WO2018098935A1 - Vecteur pour substitution de base dirigée du génome d'une plante - Google Patents

Vecteur pour substitution de base dirigée du génome d'une plante Download PDF

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WO2018098935A1
WO2018098935A1 PCT/CN2017/077133 CN2017077133W WO2018098935A1 WO 2018098935 A1 WO2018098935 A1 WO 2018098935A1 CN 2017077133 W CN2017077133 W CN 2017077133W WO 2018098935 A1 WO2018098935 A1 WO 2018098935A1
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plant
expression cassette
vector
construct
cell
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PCT/CN2017/077133
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朱健康
陆钰明
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中国科学院上海生命科学研究院
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    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • 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)
    • 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

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a vector for site-based base substitution of a plant genome and a method for site-based base substitution of a plant genome.
  • Zinc finger nuclease Zinc finger nuclease
  • TAL transcription activator-like effector nucleases
  • CRISPR/Cas9 technologies are several breakthroughs in the field of genome editing in recent years.
  • Sexual techniques 1-3 All three techniques can generate double-strand breaks in a specific site-specific cleavage DNA in an organism's genome, thereby performing site-directed editing using non-homologous end joining or homologous recombination of the organism.
  • the ZFN and Talen technologies are guided by specific proteins, the construction of protein components is relatively complicated, and the editing efficiency needs to be improved.
  • the CRISPR/Cas9 technology is guided by RNA, which is simple to construct in vitro and has high editing efficiency.
  • three techniques have been successfully applied to a variety of organisms, including E. coli, yeast, rice, Arabidopsis, soybean, corn, and other four mice and human cells.
  • the object of the present invention is to provide an efficient fixed-point base replacement tool suitable for plant cells. And stable plants can be obtained, which can be effectively applied to plant research and breeding.
  • a first aspect of the invention provides a nucleic acid construct for genetically editing a plant, the nucleic acid construct comprising:
  • the first expression cassette is a gRNA expression cassette for expressing gRNA
  • the second expression cassette is a fusion protein expression cassette for expression of a fusion protein having the structure of Formula Ia or Ib:
  • A is a cytosine deaminase
  • L1 is no or the first linker peptide (such as Xten)
  • B is Cas9 without cleavage activity or Cas9 having only single-strand cleavage activity
  • L2 is a no or second linker peptide (such as Xten);
  • U Uracil DNA glycosylase inhibitor
  • the third expression cassette is a selection marker expression cassette for expressing a selection marker
  • first expression cassette, the second expression cassette, and the third expression cassette are each independently located on the same or different carriers.
  • the first expression cassette has the structure of formula A:
  • X1 is a plant promoter, preferably a plant polymerase III driven promoter (such as U6, U3 promoter);
  • X2 is the coding sequence of gRNA
  • X3 is polyT.
  • the second expression cassette has the structure of formula B:
  • Y1 is a plant promoter, preferably UBI, CaMV35S, Actin promoter;
  • Y2 is the coding sequence of a fusion protein having the structure of formula Ia or Ib as described above;
  • Y3 is a terminator
  • the third expression cassette has the structure of formula C:
  • Z1 is a plant promoter, preferably a 35S, UBI, Actin promoter;
  • Z2 is a coding sequence encoding a screening marker
  • Z3 is the terminator.
  • the selection marker is selected from the group consisting of a hygromycin resistance gene (HYG), a G418 and kanamycin resistance gene (NPTII), and a Basta resistance gene (BAR). , puromycin resistance gene (PAC), neomycin resistance gene (NEO), or a combination thereof.
  • HAG hygromycin resistance gene
  • NPTII G418 and kanamycin resistance gene
  • BAR Basta resistance gene
  • PAC puromycin resistance gene
  • NEO neomycin resistance gene
  • first expression cassette and the second expression cassette are located on the same carrier.
  • first expression cassette, the second expression cassette, and the third expression cassette are located on the same carrier.
  • the second expression cassette and the third expression cassette are located on the same carrier.
  • the vector is an expression vector that can be transfected or transformed into a plant cell.
  • the carrier is an Agrobacterium Ti carrier.
  • the two or three expression cassettes are arranged in clusters. Thereby an expression cassette is constructed.
  • the RB sequence is contained outside the 5' end of the expression cassette, and the LB sequence is contained outside the 3' end of the expression cassette.
  • the order of arrangement of each expression cassette is selected from the group consisting of:
  • first expression cassette and the second expression cassette are located on different vectors.
  • the RB sequence is contained outside the 5' end of the expression cassette; and the LB sequence is contained outside the 3' end of the expression cassette.
  • a second aspect of the invention provides an expression vector comprising the nucleic acid construct of the first aspect of the invention, wherein the first expression cassette, the second expression cassette, and the third expression cassette They are each independently located on the same or different carriers.
  • the expression vector backbone is pCamiba1300.
  • the vector is an expression vector that can be transfected or transformed into a plant cell.
  • the carrier is an Agrobacterium Ti carrier.
  • the carrier is cyclic or linear.
  • a third aspect of the invention provides a host cell comprising the expression vector of the second aspect of the invention.
  • the host cell is Agrobacterium.
  • the host cell is a plant cell.
  • a fourth aspect of the invention provides a genetically engineered cell comprising the construct of the first aspect of the invention, or a genome thereof integrated with one or more of the constructs of the first aspect of the invention.
  • the cell is a plant cell.
  • the plant is selected from the group consisting of a gramineous plant, a leguminous plant, a cruciferous plant, or a combination thereof.
  • the plant is selected from the group consisting of rice, soybean, tomato, corn, tobacco, wheat, sorghum, Arabidopsis thaliana, barley, oats, millet, peanuts, or combinations thereof.
  • the genetically engineered cell is introduced into the cell by the method according to the first aspect of the invention by a method selected from the group consisting of Agrobacterium transformation, gene gun, microinjection, and electric shock. Method, ultrasonic method, polyethylene glycol (PEG) mediated method, or a combination thereof.
  • a fifth aspect of the invention provides a method for genetically editing a plant, comprising the steps of:
  • the introduction is introduced by Agrobacterium.
  • the introduction is by a gene gun.
  • the gene is edited as a fixed-point base substitution.
  • the site-directed mutagenesis comprises mutating C to T or C to G
  • the site-directed mutagenesis comprises site-directed mutagenesis of C in positions 4-8 of the distal end of the PAM.
  • a sixth aspect of the invention provides a method of preparing a transgenic plant cell, comprising the steps of:
  • the transfection is performed using an Agrobacterium transformation method or a gene gun bombardment method.
  • a seventh aspect of the invention provides a method of preparing a transgenic plant cell, comprising the steps of:
  • An eighth aspect of the invention provides a method of preparing a transgenic plant, comprising the steps of:
  • the transgenic plant cell prepared by the method of the sixth aspect of the invention or the method of the seventh aspect of the invention is regenerated into a plant body, thereby obtaining the transgenic plant.
  • a ninth aspect of the invention provides a transgenic plant, which is prepared by the method of the eighth aspect of the invention, or the plant cell of the plant comprises the construct of the first aspect of the invention or The genome is integrated with one or more of the constructs of the first aspect of the invention.
  • Figure 1 is a schematic diagram of site-based base substitution based on cytosine deaminase.
  • the DNA sequence expressing Cas is ligated to a DNA sequence expressing cytosine deaminase, transformed into a plant receptor, and the DNA in the transformed plant cell expresses the fusion protein and guide RNA.
  • Figure 2 uses a fixed-base replacement vector of APOBEC1 and CRISPR/Cas9.
  • A Schematic representation of the vector pCSGAPO1.
  • B Schematic diagram of the deamination region.
  • FIG. 1 Results of fixed-base substitution of rice callus.
  • A Schematic diagram of base substitution of rice NRT1.1B gene.
  • B Schematic diagram of base substitution of rice SLR1 gene.
  • C High-throughput sequencing results.
  • FIG. 1 Base substitution results of the NRT1.1B gene in rice transgenic plants.
  • A Sanger sequencing results of Indel plants (#35) and base replacement plants (#15).
  • B Single colony sequencing results of PCR products of base replacement plants (#15).
  • Figure 5 shows the results of base substitution of the SLR1 gene in rice transgenic plants.
  • A Schematic and results of StyI digestion assay.
  • B Sanger sequencing results of Indel plants (#07) and base substitution plants (#07, #10, #18, #23, #28, and #37).
  • C Single colony sequencing results of base replacement plants (#07).
  • D T0 generation plant phenotype of wild type (WT), knockout (SLR#09) and base substitution (SLR#07) of SLR1 gene.
  • the present inventors have extensively and intensively studied to screen out optimized promoters for specific sites by extensive screening, and by using a nucleic acid construct of a specific structure, the present invention successfully achieves RNA-directed bases in plants for the first time.
  • Site-directed mutagenesis (such as C mutation to T or G), and mutation efficiency can be as high as 11.5%, and up to 60% of cells in a mutant plant have been replaced.
  • the present invention finds for the first time that cytosine deaminase can cleave the cytosine on the target genome of a plant cell under the guidance of CRISPR/Cas9, mutate it to T or G, and complete on this basis.
  • the invention has been made.
  • base substitution base mutation
  • base mutation are used interchangeably and refer to the mutation of a base to another base, such as a C mutation to T or a C mutation to G.
  • screening marker gene refers to a gene used for screening a transgenic cell or a transgenic animal in a transgenic process
  • the screening marker gene useful in the present application is not particularly limited, and includes various screening marker genes commonly used in the transgenic field, representative examples. Including (but not limited to): hygromycin resistance gene (Hyg), kanamycin resistance gene (NPTII), neomycin gene, puromycin resistance gene, hygromycin resistance gene (HYG , G418 and kanamycin resistance gene (NPTII), Basta resistance gene (BAR), puromycin resistance gene (PAC), and/or neomycin resistance gene (NEO).
  • Hyg hygromycin resistance gene
  • NPTII kanamycin resistance gene
  • NPTII kanamycin resistance gene
  • NPTII puromycin resistance gene
  • BAR Basta resistance gene
  • PAC puromycin resistance gene
  • NEO neomycin resistance gene
  • the term "expression cassette” refers to a stretch of polynucleotide sequences comprising a gene to be expressed and a sequence component that expresses the desired element.
  • the term “screening marker expression cassette” refers to a polynucleotide sequence comprising a sequence encoding a selection marker and a sequence component expressing the desired element.
  • the components required for expression include a promoter and a polyadenylation signal sequence.
  • the selection marker expression cassette may or may not contain other sequences including, but not limited to, enhancers, secretion signal peptide sequences, and the like.
  • the promoter suitable for the exogenous gene expression cassette and the selection marker gene expression cassette may be any of the common promoters, and it may be a constitutive promoter or an inducible promoter.
  • the promoter is a constitutive promoter, such as a UBI promoter, and other plant promoters suitable for eukaryotic expression.
  • plant promoter refers to a nucleic acid sequence capable of initiating transcription of a nucleic acid in a plant cell.
  • the plant promoter may be derived from a plant, a microorganism (such as a bacterium, a virus) or an animal, or a synthetic or engineered promoter.
  • plant terminator refers to a terminator capable of stopping transcription in a plant cell.
  • the plant transcription terminator may be derived from a plant, a microorganism (such as a bacterium, a virus) or an animal, or a synthetic or engineered terminator. Representative examples include (but are not limited to): Nos terminator.
  • 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 mutated Cas9 protein, specifically, a mutant Cas9 protein having no cleavage activity or only single-strand cleavage activity.
  • the term "coding sequence of a Cas protein” refers to a nucleotide sequence that encodes a Cas protein.
  • the skilled artisan will recognize that because of the degeneracy of the codon, a large number of polynucleotide sequences can encode the same polypeptide. .
  • the skilled person will also recognize that different species have a certain preference for codons, and may optimize the codons of the Cas protein according to the needs of expression in different species. These variants are all referred to by the term "Cas protein.
  • the coding sequence is specifically covered.
  • the term specifically encompasses a full-length sequence substantially identical to the Cas gene sequence, as well as a sequence encoding a protein that retains the function of the Cas protein.
  • plant includes whole plants, plant organs (such as leaves, stems, roots, etc.), seeds and plant cells, and progeny thereof.
  • the kind of plant which can be used in the method of the present invention is not particularly limited, and generally includes any higher plant type which can be subjected to transformation techniques, including monocots, dicots, and gymnosperms.
  • nucleic acid construct for gene editing of a plant comprising:
  • the first expression cassette is a gRNA expression cassette for expressing gRNA
  • the second expression cassette is a fusion protein expression cassette for expression of a fusion protein having the structure of Formula Ia or Ib:
  • A is a cytosine deaminase
  • L1 is no or the first linker peptide (such as Xten)
  • B is Cas9 without cleavage activity or Cas9 having only single-strand cleavage activity
  • L2 is a no or second linker peptide (such as Xten);
  • U Uracil DNA glycosylase inhibitor
  • the third expression cassette is a selection marker expression cassette for expressing a selection marker
  • first expression cassette, the second expression cassette, and the third expression cassette are each independently located on the same or different carriers.
  • the construction of the present invention is carried out by inserting the construct of the present invention into an exogenous vector, especially a vector suitable for transgenic plant manipulation.
  • the transgenic plant cells are prepared by transforming the vector of the present invention into plant cells to mediate the integration of the plant cell chromosomes by the vector of the present invention.
  • transgenic plant cells of the present invention are regenerated into plant bodies to obtain transgenic plants.
  • the above nucleic acid construct constructed by the present invention can be introduced into a plant cell by a conventional plant recombination technique (for example, Agrobacterium transfer technology) to obtain a nucleic acid construct (or a vector carrying the nucleic acid construct). Plant cells, or plant cells in the genome in which the nucleic acid construct is integrated.
  • a conventional plant recombination technique for example, Agrobacterium transfer technology
  • the main feature of this vector is the linkage of cytosine deaminase to the Cas protein in the CRISPR/Cas system to form a fusion protein, which is directed by the guide RNA to a target site in the genome. Since cytosine deaminase directly deacetylates cytosine C to form U, DNA double-strand cleavage activity of Cas protein is not required. Therefore, in the present invention, the Cas protein is a mutant Cas protein having no cleavage activity. After deamination of cytosine C to form U, plant cells initiate base mismatch repair and repair U-G to T-A or C-G.
  • the DNA single-strand nicking activity of the Cas protein can be retained, that is, the single-strand nicking is caused in the non-edited single strand (editing the matching strand of the target C). Therefore, preferably, the cytosine deaminase is fused to a Cas protein having single-strand nicking activity to form a fusion protein.
  • the proteins are usually linked by some flexible short peptides, namely Linker.
  • the Linker can use XTEN.
  • a strong promoter suitable for plant cells such as the CaMV 35S promoter or the UBI promoter, is generally selected.
  • the expression cassette of the guide RNA suitable for plant cells is selected and constructed in the same vector as the fusion protein expression cassette described above. Obviously, these two expression cassettes can also be on different DNA molecules.
  • the region of action of the deaminase is generally fixed.
  • the deamination region is the 4-8 base region of the guilde RNA, which is Amino window area" ( Figure 2).
  • the cytosine to be edited is in the "deamination window" when designing the target.
  • the above vector is introduced into a plant recipient by a suitable method.
  • the introduction methods include, but are not limited to, gene gun method, microinjection method, electric shock method, ultrasonic method, and polyethylene glycol (PEG)-mediated method.
  • Receptor plants include, but are not limited to, rice, soybean, tomato, corn, tobacco, wheat, sorghum, and the like.
  • the DNA in the transformed plant cell expresses the fusion protein and the guide RNA.
  • the Cas protein fused with cytosine deaminase, under the guidance of its guide RNA, deamination of cytosine C at the target site to uracil U, and then repairs U to T or G under the action of the plant cell repair system. Further, the plants after base substitution are obtained by tissue culture.
  • the invention can be used in the field of plant genetic engineering for plant research and breeding, especially genetic improvement of economically valuable crops and forestry crops.
  • the present invention provides, for the first time, an efficient method for realizing site-directed base mutations in plants, which can be widely used for plant research and breeding.
  • the method of site-directed base mutation of the present invention can efficiently perform base mutation (e.g., C mutation to T or G) at a specific position of a plant cell.
  • base mutation e.g., C mutation to T or G
  • the method of the present invention can effectively avoid random integration of foreign DNA when performing precise base substitution on plant cells without additionally introducing donor DNA.
  • mouse cytosine deaminase APOBEC1 was synthesized in vitro and ligated to the N-terminus of the mutated Cas9 (D10A), and the fusion protein was formed by 16aa XTEN flexible short peptide, and its nucleotide sequence is SEQ ID NO.:1. Shown as follows:
  • This sequence was constructed into the rice pCambia1300 vector (purchased from the Camiba Institute, Australia) and initiated by the plant strong promoter UBI. At the same time, the expression cassette of the guide RNA was also constructed into the vector to generate the vector pCSGAPO1 (Fig. 2A). This vector was applied to the following examples.
  • NRT1.1B can control the absorption of nutrient nitrogen in rice and increase yield
  • SLR1 controls the synthesis of gibberellin and affects the height of rice plants.
  • the amino acid to be edited in rice NRT1.1B gene is Thr327, and the amino acid to be edited in SLR1 gene is Ser97.
  • the targets separately.
  • the cytosine C to be replaced is all within the "deamination window" of the target. Therefore, we constructed the guide RNA expression cassettes (SEQ ID NO.: 2 and SEQ ID NO.: 3) of these two targets to the vector pCSGAPO1 (obtained from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences) to obtain base substitution.
  • the above two vectors were transformed into Agrobacterium EAH105, and then the callus of Flower 11 in rice was transformed. After 4 weeks of screening culture, 100-200 resistant calli granules were selected and added to the new medium for 2 weeks. Finally, all the resistant callus granules were picked out, and the genomic DNA was extracted and mixed for base substitution detection.
  • the two base substitution vectors pCSGAPO1-NRT and pCSGAPO1-SLR1 in Example 1 were transformed into Agrobacterium EHA105, and 37 transgenic plants editing NRT1.1B and 45 transgenes editing SLR1 were obtained by conventional rice genetic transformation. Plant (Table 2).
  • the base substitution of the site can be detected by restriction enzyme polymorphism.
  • StyI can digest it into two bands of 90 bp + 314 bp.
  • the results showed that 6 of the 45 transgenic plants editing SLR1 produced C ⁇ T base substitution at position 6 of their guide RNA.
  • the Sanger sequencing analysis of these 45 plants further verified the experimental results.
  • the animal promoter cannot perform site-based base substitution on the plant of the present invention (such as rice), and only the plant promoter of the present invention (especially a rice-specific strong promoter) can be used for the plant of the present invention (for example, rice) performs site-based base substitution, and the base substitution efficiency is up to 11.5%, and up to 60% of cells in a mutant plant have base substitutions.
  • TALE transcription activator-like effector

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Abstract

L'invention concerne une construction d'acide nucléique ayant une structure particulière, et un procédé pour une mutation de base ou une substitution de base sur un site particulier d'un génome de plante à l'aide de la construction. La construction d'acide nucléique comprend une première cassette d'expression, une deuxième cassette d'expression et une troisième cassette d'expression facultative, la première cassette d'expression étant une cassette d'expression pour exprimer des ARNg, la deuxième cassette d'expression étant une cassette d'expression pour exprimer une protéine de fusion comprenant une CAS9 et une cytosine désaminase, et la troisième cassette d'expression étant une cassette d'expression pour exprimer un marqueur de sélection.
PCT/CN2017/077133 2016-12-02 2017-03-17 Vecteur pour substitution de base dirigée du génome d'une plante WO2018098935A1 (fr)

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BRIAN S. PLOSKY: "CRISPR-Mediated Base Editing without DNA Double-Strand Breaks", MOLECULAR CELL, vol. 62, no. 4, 19 May 2016 (2016-05-19), pages 477 - 478, XP029552452, ISSN: 1097-2765, DOI: doi:10.1016/j.molcel.2016.05.006 *
YUAN ZONG: "Precise base editing in rice, wheat and maize with a Cas9- cytidine deaminase fusion", NATURE BIOTECHNOLOGY, vol. 35, no. 5, 27 February 2017 (2017-02-27), pages 1 - 5, XP055482699, ISSN: 1546-1696 *

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Publication number Priority date Publication date Assignee Title
US11447785B2 (en) 2016-11-14 2022-09-20 Institute of Genetics and Developmental Biology, Chinese Academv of Sciences Method for base editing in plants

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