步骤：
    将权利要求10所述方法制备的所述经基因编辑的植物细胞再生为植物体，从而获得所述经基因编辑的植物。
  13. 一种提高植物中的碱基编辑效率的方法，其特征在于，所述方法包括利用 权利要求1-3任一所述的核酸构建物、权利要求4所述的载体、权利要求5所述的宿主细胞、权利要求6或7所述的试剂组合，或权利要求8所述的试剂盒对植物进行碱基编辑的步骤。
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