WO2020177751A1 - Construction d'acide nucléique pour édition de gènes - Google Patents

Construction d'acide nucléique pour édition de gènes Download PDF

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WO2020177751A1
WO2020177751A1 PCT/CN2020/078079 CN2020078079W WO2020177751A1 WO 2020177751 A1 WO2020177751 A1 WO 2020177751A1 CN 2020078079 W CN2020078079 W CN 2020078079W WO 2020177751 A1 WO2020177751 A1 WO 2020177751A1
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coding sequence
nucleic acid
acid construct
sequence
gene
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PCT/CN2020/078079
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Chinese (zh)
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李峰
梁亚峰
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山东舜丰生物科技有限公司
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Priority claimed from CN201910838286.3A external-priority patent/CN110526993B/zh
Priority claimed from CN201910839046.5A external-priority patent/CN110527695B/zh
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    • 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
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • 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
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/20—Brassicaceae, e.g. canola, broccoli or rucola
    • 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
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • 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
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/54—Leguminosae or Fabaceae, e.g. soybean, alfalfa or peanut
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)

Definitions

  • the present invention relates to the field of biotechnology, in particular, to a nucleic acid construct for gene editing.
  • CRISPR-Cas9 gene editing technology has been widely used in the research of gene editing in animals and plants. Currently, it mainly includes cytosine base editor (CBE) and adenine base editor (ABE).
  • CBE cytosine base editor
  • ABE adenine base editor
  • the base editor can be performed in the genome Accurate base changes (such as substitutions) without causing DNA double-strand breaks (DSB).
  • the early developed CBE and ABE systems consist of rat cytidine deaminase APOBEC1, which is derived from lamprey’s cytidine deaminase PmCDA1 and TadA, which is derived from tRNA adenine deaminase, and have been applied to many plant species.
  • Medium such as rice, wheat, corn, tomato, Arabidopsis and Brassica napus.
  • Kang et al. optimized the promoter
  • Zong et al. optimized the deaminase.
  • Cas9 protein variants can recognize PAM sequences that are different from the classic NGG motif.
  • the efficiency of single-base editing is still very low.
  • the base editors reported so far can only recognize a limited number of PAM sequences, which greatly limits the editable range of plant genomes.
  • the purpose of the present invention is to provide a new nucleic acid construct for gene editing that can efficiently achieve A-G transformation in a large range and accurately in plant cells while significantly reducing the risk of insertion or deletion mutations.
  • the first aspect of the present invention provides a nucleic acid construct having a 5'-3' (5' to 3') formula I structure:
  • I1 is the first integrated component
  • I2 is the second integrated component
  • Z1 is the first expression cassette
  • Z2 is the second expression cassette
  • one of the expression cassettes of Z1 and Z2 has the structure of Ia or Ia', and the other expression cassette has the structure of formula Ib:
  • P1, S1, X1, L1, X2, X4, L2, X3, P2, Y1 are elements used to form the construct, respectively;
  • P1 is a first promoter, and the first promoter is an RNA polymerase II dependent promoter;
  • S1 is the coding sequence of the first nuclear localization signal
  • X1 is the coding sequence of adenine deaminase (such as wild-type and/or mutant TadA) and/or the coding sequence of cytosine deaminase;
  • L1 is the coding sequence of no or first connecting peptide
  • X2 is the coding sequence of Cas9 nuclease, which has no cleavage activity or single-stranded cleavage activity;
  • X4 is the coding sequence of no or uracil glycosidase inhibitor UGI;
  • L2 is the coding sequence of no or second connecting peptide
  • X3 is the coding sequence of the second nuclear localization signal
  • P2 is the second promoter
  • Y1 is the coding sequence of gRNA
  • each "-" is a bond or a nucleotide connection sequence
  • the additional condition is that when X1 is the coding sequence of adenine deaminase, X4 is none, when X1 is the coding sequence of cytosine deaminase, and X4 is the coding sequence of the uracil glycosidase inhibitor UGI.
  • the gRNA includes crRNA, tracrRNA, and sgRNA.
  • the first promoter is derived from one or more plants selected from the group consisting of corn, rice, soybean, Arabidopsis or tomato.
  • the first promoter is derived from one or more microorganisms selected from the group consisting of Streptomyces and Escherichia coli.
  • the first promoter is derived from one or more viruses selected from the group consisting of tobacco mosaic virus, yellow leaf curl virus, cauliflower mosaic virus, and cotton leaf curl virus.
  • the first promoter includes a maize ubiquitin promoter.
  • the ubiquitin promoter includes UBI promoter.
  • the first promoter is selected from the group consisting of UBI, UBQ, 35S, Actin, SPL, CmYLCV, YAO, CDC45, rbcS, rbcL, PsGNS2, UEP1, TobRB7, Cab, or a combination thereof.
  • the length of the nucleotide sequence of L1 and L2 is independently 3-120 nt, preferably 3-96 nt, and preferably a multiple of 3.
  • the lengths of the amino acid sequences encoded by L1 and L2 are 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 Cas9 nuclease is selected from the group consisting of nCas9, Cas9NG, nCas9NG, or a combination thereof.
  • the Cas9 nuclease is selected from the group consisting of nSpCas9 (D10A), nSpCas9NG, nSaCas9, nScCas9, nSqCas9, nXCas9, or a combination thereof.
  • the Cas9 nuclease includes a mutant Cas9 nuclease.
  • the identity of the Cas9 nuclease and the mutant Cas9 nuclease is ⁇ 80%, preferably ⁇ 90%; more preferably ⁇ 95%, more preferably, ⁇ 98% Or 99%.
  • the activity of the mutant Cas9 nuclease is equivalent to or significantly better than that of the wild-type Cas9 enzyme.
  • the mutant Cas9 nuclease is passed through one or more of the wild-type Cas9 nuclease, preferably 1-15, preferably 1-10, preferably 1- 7, more preferably 2-5, amino acid substitution, deletion; and/or after 1-5, preferably 1-4, more preferably 1-3, most preferably 1-2 amino acid addition Forming.
  • the mutation site is at position D10A of Cas9 nuclease, and its amino acid sequence is shown in SEQ ID NO.:2.
  • the X2 element is mutated at one or more sites selected from the following group in the Cas9 nuclease corresponding to SEQ ID NO.: 2:
  • Threonine (T) at position 1337.
  • the arginine (R) at position 1335 is mutated to alanine (A); and/or
  • Leucine (L) at position 1111 is mutated to arginine (R); and/or
  • Threonine (T) at position 1337 was mutated to arginine (R).
  • the mutation is selected from the following group: R1335A; L1111R; D1135V; G1218R; E1219F; A1322R; T1337R.
  • amino acid sequence of the X2 element is shown in SEQ ID NO.: 3.
  • the X2 element is derived from bacteria.
  • the source of the X2 element is selected from the group consisting of Streptococcus pyogenes, Staphylococcus aureus, Streptococcus canis, or a combination thereof.
  • the coding sequence of the first connecting peptide and the coding sequence of the second connecting peptide each independently include XTEN.
  • the coding sequence of the first connecting peptide and the coding sequence of the second connecting peptide are shown in SEQ ID NO.: 4 or 7.
  • the nuclear localization signal is a codon-optimized nuclear localization signal.
  • the nuclear localization signal is a plant codon optimized nuclear localization signal.
  • the nuclear localization signal includes bpNLS.
  • the nuclear localization signal is bpNLS.
  • nucleotide sequences of the S1 element and the X3 element are shown in SEQ ID NO.: 5 or 20, respectively.
  • 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 tandem adenine deaminase, and the structure of the tandem adenine deaminase is shown in formula II:
  • Z8 is the amino acid sequence of wild-type adenine deaminase TadA
  • L8 is an optional connecting peptide sequence
  • Z9 is the amino acid sequence of the mutant adenine deaminase TadA7-10.
  • the adenine deaminase is a codon-optimized adenine deaminase.
  • the adenine deaminase is a plant codon optimized adenine deaminase.
  • the coding sequence of the adenine deaminase is selected from the following group:
  • the coding sequence of the adenine deaminase is shown in SEQ ID NO.1.
  • amino acid sequence of the adenine deaminase is shown in SEQ ID NO.: 8.
  • 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.
  • the cytosine deaminase is a codon-optimized cytosine deaminase.
  • the cytosine deaminase is a plant codon optimized cytosine deaminase.
  • the coding sequence of the cytosine deaminase is selected from the following group:
  • the coding sequence of the cytosine deaminase is shown in SEQ ID NO.19.
  • amino acid sequence of the cytosine deaminase is shown in SEQ ID NO.:21.
  • nucleotide sequence of the X4 element is shown in SEQ ID NO.: 22.
  • the second promoter is derived from one or more plants selected from the group consisting of rice, corn, soybean, Arabidopsis or tomato.
  • the second promoter is derived from one or more microorganisms selected from the group consisting of Streptomyces and Escherichia coli.
  • the second promoter is derived from one or more viruses selected from the group consisting of tobacco mosaic virus, yellow leaf curl virus, cauliflower mosaic virus, and cotton leaf curl virus.
  • 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.
  • the second promoter is selected from the group consisting of OsU6, OsU3, OsU6a, OsU6b, OsU6c, AtU6-1, AtU3b, AtU3d, AtU6-1, AtU6-26, AtU6-29, or combination.
  • the "no cleavage activity or single-strand cleavage activity" refers to the Cas9 nuclease having no cleavage activity for the single-stranded target site T.
  • the "no cleavage activity or single-strand cleavage activity" refers to the Cas9 nuclease has no cleavage activity for the single-stranded target site G.
  • nucleotide elements of the present invention are connected in-frame to express a fusion protein with the correct amino acid sequence.
  • the construct has a structure of formula IIa or IIa' or formula IIb or IIb':
  • both the first expression cassette and the second expression cassette have a terminator.
  • first expression cassette and the second expression cassette 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.
  • nucleotide sequence of the terminator is shown in SEQ ID NO.:6.
  • the first integration element includes a 5'homology arm sequence.
  • the second integration element includes a 3'homology arm sequence.
  • the length of the nucleic acid construct is 3000-10000 bp, preferably 4000-8500 bp, more preferably 4000-6000 bp.
  • one or more additional expression cassettes are additionally inserted.
  • the additional expression cassette is independent of the first expression cassette and the second expression cassette.
  • the additional expression cassette expresses a substance selected from the following group:
  • 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 fusion protein, the fusion protein includes (a) adenine deaminase and/or cytosine deaminase; and (b) Cas9 nuclease, and the fusion protein is composed of the present invention
  • the nucleic acid construct described in the first aspect encodes.
  • the third 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 fourth aspect of the present invention provides a genetically engineered 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 plants include: Arabidopsis, wheat, barley, oats, corn, rice, sorghum, millet, soybean, peanut, tobacco, tomato, cabbage, rape, spinach, lettuce, cucumber, chrysanthemum , Water spinach, celery, lettuce, or combinations thereof.
  • the genetically engineered cell is introduced into the cell with the nucleic acid construct of claim 1 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) mediation 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) mediation method.
  • the fifth aspect of the present invention provides a reagent combination for gene editing, including:
  • P1 is a first promoter, and the first promoter is an RNA polymerase II dependent promoter;
  • S1 is the coding sequence of the first nuclear localization signal
  • X1 is the coding sequence of adenine deaminase (such as wild-type and/or mutant TadA) and/or the coding sequence of cytosine deaminase;
  • L1 is the coding sequence of no or first connecting peptide
  • X2 is the coding sequence of Cas9 nuclease, said Cas9 nuclease has no cleavage activity or single-stranded cleavage activity;
  • X4 is the coding sequence of no or uracil glycosidase inhibitor UGI;
  • L2 is the coding sequence of no or second connecting peptide
  • X3 is the coding sequence of the nuclear localization signal
  • the additional condition is that when X1 is the coding sequence of adenine deaminase, X4 is none, when X1 is the coding sequence of cytosine deaminase, and X4 is the coding sequence of the uracil glycosidase inhibitor UGI; and
  • 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 sixth aspect of the present invention provides a kit containing the reagent combination according to the fifth aspect of the present invention.
  • the kit further contains a label or instructions.
  • the seventh 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 third aspect of the present invention, or the reagent combination according to the fifth 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 introduction.
  • 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 monocots, dicots and gymnosperms.
  • the plant is selected from the group consisting of gramineous plants, legumes, cruciferous plants, Solanaceae, Umbelliferae, or a combination thereof.
  • the plants include: Arabidopsis, wheat, barley, oats, corn, rice, sorghum, millet, soybean, peanut, tobacco, tomato, cabbage, rape, spinach, lettuce, cucumber, chrysanthemum , Water spinach, celery, lettuce, or combinations thereof.
  • the eighth aspect of the present invention provides a method for preparing gene-edited plant cells, including the steps:
  • nucleic acid construct of the first aspect of the present invention, the vector of the third aspect of the present invention, or the combination of the reagent of the fifth aspect of the present invention are transfected into plant cells, so that the chromosomes in the plant cells undergo site-directed replacement ( Or mutation), thereby preparing the gene-edited plant cell.
  • the transfection adopts the Agrobacterium transformation method or the gene gun bombardment method.
  • the ninth aspect of the present invention provides a nucleic acid construct according to the first aspect of the present invention, a fusion protein according to the second aspect of the present invention, a vector according to the third aspect of the present invention, and a gene according to the fourth aspect of the present invention
  • the engineered cell, the reagent combination according to the fifth aspect of the present invention, and the use of the kit according to the sixth aspect of the present invention are used for gene editing of plants.
  • the tenth 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 eighth aspect of the present invention or the ninth aspect of the present invention is regenerated into a plant body, thereby obtaining the gene-edited plant.
  • the eleventh aspect of the present invention provides a gene-edited plant prepared by the method of the tenth aspect of the present invention.
  • the twelfth aspect of the present invention provides a composite comprising the following two components:
  • nucleic acid component the nucleic acid is gRNA
  • Figure 1 shows the editing efficiency of ABE-nCas9 and ABEmax-nCas9 on ALS genes.
  • Figure 2 shows the editing efficiency of ABE-nCas9, ABEmax-nCas9 and ABEmax-nCas9NG on ALS genes at different PAM sites.
  • Figure 3 shows the base editing efficiency of CBE2.0-nCas9 for replacing the target site C with T in the rice NRT1.1B gene.
  • the abscissa represents the sgRNA-PAM sequence, and the ordinate represents the substitution efficiency.
  • Figure 4 shows the ratio of homozygous to other non-homozygous in rice NRT1.1B gene mutant plants.
  • FIGS 5 and 6 show the difference in traits between rice SLR1 gene mutant plants and wild-type plants.
  • Figure 7 shows the base editing efficiency of CBE2.0-nCas9 for replacing the target site C of the rice SLR1 gene with T.
  • the abscissa represents the sgRNA-PAM sequence, and the ordinate represents the substitution efficiency.
  • Figure 8 shows the ratio of homozygous to other non-homozygous mutation types in rice SLR1 gene mutants.
  • Figure 9 shows the base editing efficiency of CBE2.0-nCas9 for replacing the target site C with T in the rice ALS gene.
  • Figure 10 shows the ratio of homozygous to other non-homozygous plants in mutant plants of the rice ALS gene.
  • Figure 11 shows the base editing efficiency of CBE2.0-nCas9NG for replacing the target site C with T in rice ALS gene.
  • Figure 12 shows the difference in the growth phenotype of rice ALS gene mutant plants and wild-type plants sprayed with imidazolium herbicide.
  • Figure 13 shows the mutation sites of rice ALS gene mutant plants relative to wild-type plants.
  • Figure 14 shows the mutation sites of the rice EPSPS gene.
  • Figure 15 shows the editing efficiency of CBE2.0-nCas9 and CBE2.0-nCas9NG for different target genes.
  • adenine base editor elements and/or cytosine base editor elements After extensive and in-depth research, the inventors have optimized the quality and quantity of adenine base editor elements and/or cytosine base editor elements, using binuclear localization signals, optimized adenine deaminase and/or cytosine Pyrimidine deaminase and different Cas9 nucleases construct an adenine and/or cytosine base editing tool that is more efficient and has a wider recognition range in plant gene editing.
  • the present invention successfully implements sgRNA in plants for the first time Guided base site-directed mutation (such as A mutation to G or C mutation to T), and the mutation efficiency of the adenine base editor element is very high (up to ⁇ 40% or higher), which can identify more PAM positions At the same time, the indel ratio is very low.
  • the inventors have also improved the mutation efficiency significantly (up to ⁇ 80% or higher) by optimizing the cytosine deaminase. By optimizing the Cas9 protein, it can recognize more Many PAM sites (including NGG, NG), and the indel ratio is very low, ⁇ 7%, which improves the accuracy of editing.
  • the present invention has been completed on this basis.
  • homologous arm refers to the flanking sequences that are identical to the genome 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 nucleic acid in plant cells.
  • 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).
  • Cas protein refers to a nuclease.
  • a preferred Cas protein is the Cas9 protein.
  • Typical Cas9 proteins include (but are not limited to): Cas9 derived from Streptococcus pyogenes.
  • the Cas9 protein is a mutant Cas9 protein, specifically, a mutant Cas9 protein that has no cleavage activity or only a single-stranded cleavage activity.
  • the Cas9 protein of the present invention includes SpCas9n (D10A), nSpCas9NG, SaCas9n, ScCas9n, XCas9n.
  • the term "coding sequence of Cas protein” refers to a nucleotide sequence encoding Cas protein.
  • the skilled person will realize that because of the degeneracy of the codon, 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 encoding 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.
  • nucleotide sequence is from 5'to 3', unless otherwise specified.
  • adenine deaminase refers to TadA adenine deaminase, derived from Escherichia coli, originally acting on tRNA and capable of deaminating specific adenines in tRNA.
  • the applicable TadA includes both the wild-type form and its 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 the adenine deaminase of the present invention is codon-optimized, so that it can be expressed in plants more efficiently.
  • cytosine deaminase refers to the cytosine deaminase APOBEC, derived from Escherichia coli, originally acting on tRNA, capable of deaminating specific cytosines in tRNA.
  • 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 reaction with DNA as a substrate.
  • the coding sequence of the cytosine deaminase of the present invention is codon-optimized, so that it can be expressed in plants more efficiently.
  • the preferred cytosine deaminase is CBE2.0.
  • amino acid sequence of CBE2.0 is shown in SEQ ID NO.: 23.
  • the amino acid sequence of CBE2.1 is shown in SEQ ID NO.: 24.
  • the amino acid sequence of CBE2.2 is shown in SEQ ID NO.: 25.
  • amino acid sequence of CBE2.3 is shown in SEQ ID NO.: 26.
  • the amino acid sequence of CBE2.4 is shown in SEQ ID NO.: 27.
  • the present invention provides a nucleic acid construct for gene editing of plants, the nucleic acid construct has a 5'-3' formula I structure:
  • I1 is the first integrated component
  • I2 is the second integrated component
  • Z1 is the first expression cassette
  • Z2 is the second expression cassette
  • one of the expression cassettes of Z1 and Z2 has the structure of Ia or Ia', and the other expression cassette has the structure of formula Ib:
  • I1, P1, S1, X1, L1, X2, X4, L2, X3, P2, Y1, I2 are elements used to form the construct, respectively, and the definitions are as described in the first aspect of the present invention
  • each "-" is a bond or a nucleotide connection sequence
  • the additional condition is that when X1 is the coding sequence of adenine deaminase, X4 is none, when X1 is the coding sequence of cytosine deaminase, and X4 is the coding sequence of the uracil glycosidase inhibitor UGI.
  • 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 (that is, the nucleotide sequence from P1 to Y1) into In the genome of plant cells.
  • 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 construct 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 methods, fully artificial chemical synthesis methods, and enzyme digestion methods, and then connected together by well-known DNA ligation techniques 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 chromosomes of plant cells and express in the plant body to prepare gene-edited plant cells.
  • 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 the vector carrying the nucleic acid construct) Or obtain a plant cell with the nucleic acid construct 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 routine screening or other means known in the art, thereby obtaining a gene-edited plant without the nucleic acid construct body.
  • the present invention is to construct an optimized adenine deaminase expression cassette and/or cytosine deaminase expression cassette in the CRISPR/Cas9 system of plants.
  • Adenine deaminase can convert the target DNA Adenine (A) is deaminated and converted to inosine (I). Inosine can be paired with cytosine, and it is read and copied as guanine (G) at the DNA level to realize A to G mutation within the mutation window;
  • Cytosine deaminase can deaminate the cytosine (C) in the target DNA into uracil (U), and uracil will be recognized as T during the DNA replication process, realizing the mutation from C to T.
  • the basic structure of the nucleic acid construct of the adenine base editor element is as follows:
  • the deaminase expression cassette is ZmUbi-ecTadA-32aa linker-ecTadA(7.10)-32aa linker-nSpCas9/nSaCas9-SV40NLS-NOS
  • the basic structure of the nucleic acid construct of the cytosine base editor element is as follows:
  • the deaminase expression cassette is ZmUbi-rAPOBEC1-XTEN-nCas9-UGI-SV40NLS-NOS
  • the main feature of the vector is to link adenine deaminase and/or cytosine deaminase with the Cas protein in the CRISPR/Cas system and the coding sequence of the nuclear localization signal bpNLS to form the coding sequence of the fusion protein.
  • the fusion protein encoded by the coding sequence is expressed in the cytoplasm, the fusion protein can be transferred to the nucleus very efficiently, and guided to the target site in the genome by the guide RNA encoded by the formula I construct, Therefore, the base substitution from AT to GC or CG to TA is performed at the target site, and the risk of insertion/deletion is basically avoided or eliminated.
  • the Cas protein is a mutant Cas protein with no cleavage activity or a single-strand cleavage activity.
  • the Cas protein of the present invention may be Cas9 (D10A), and its amino acid sequence is shown in SEQ ID NO.:2.
  • the Cas protein of the present invention may be nCas9NG, and its amino acid sequence is shown in SEQ ID NO.:3.
  • the proteins are usually connected by some flexible short peptides, namely Linker (connecting peptide sequence).
  • Linker connecting peptide sequence
  • the Linker can use XTEN, and its coding sequence is shown in SEQ ID NO.:4.
  • suitable promoters include constitutive and/or inducible promoters.
  • a strong promoter suitable for plant cells can be selected. Representative examples include (but not limited to): CaMV 35S promoter or UBI promoter or Actin promoter.
  • the action area of the deaminase is fixed.
  • adenine deaminase TadA or mutant adenine deaminase TadA7-10 protein for example, (a) adenine deaminase TadA or mutant adenine deaminase TadA7-10 protein; and/or (b) cytosine deaminase APOBEC or mutant cytosine deaminase (CBE2. 0)
  • the experimental results obtained by the present invention show that the editing windows of the various Cas proteins of the present invention have little difference, and they are all within the first 20 bases of the PAM site Within, the preferred hot spot area is in the range of 3-10 bases.
  • 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 conventional methods, for example, the construct of formula I or the corresponding vector is introduced into plant cells 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 Cas protein fused with adenine deaminase under the guidance of the corresponding gRNA, mutates the A at the target position to G (thereby mutates the T of the complementary strand to C) or mutates the C at the target position to T (and thus makes the complementary
  • the G mutation of the chain is A).
  • codon optimization is performed on the coding sequence of adenine deaminase and/or cytosine deaminase, especially plant codon optimization.
  • codon optimization There are 64 genetic codes, but most of them tend to use some of these codons. Those most frequently used are called preferred codons or optimal codons, and those that are not frequently used are called rare or low-utilized codons. In fact, every organism that uses protein expression or production shows a certain degree of difference or preference in codon usage. Use preferred codons and avoid inefficient or rare codons for gene synthesis. This kind of gene resetting is called codon optimization.
  • codon optimization is performed on the amino acid sequence of adenine deaminase and/or cytosine deaminase and nuclear localization signal, and the codons preferred by eukaryotic cells are used for optimization.
  • the preferred codons of animal cells are used.
  • the codons are optimized, and more preferably, the codons preferred by plant cells are used for optimization.
  • 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 Cas9 nuclease (such as nCas9 or nCas9NG) with optimized adenine deaminase and binuclear localization signal to form a fusion protein for the first time, and successfully realizes sgRNA-guided base-directed mutation (such as A mutation to G), and the mutation efficiency is very high (up to ⁇ 40% or higher), while significantly reducing or substantially eliminating the risk of insertion and/or deletion (indel) at the target site.
  • Cas9 nuclease such as nCas9 or nCas9NG
  • adenine deaminase and binuclear localization signal to form a fusion protein for the first time
  • sgRNA-guided base-directed mutation such as A mutation to G
  • the present invention combines Cas9 nuclease (such as nCas9 or nCas9NG) with optimized cytosine deaminase, UGI, and binuclear localization signal to form a fusion protein for the first time, and successfully realizes sgRNA-guided base-directed mutation (such as C
  • the mutation is T
  • the mutation efficiency is very high (up to ⁇ 80% or higher), while significantly reducing or substantially eliminating the risk of insertion and/or deletion (indel) at the target site, and can reduce the indel ratio It is ⁇ 7%.
  • the present invention can expand the range of targeted editing in the plant genome by using different forms of Cas9.
  • the present invention finds for the first time that the nCas9NG of the present invention can recognize non-NGG (such as NG) PAM, and can obtain very efficient base editing efficiency.
  • non-NGG such as NG
  • ALS-sg3 CGCATTCAAGGACATGATCCTGG (SEQ ID NO.: 9)
  • ALS-sg1 GCGCCCCCACTTGGGATCATAGG (SEQ ID NO.: 10)
  • the nucleic acid construct (pCambia1300) containing the base editor and hygromycin resistance gene was introduced into Agrobacterium EHA105, and then transformed into rice callus.
  • the transformation, tissue culture, and plant growth of rice were performed according to the procedures recorded in the literature. (Nishimura et al., 2006; Wang et al., 2015).
  • the efficiency of ABE-nCas9 to generate A to G mutations at the target site is about 20%, while the efficiency of ABEmax-nCas9 is about 40%-50%.
  • the specific results are shown in Figure 1.
  • the editing efficiency of ABEmax-nCas9 is as high as 40%, which is double the editing efficiency of ABE-nCas9.
  • the results show that the optimized ABEmax-nCas9 base editor can be efficiently applied to site-specific base replacement in plant genomes.
  • Adopt Mut MultiS multiple site-directed mutagenesis kit obtained nSpCas9-NG on the basis of nSpCas9 (D10A), which contains 7 amino acid substitutions R1335A/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R.
  • the mutated sequence replaced the nSpCas9(D10A) fragment in the ABEmax-nCas9 editor with BamHI and SpeI restriction enzyme sites to obtain the ABEmax-nCas9NG editor.
  • Example 3 The base editing effect of CBE2.0-nCas9 on rice NRT1.1B and SLR1 genes
  • NRT1.1B and SLR1 genes were selected, and the previously tested sgRNA was used for editing.
  • NRT1.1B controls the absorption of nitrogen as a nutrient element in rice.
  • the change of amino acid at position 327 from Thr to Met can increase yield;
  • SLR1 controls the synthesis of gibberellin and affects rice plant height.
  • OsU6-sgRNA NRT1.1B The sequence of OsU6-sgRNA (NRT1.1B) is shown in SEQ ID NO.: 28.
  • OsU6-sgRNA SLR1
  • SLR1 The sequence of OsU6-sgRNA (SLR1) is shown in SEQ ID NO.: 29.
  • the nucleic acid construct (pCambia1300) containing the base editor and hygromycin resistance gene was introduced into Agrobacterium EHA105, and then transformed into rice callus.
  • the transformation, tissue culture, and plant growth of rice were performed according to the procedures recorded in the literature. (Nishimura et al., 2006; Wang et al., 2015).
  • the base replacement efficiency of the first generation of CBE base editor at the above two sites is only 2.7% (NRT1.1B) and 13.3% (SLR1) (Lu and Zhu, 2017), compared with the present invention
  • the efficiency of the optimized CBE base editor has been increased by 26 times and 6 times respectively.
  • Example 4 The base editing effect of CBE2.0-nCas9 on rice acetolactate synthase gene (ALS)
  • Acetolactate synthase is a key enzyme for the synthesis of valine, leucine and isoleucine in plants.
  • ALS inhibitors are often used as herbicides. However, in addition to inhibiting the growth of weeds, these herbicides also Can inhibit the growth of crops. Studies have shown that the mutation of Ser at position 627 of the protein sequence of ALS to Asn (corresponding to the mutation of G at position 1880 in the DNA sequence to A) confers tolerance to imidazolinone herbicides (Piao et al., 2018) ).
  • Example 5 The base editing effect of CBE2.0-nCas9NG on acetolactate synthase gene (ALS) and 5-enolpyruvylshikimate-3-phosphate synthase gene (EPSPS) in rice
  • ALS acetolactate synthase gene
  • EPSPS 5-enolpyruvylshikimate-3-phosphate synthase gene
  • Adopt Mut MultiS multiple site-directed mutagenesis kit purchased from Nanjing Novozan obtained nSpCas9-NG on the basis of nSpCas9 (D10A), which contains 7 amino acid substitutions R1335A/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R.
  • the mutated sequence replaced the nSpCas9(D10A) fragment in the CBE2.0-nCas9 editor with BamHI and SpeI restriction enzyme sites to obtain the CBE2.0-nCas9NG editor.
  • CBE2.0-nCas9NG base editor can recognize the PAM motif of NGN.
  • the sgRNA sequence for ALS has been redesigned (ALSsg2, with AGC as the PAM motif: CCCCACTTGGGATCATAGGCAGC (SEQ ID NO.: 44))
  • ALSsg2 with AGC as the PAM motif: CCCCACTTGGGATCATAGGCAGC (SEQ ID NO.: 44)

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Abstract

L'invention concerne une construction d'acide nucléique pour l'édition de gènes, la construction d'acide nucléique ayant une structure spécifique pouvant permettre la mutagenèse dirigée sur l'ARNg et dirigée sur le site de bases dans des plantes.
PCT/CN2020/078079 2019-03-06 2020-03-05 Construction d'acide nucléique pour édition de gènes WO2020177751A1 (fr)

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