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WO2018099475A1 - Procédé d'édition dirigée sur un site pour génome végétal sur la base de cpf1 - Google Patents

Procédé d'édition dirigée sur un site pour génome végétal sur la base de cpf1 Download PDF

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WO2018099475A1
WO2018099475A1 PCT/CN2017/114259 CN2017114259W WO2018099475A1 WO 2018099475 A1 WO2018099475 A1 WO 2018099475A1 CN 2017114259 W CN2017114259 W CN 2017114259W WO 2018099475 A1 WO2018099475 A1 WO 2018099475A1
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sequence
expression cassette
cpf1
vector
nls
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朱健康
王木桂
毛妍斐
陆钰明
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Shanghai Institutes for Biological Sciences SIBS of CAS
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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
    • 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
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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)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/8202—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
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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
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology

Definitions

  • the present invention relates to the field of biotechnology, and in particular to an RNA-guided plant genome fixed-point editing method.
  • CRISPR Clustered regular interspaced short palindromic repeats
  • CRISPR-Cas9 is a nucleic acid sequence separated by a short repeat sequence, which transcribes CRISPR RNA (crRNA) and another re-acting crRNA (trans-activating crRNA).
  • tracrRNA tracrRNA partial regions paired to form a binary complex, which then directs the Cas protein with non-specific nuclease activity to cleave the DNA sequence that matches the crRNA, so the engineered CRISPR-Cas9 system also needs to TracrRNAs fuse together to form a single chimeric RNA (chiRNA) and require the help of host RNase to function.
  • a PAM protospacer-adjacent motif
  • the Cas9 cut genome forms a blunt end, which facilitates gene knockout but is not conducive to the targeted insertion of foreign genes.
  • Cpf1 CRISPR from Prevotella and Francisella 1
  • Fn Cpf1 Francisella novicida U112 Cpf1
  • the Cpf1 enzyme of different bacteria finally found two Cpf1 proteins: AsCpf1 (Acidaminococcus sp. BV3L6 Cpf1) and LbCpf1 (Lachnospiraceae bacterium ND2006 Cpf1), which were able to directionalally cleave human DNA in vivo (Zetsche et al., 2015).
  • Cpf1 protein combines the functions of DNA cleavage enzyme and RNA pruning enzyme to not only target DNA double strands, but also to cut the corresponding immature crRNA (pre-crRNA) into mature crRNA (Fonfara et al. , 2016).
  • the AsCpf1 and LbCpf1 systems have been used in human and animal cells (Kim et al., 2016a; Kim et al., 2016b; Tóth et al., 2016).
  • the LbCpf1 system has also been reported for use in rice, its mutation efficiency is not high and it relies on long-chain immature direct repeats (DR), while short-chain mature DR sequences have no mutations (Xu et al). ., 2016).
  • the object of the present invention is to provide a set of plant genome fixed-point editing methods based on Cpf1 (including AsCpf1, FnCpf1 and LbCpf1), which can be simple and efficient to achieve single gene knockout, multi-gene knockout and homologous recombination in a single dicot.
  • the source clip is tapped at a fixed point.
  • nucleic acid construct for use in point-to-point editing of a plant genome, the nucleic acid construct comprising a first expression cassette and optionally a second expression cassette;
  • the first expression cassette is a Cpf1-NLS fusion protein expression cassette, wherein the Cpf1-NLS fusion protein has the structure of formula I:
  • P1 is the first promoter
  • A is a no, signal peptide, and/or protein tag sequence
  • B1 is a no or nuclear localization signal sequence NLS
  • B2 is a no or nuclear localization signal sequence NLS
  • B3 is a no-or nuclear positioning signal sequence NLS
  • B1, B2 and B3 are none; or B1 and B3 are none, and B2 is a nuclear localization signal sequence NLS;
  • C is an N-terminal fragment element of Cpf1;
  • D is a C-terminal fragment element of Cpf1
  • E1 is the first terminator
  • N-terminal fragment element and the C-terminal fragment element together constitute a complete Cpf1 protein
  • the second expression cassette is a crRNA expression cassette
  • the crRNA expression cassette contains a coding sequence corresponding to the mature type crRNA or the immature type pre-crRNA.
  • "-" represents a key
  • the nucleic acid construct comprises a first expression cassette and a second expression cassette.
  • first expression cassette and the second expression cassette are each located on a different expression vector.
  • first expression cassette and the second expression cassette are on the same expression vector.
  • B1 and B3 are absent, and B2 is the nuclear localization signal sequence NLS; or
  • B1 is none, and B2 and B3 are nuclear positioning signal sequences NLS; or
  • B2 is none, and B1 and B3 are nuclear positioning signal sequences NLS; or
  • B3 is none, and B1 and B2 are nuclear positioning signal sequences NLS; or
  • B1, B2 and B3 are nuclear localization signal sequences NLS.
  • the nuclear localization signal sequence NLS is selected from or derived from the group consisting of: SV40, KRP2 (Kip-related protein gene NO. 2), MDM2, CDc25C, DPP9, MTA1, CBP80, AreA, M9, Rev , hTAP, MyRF, EBNA-6, TERT, Tfam, or a combination.
  • the C corresponds to the amino acid sequence from position 1 to position n of the Cpf1 protein, wherein n is 627-673 (AsCpf1), 691-718 (FnCpf1) or 615-633 (LbCpf1) Positive integer.
  • n is any positive integer of 628-672 (AsCpf1), 692-717 (FnCpf1) or 616-632 (LbCpf1).
  • n is 652 (AsCpf1), 709 (FnCpf1) or 627 (LbCpf1).
  • said D corresponds to the sequence from the n+1th to the mth position of the Cpf1 protein, wherein n is as defined above, and m is the position number of the last amino acid of the full length Cpf1.
  • the B2 is located in a non-conserved region of the Cpfl polypeptide.
  • the second expression cassette has a structural crRNA expression cassette of formula II:
  • P2 is the second promoter
  • Each R independently corresponds to a mature or immature direct repeat (DR)
  • Each S is independently a leader sequence or a target site sg;
  • q is a positive integer ⁇ 1;
  • T is the no or termination sequence.
  • the termination sequence is selected from the group consisting of polyT, NOS, polyA, or a combination thereof.
  • q is 1.
  • the second expression cassette is a crRNA expression cassette having the structure of formula III or IV:
  • q is from 2 to 50, preferably from 2 to 10, preferably from 3 to 15.
  • q is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.
  • each S is an sg sequence.
  • the sg sequence has a length of 17-35 nt, preferably 18-28 nt, more preferably 18-24 nt.
  • the first promoter is a promoter of the Pol II type. In another preferred embodiment, the first promoter is selected from the group consisting of Ubi, Actin, CmYLCV, UBQ, 35S, SPL, tissue specific promoter YAO, CDC45, rbcS, inducible promoter XEV, or a combination thereof.
  • the second promoter is a promoter of the Pol II type or the Pol III type.
  • the second promoter is selected from the group consisting of OsU3, OsU6a, OsU6b, OsU6c, AtU6-1, AtU3b, AtU3d, AtU6-1, AtU6-29, Actin, 35S, Ubi, UBQ, SPL, CmYLCV and tissue specific promoter YAO, CDC45, rbcS, inducible promoter XEV or a combination thereof.
  • the second expression cassette is a structural crRNA expression cassette having the formula IV:
  • the first terminator is selected from the group consisting of a NOS terminator, a Poly A terminator.
  • the construct is a vector.
  • the construct is a plasmid.
  • the construct is a linear nucleic acid.
  • a vector or combination of vectors comprising the nucleic acid construct of the first aspect of the invention.
  • the nucleic acid construct comprises a first expression cassette and a second expression cassette.
  • first expression cassette and the second expression cassette are on the same expression vector.
  • first expression cassette and the second expression cassette are located on the same expression vector, and are provided on the flanks (ie, left and right sides) of the first expression cassette and the second expression cassette.
  • LB sequence and RB sequence are provided on the flanks (ie, left and right sides) of the first expression cassette and the second expression cassette.
  • first expression cassette and the second expression cassette are located in different first expression vectors and second expression vectors, respectively.
  • first expression cassette and the second expression cassette are respectively located in different first expression vectors and second expression vectors, then in the first expression vector in the first expression cassette The LB sequence and the RB sequence are flanked, and LB sequences and RB sequences are flanked by the second expression cassette in the second expression vector.
  • the vector combination further comprises an auxiliary vector carrying a donor DNA expression cassette.
  • the vector further comprises at least a donor DNA expression cassette.
  • a reagent combination comprising:
  • the reagent combination further comprises donor DNA.
  • a method of genetically editing a plant comprising the steps of:
  • the method further includes:
  • step (iii) regenerating or cultivating the plant cells, plant tissues or plants in which the gene editing has been identified in step (ii).
  • the gene editing comprises gene knockout, site-specific insertion, gene replacement, or a combination thereof.
  • the targeted insertion comprises a site-directed insertion based on homologous recombination or non-homologous recombination end joining.
  • the gene editing comprises gene editing at one or more sites.
  • the plurality of sites comprise different sites on the same gene, sites on different genes of the same gene family, and sites on different genes in the same signaling pathway.
  • the introduction is introduced by Agrobacterium.
  • the introduction is by a gene gun.
  • the introduction is by microinjection, electroporation, sonication, and polyethylene glycol (PEG) mediated introduction.
  • PEG polyethylene glycol
  • the plant is selected from the group consisting of a gramineous plant, a leguminous plant, and a cruciferous plant.
  • the plant comprises: Arabidopsis thaliana, wheat, barley, oats, corn, rice, sorghum, millet, soybean, peanut, tobacco, and tomato.
  • Figure 1 is a schematic diagram showing the construction of a rice single-cell knockout CRISPR-AsCpf1 (ncNLS) expression vector.
  • FIG 2 is a schematic diagram showing the construction of the Arabidopsis single point knockout CRISPR-AsCpf1 expression vector.
  • NLS is designed in three ways: it is placed at the 5' end and the 3' end of the AsCpf1 coding sequence (ncNLS); it is placed only at the 3' end of AsCpf1 (cNLS) or in a non-conserved region (mNLS) of the AsCpf1 coding sequence.
  • AsDR refers to the direct repeat of AsCpf1.
  • Figure 3 is a schematic diagram showing the construction of a rice single-point knockout CRISPR-AsCpf1 (cNLS) expression vector (without the protein tag and nuclear localization signal sequence NLS at the 5' end of the AsCpf1 coding sequence).
  • cNLS CRISPR-AsCpf1
  • Figure 4 is a schematic diagram showing the construction of a rice single-point knockout CRISPR-FnCpf1 (ncNLS) expression vector.
  • FIG. 5 is a schematic diagram showing the construction of a rice single-point knockout CRISPR-FnCpf1 (cNLS) expression vector (without the protein tag and nuclear localization signal sequence NLS at the 5' end of the FnCpf1 coding sequence).
  • cNLS CRISPR-FnCpf1
  • Figure 6 is a schematic diagram showing the construction of a rice single-point knockout CRISPR-LbCpf1 (ncNLS) expression vector.
  • Figure 7 is a schematic diagram showing the construction of a multi-site knockout CRISPR-AsCpf1 expression vector in rice.
  • Figure 8 is a schematic diagram showing the construction of a multi-site knockout CRISPR-FnCpf1 expression vector in rice.
  • Figure 9 is a schematic diagram showing the construction of a multi-site knockout CRISPR-LbCpf1 expression vector in rice.
  • Figure 10 is a schematic diagram showing the construction of a multi-site knockout CRISPR-FnCpf1 expression vector in Arabidopsis thaliana.
  • Figure 11 shows the sticky end of the YP reporter gene test Cpf1 cleavage mediated insertion of the exogenous fragment.
  • Figure 12 shows the GUUS and YFFP reporter gene systems.
  • the GU-US and YF-FP genes do not detect signals.
  • GU-US and YF-FP are repaired into complete ones by single strand annealing (SSA).
  • SSA single strand annealing
  • the GUS gene and the YFP gene are thus detectable by GUS staining or fluorescence microscopy.
  • the gRNA target site region is also referred to as MRS in the present invention.
  • Figure 13 is a CRISPR-AsCpf1 cleavage activity tested by the YFFP reporter system for different NLS placements, each transformation being repeated twice.
  • Figure 14 is a comparison of the cleavage activity of AsCpf1 (ncNLS), FnCpf1 (ncNLS) and LbCpf1 (ncNLS) in the YFFP reporter system.
  • FIG. 15 shows GUS staining of T1 plants after transformation of AsCpf1 (ncNLS) with Arabidopsis thaliana with GUUS reporter gene system.
  • A, E Two independent T1 generation stably transformed plants.
  • B, C, D GUS-stained blue spots magnified in leaves and roots.
  • Figure 18 is a Pol II promoter driven CRISPR array and Cpf1 expression cassette.
  • Figure 19 is a double DR framework of the crRNA expression cassette.
  • Figure 20 is a crRNA expression cassette of the H-HDV framework.
  • the present inventors After extensive and intensive research, the present inventors have for the first time constructed a highly versatile and highly specific CRISPR/Cpf1 plant genome fixed-point editing system based on Cpf1, and a nucleic acid construct, vector or vector for plant genome fixed-point editing. Combination, as well as plant genome fixed point editing methods. Specifically, based on the method of the present invention, single gene knockout, polygenic knockout or homologous recombination and directional insertion of exogenous fragments can be performed simply and efficiently at a predetermined plant genomic locus. On the basis of this, the present invention has been completed.
  • the CRISPR/Cpf1 editing system of the present invention adopts a mature crRNA, and since the mature crRNA is shorter, is convenient for artificial synthesis, and is more easily transformed into cells, development based on mature crRNA
  • the CRISPR-Cpf1 system will have an advantage.
  • a technical scheme for placing a nuclear localization signal sequence (NLS) at the N-terminus, C-terminus of the Cpf1 protein and a non-conserved region of the Cpf1 polypeptide was also developed.
  • the experimental results show that the genetic editing of Arabidopsis thaliana and rice has been successfully completed by the above technical scheme, and the efficiency is as high as 60%-70%.
  • the design of the non-conserved region (mNLS) of the nuclear localization signal sequence (NLS) built into the Cpf1 coding sequence has relatively high activity.
  • containing includes “including”, “consisting essentially of”, “consisting essentially of”, and “consisting of.”
  • operably linked refers to a condition in which portions of a linear DNA sequence are capable of modulating or controlling the activity of other portions of the same linear DNA sequence. For example, if a promoter controls the transcription of a sequence, then it is operably linked to the coding sequence.
  • the invention provides a nucleic acid construct for site-directed editing of a plant genome, the nucleic acid construct comprising a first expression cassette and optionally a second expression cassette;
  • the first expression cassette is a Cpf1-NLS fusion protein expression cassette, wherein the Cpf1-NLS fusion protein has the structure of formula I:
  • P1 is the first promoter
  • A is a no, signal peptide, and/or protein tag sequence
  • B1 is a no or nuclear localization signal sequence NLS
  • B2 is a no or nuclear localization signal sequence NLS
  • B3 is a no-or nuclear positioning signal sequence NLS
  • B1, B2 and B3 are none; or B1 and B3 are none, and B2 is a nuclear localization signal sequence NLS;
  • C is an N-terminal fragment element of Cpf1;
  • D is a C-terminal fragment element of Cpf1
  • E1 is the first terminator
  • N-terminal fragment element and the C-terminal fragment element together constitute a complete Cpf1 protein
  • the second expression cassette is a crRNA expression cassette, and the crRNA expression cassette has a corresponding The coding sequence for mature crRNA or immature pre-crRNA.
  • each of the above elements can be produced by a conventional method (e.g., PCR method, artificial total synthesis), and then ligated by a conventional method to form a nucleic acid construct of the present invention. If necessary, the digestion reaction may optionally be carried out before the ligation reaction.
  • a conventional method e.g., PCR method, artificial total synthesis
  • nucleic acid constructs of the invention may be linear or cyclic.
  • the nucleic acid constructs of the invention may be single-stranded or double-stranded.
  • the nucleic acid construct of the present invention may be DNA, RNA, or DNA/RNA hybrid.
  • nNLS refers to the nuclear localization signal sequence NLS located at the 5' end of the Cpf1 protein coding sequence
  • cNLS means that the nuclear localization signal sequence NLS is located at the 3' end of the Cpf1 protein coding sequence
  • ncNLS refers to the 5' of the Cpf1 protein coding sequence.
  • the nuclear localization signal sequence NLS is set at both the terminal and the 3' end
  • mNLS means that the NLS nuclear localization signal sequence is built in the non-conserved region of the Cpf1 protein coding sequence.
  • foreign gene refers to an exogenous DNA molecule that acts in a phased manner.
  • the foreign gene which can be used in the present application is not particularly limited, and includes various exogenous genes commonly used in the field of transgenic animals. Representative examples include, but are not limited to, a ⁇ -glucuronidase gene, a red fluorescent protein gene, a green fluorescent protein gene, a lysozyme gene, a salmon calcitonin gene, a lactoferrin, or a serum albumin gene.
  • 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. These include, but are not limited to, the hygromycin resistance gene (Hyg), the kanamycin resistance gene (NPTII), the neomycin gene, or the puromycin 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.
  • 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.
  • Cpf1 protein refers to a nuclease.
  • Typical Cpf1 proteins include, but are not limited to, FnCpf1 (Francisella novicida U112 Cpf1), AsCpf1 (Acidaminococcus sp. BV3L6 Cpf1), and LbCpf1 (Lachnospiraceae bacterium ND2006 Cpf1).
  • coding sequence of a Cpf1 protein refers to a nucleotide sequence that encodes a Cpf1 protein having cleavage activity.
  • the inserted polynucleotide sequence is transcribed and translated to produce a functional Cpf1 protein
  • 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 Cpf1 protein according to the needs of expression in different species. These variants are all referred to by the term "Cpf1 protein.
  • the coding sequence is specifically covered.
  • the term specifically encompasses a full-length sequence substantially identical to the Cpfl gene sequence, as well as a sequence encoding a protein that retains the function of the Cpfl protein.
  • the C corresponds to the amino acid sequence of the 1st to the nth position of the Cpf1 protein, wherein n is 627-673 (when C is the N-terminus of AsCpf1 or a similar protein), 691-718 (when C is a positive integer for the N-terminus of FnCpf1 or a similar protein, 615-633 (when C is the N-terminus of LbCpf1 or a similar protein).
  • the second expression cassette has a structural crRNA expression cassette of formula II:
  • P2 is the second promoter
  • Each R independently corresponds to a mature or immature direct repeat (DR)
  • Each S is independently a leader sequence or a target site sg;
  • q is a positive integer ⁇ 1;
  • T is a no or polyT or Nos or polyA sequence.
  • the invention also provides a vector or combination of vectors comprising a nucleic acid construct of the invention.
  • the vector combination of the invention further comprises an auxiliary vector carrying a donor DNA expression cassette.
  • some elements are operably linked.
  • a promoter when operably linked to a coding sequence, it is meant that the promoter is capable of initiating transcription of the coding sequence.
  • the present invention also provides reagent combinations and kits comprising the above vectors or combinations of vectors, which are useful in the plant gene editing method of the present invention.
  • the invention also provides a method for genetically editing a plant, comprising the steps of:
  • the gene editing includes gene knockout, site-directed insertion, gene replacement, or a combination thereof.
  • the plant gene editing method of the invention can be used for improving various plants, especially for improving crops.
  • plant includes whole plants, plant organs (eg, leaves, stems, roots, etc.), seeds. And plant cells and their progeny.
  • 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.
  • the invention can be used in the field of plant genetic engineering, such as plant gene function research and crop genetic improvement.
  • the CRISPR/Cpf1 system Compared to the traditional CRISPR/Cas9 gene editing system, the CRISPR/Cpf1 system has the following advantages:
  • crRNA Only the crRNA is required, and the Cpf1 protein is required to cleave the target DNA sequence by fusion with another re-acting crRNA (tracrRNA);
  • the PAM sequence is TTN or TTTN, which is convenient for editing AT-rich sequences
  • the PAM sequence is located at the 5' end of sg, and the cleavage site is 15-18 bases away from PAM, providing greater flexibility in the selection of target sites;
  • the Cpf1 cleavage genome forms a sticky end, which makes the targeted insertion of the foreign gene more convenient and controllable;
  • Cpf1 itself has RNase activity and can be self-sheared to form a mature crRNA sequence, such that a crRNA expression cassette can cascade multiple DR-sg sequences to facilitate multi-site knockout.
  • the protein coding sequences of AsCpf1, FnCpf1 and LbCpf1 are human-optimized coding sequences, and the specific sequences can be viewed at https://benchling.com/s/wXO8WZJ7/edit, https://benchling.com/s/0xgyNBMK /edit, https://benchling.com/s/HVIyGqQs/edit.
  • the HindIII restriction site is underlined to facilitate cloning into the pCambia expression vector; the black shading indicates the mature DR sequence corresponding to AsCpf1; the two BsaI restriction site-related sequences are inserted in the box to construct the gene knockout vector.
  • the sequence is replaced by the sg sequence; the bold letter is the transcription terminator sequence; the rest is the sequence of the OsU6 promoter.
  • the HindIII restriction site is underlined to facilitate cloning into the pCambia expression vector; the black shading indicates the mature DR sequence corresponding to FnCpf1; the two BsaI restriction site-related sequences are inserted in the box to construct the gene knockout vector.
  • the sequence is replaced by the sg sequence; the bold letter is the transcription terminator sequence; the rest is the sequence of the OsU6 promoter.
  • the HindIII restriction site is underlined to facilitate cloning into the pCambia expression vector; the black shading indicates the mature DR sequence corresponding to LbCpf1; the two BsaI restriction site-related sequences are inserted in the box to construct the gene knockout vector.
  • the sequence is replaced by the sg sequence; the bold letter is the transcription terminator sequence; the rest is the sequence of the OsU6 promoter.
  • the HindIII restriction site is underlined to facilitate cloning into the pCambia expression vector; the black shading indicates the mature DR sequence corresponding to FnCpf1; the sg sequence of the four targeting sites in the box is 5' to 3' The order is 798sg, 799sg, 802sg and 803sg; the bold letters are transcription terminator sequences; The rest is the sequence of the OsU6 promoter.
  • the HindIII restriction site is underlined to facilitate cloning into the pCambia expression vector; the black shading indicates the mature DR sequence corresponding to LbCpf1; the sg sequence of the four targeting sites in the box is 5' to 3' The order is 240sg, 260sg, 230sg and 250sg; the bold letters are transcription terminator sequences; the rest are the sequences of the OsU6 promoter.
  • Z1 is N 6 and Z2 is CTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTC (SEQ ID NO. 50)
  • HDV Hepatitis deltavirus
  • the crRNA expression cassette has the following four elements from 5'-3': the OsU6 or OsU3 promoter, the mature direct repeat (DR) corresponding to AsCpf1, and two BsaI cleavage sites.
  • the sg sequence was subjected to a seamless cloning, transcription terminator sequence (TTTTTTT) (sequence 1).
  • the AsCpf1 expression cassette has the following elements from 5'-3': Ubi promoter derived from maize, 3 ⁇ Flag or 3 ⁇ HA protein tag, NLS nuclear localization signal sequence (nNLS), coding sequence of AsCpf1, second NLS core Positioning signal sequence (cNLS), NOS transcription terminator sequence (Fig. 1).
  • Ubi promoter derived from maize, 3 ⁇ Flag or 3 ⁇ HA protein tag
  • nNLS NLS nuclear localization signal sequence
  • cNLS second NLS core Positioning signal sequence
  • NOS transcription terminator sequence Fig. 1
  • the corresponding OsU6 or OsU3 promoter was replaced with the AtU6 or AtU3 promoter
  • the Ubi promoter was replaced with the UBQ promoter.
  • the other components were identical ( Figure 2).
  • the sequence at the 5' end is relatively conservative, so another design of the present invention is to remove the protein tag and the NLS nuclear localization signal sequence at the 5' end of the AsCpf1 coding sequence based on the above structure, and the other elements are retained (Fig. 3), or directly put the NLS sequence into a non-conserved region (mNLS) in the AsCpf1 coding sequence (Fig. 2), in order to increase the cleavage activity of AsCpf1 in vivo.
  • mNLS non-conserved region
  • the crRNA expression cassette has the following four elements from 5'-3': OsU6 or OsU3 promoter, FnCpf1 corresponding mature direct repeat (DR), and two BsaI cleavage sites for The sg sequence, the transcription terminator sequence (TTTTTTT) (sequence 2) was cloned seamlessly.
  • the FnCpf1 expression cassette has the following elements from 5'-3': Ubi promoter derived from maize, 3 ⁇ Flag or 3 ⁇ HA protein tag, NLS nuclear localization signal sequence (nNLS), coding sequence of FnCpf1, second NLS core Localization signal sequence (cNLS), NOS transcription terminator sequence ( Figure 4).
  • nNLS NLS nuclear localization signal sequence
  • cNLS second NLS core Localization signal sequence
  • Figure 4 NOS transcription terminator sequence
  • the crRNA expression cassette has the following four elements from 5'-3': OsU6 or OsU3 promoter, LbCpf1 corresponding mature direct repeat (DR), and two BsaI cleavage sites for The sg sequence, transcription terminator sequence (TTTTTTT) (sequence 3) was cloned seamlessly.
  • the LbCpf1 expression cassette has the following elements from 5'-3': Ubi promoter derived from maize, 3 ⁇ Flag or 3 ⁇ HA protein tag, NLS nuclear localization signal sequence (nNLS), the coding sequence of LbCpf1, the second NLS nuclear localization signal sequence (cNLS), and the NOS transcription terminator sequence (Fig. 6).
  • the sequence at the 5' end thereof is relatively conservative, so another design of the present invention is to remove the protein tag and the NLS nuclear localization signal sequence at the 5' end of the LbCpf1 coding sequence based on the above structure, and the other elements are retained, or The NLS sequence was directly placed in a non-conserved region (mNLS) in the LbCpf1 coding sequence to increase the cleavage activity of LbCpf1 in vivo.
  • mNLS non-conserved region
  • Cpf1 itself has RNase activity, it can self-cleave the transcribed precursor crRNA sequence. It is speculated that if a crRNA expression cassette is ligated in series with multiple DR-sg sequences, it can be cleavable by Cpf1 into a single DR-sg sequence. Achieve multi-site knockout.
  • This design requires the construction of a multi-site crRNA expression cassette, in the case of rice, from 5'-3' with the following elements: OsU6 or OsU3 promoter, each Cpf1 corresponding crRNA sequence (including DR sequence), corresponding to target site 1
  • the Cpf1 expression cassette is identical to the construction of the single point knockout.
  • the above two expression cassettes were cloned into the pCambia expression vector (Figs. 7, 8, 9).
  • the permutation-related element is used for Arabidopsis multi-site knockout ( Figure 10).
  • the Cpf1 cleavage genome forms sticky ends and is theoretically more suitable for directional insertion of exogenous fragments.
  • CRISPR-Cpf1 to create a DSB near the target site and simultaneously introducing a large number of exogenous fragments by means of gene gun bombardment or DNA viral replication, gene homologous recombination or directed insertion of plant cells can be efficiently achieved.
  • a YP reporter gene was used as a test system to verify that the sticky end generated by Cpf1 cleavage mediates the design of the site insertion of the foreign fragment is shown in FIG.
  • OsEPSPS Three rice endogenous loci were selected: OsEPSPS, OsBEL, OsPDS, and two CRISPR-AsCpf1 targeting site sequences (sg) were designed for each gene and ligated to the pCambia-CRISPR-AsCpf1 expression vector by BsaI restriction sites. .
  • the constructed vector was transformed into Agrobacterium tumefaciens EHA105, and then the callus of the rice variety Nyza sativa ssp japonica cv. Nipponbare was infested by the strain. After 3 days of co-cultivation, the transformed callus was transferred to hygromycin-containing.
  • Example 3 uses the CRISPR-FnCpf1 (ncNLS) system to perform single-point knockout of rice endogenous genes
  • OsEPSPS Three rice endogenous loci were selected: OsEPSPS, OsBEL, OsPDS, and two CRISPR-FnCpf1 targeting site sequences (sg) were designed for each gene, which were ligated to the pCambia-CRISPR-FnCpf1 expression vector by BsaI restriction sites. .
  • the constructed vector was transformed into Agrobacterium tumefaciens EHA105, and then the callus of the rice variety Nipponbare was infested by the strain. After 3 days of co-cultivation, the transformed callus was transferred to a screening culture containing hygromycin, and after 15 days of culture, the transfer was carried out.
  • Example 4 A single point knockout of rice endogenous genes was performed using the CRISPR-LbCpf1 (ncNLS) system.
  • OsEPSPS Three rice endogenous loci were selected: OsEPSPS, OsBEL, OsPDS, and two CRISPR-LbCpf1 targeting site sequences (sg) were designed for each gene, which were ligated to the pCambia-CRISPR-LbCpf1 expression vector by BsaI restriction sites. .
  • the constructed vector was transformed into Agrobacterium tumefaciens EHA105, and then the callus of the rice variety Nipponbare was infested by the strain. After 3 days of co-cultivation, the transformed callus was transferred to a screening culture containing hygromycin, and after 15 days of culture, the transfer was carried out. Regenerated plants in a differentiation medium containing hygromycin.
  • Fig. 16 albino leaves
  • Fig. 17 A population of regenerated plants produced by each transformation was randomly sampled to extract DNA and the target sites were amplified and sequenced. The results showed (Table 3) that the system was able to target mutations in rice cells to produce mutations very efficiently and to generate mutant plants with a maximum mutation rate of more than 70% in the six sites tested.
  • Example 5 CRISPR-FnCpf1 vector containing a DR-sg sequence string for multi-site knockout of rice
  • OsRLK-798 (LOC_Os02g04430), OsRLK-799 (LOC_Os02g07960), OsRLK-802 (LOC_Os01g39600) and OsRLK-803 (LOC_Os06g04370).
  • a FnCpf1 targeting site sequence (sg) was designed for each locus, joined by the mature DR sequence of FnCpf1, and is under the control of the same OsU6 promoter (sequence four SEQ ID NO. 4).
  • the expression cassette was then ligated to the FnCpfl expression cassette and placed in the LB and RB sequences of pCambia1300.
  • the constructed vector was transformed into Agrobacterium tumefaciens EHA105, and then the callus of the rice variety Nipponbare was infested by the strain. After 3 days of co-cultivation, the transformed callus was transferred to a screening culture containing hygromycin, and after 15 days of culture, the transfer was carried out. Regenerated plants in a differentiation medium containing hygromycin. DNA was randomly sampled from the regenerated plant population and amplified and sequenced at each target site. The results showed that the system was able to perform multi-gene editing in rice cells very efficiently, achieving a mutation rate of 50%-87.5% in the four sites tested (Table 4).
  • Example 6 CRISPR-LbCpf1 vector containing a DR-sg sequence string for multi-site knockout of rice
  • OsBEL-230 (LOC_Os03g55230), OsBEL-240 (LOC_Os03g55240), OsBEL-250 (LOC_Os03g55250) and OsBEL-260 (LOC_Os03g55260)
  • LbCpf1 target was designed for each gene.
  • the sequence of sites (sg) is ligated by the mature DR sequence of LbCpfl and is under the control of the same OsU6 promoter (sequence 5 SEQ ID NO. 5). This expression cassette was then ligated to the LbCpfl expression cassette and placed in the LB and RB sequences of pCambia1300.
  • the constructed vector was transformed into Agrobacterium tumefaciens EHA105, and then the callus of the rice variety Nipponbare was infested by the strain. After 3 days of co-cultivation, the transformed callus was transferred to a screening culture containing hygromycin, and after 15 days of culture, the transfer was carried out. Regenerated plants in a differentiation medium containing hygromycin. DNA was randomly sampled from the regenerated plant population and amplified and sequenced at each target site. The results showed that the system was able to perform multi-gene editing in rice cells very efficiently, achieving a mutation rate of 40%-60% in the four sites tested (Table 5).
  • Example 7 CRISPR-Cpf1 uses a modified mature DR sequence for rice single point knockout
  • Cpf1 derived from different strains has its corresponding DR sequence, and each DR sequence is not the same.
  • the Cpf1 protein binds to the DR-sg sequence, it has structural specificity for the DR-sg formed, rather than the unique sequence specific requirement, and due to the conservation of the Cpf1 protein family, each DR sequence is also highly homologous. It is speculated that Cpf1 can bind to non-self-derived DR sequences, that is, the DR sequence has a certain elasticity, and can be modified to some extent. This feature is used to further optimize the design of the DR sequence, especially when multiple DR-sg sequences are serially used for multi-gene locus editing, avoiding the reuse of the same DR sequence.
  • LbCpf1 and FnCpf1 which are highly active in rice gene editing, and exchanged their mature DR sequences for single gene editing of rice OsPDS-1 and OsBEL-2 loci.
  • the constructed vector was transformed into Agrobacterium tumefaciens EHA105, and then the callus of the rice variety Nipponbare was infested by the strain. After 3 days of co-cultivation, the transformed callus was transferred to a screening culture containing hygromycin, and after 15 days of culture, the transfer was carried out. Regenerated plants in a differentiation medium containing hygromycin.
  • Example 8 Using a Pol II type promoter to drive a long DR-guide array in a CRISPR-Cpf1 system for multi-site knockout of rice
  • Example 5 and Example 6 the DR-guide array driven by the OsU6 promoter can achieve high-efficiency knockout of four loci in rice, but U6 belongs to the Pol III type promoter and has limited ability to drive long chains. Moreover, the Pol III type promoter does not have condition-specific or tissue-specific activation ability, but the Pol II type promoter can effectively overcome the above defects.
  • the inventors took FnCpf1 as an example, and selected two Pol II type promoters, 2 ⁇ 35S and Actin, respectively, to drive a long DR-guide array to perform multi-gene locus knockout on rice.
  • LOC_Os01g43530 Eight genes related to Late Embryogenesis Abundant protein genes (LEA) were selected as targets, and DR-guide arrays were constructed in the following order: LOC_Os01g43530, LOC_Os05g50710, LOC_Os01g12580, LOC_Os03g62620, LOC_Os04g49980, LOC_Os06g02040, LOC_Os08g23870 and LOC_Os06g21910 .
  • LOC_Os01g43530 Eight genes related to Late Embryogenesis Abundant protein genes (LEA) were selected as targets, and DR-guide arrays were constructed in the following order: LOC_Os01g43530, LOC_Os05g50710, LOC_Os01g12580, LOC_Os03g62620, LOC_Os04g49980, LOC_Os06g02040, LOC_Os08g238
  • a FnCpf1 targeting site sequence (sg) was designed for each locus, which was ligated by the mature DR sequence of FnCpf1, and the DR sequence was ligated at both ends of the array, driven by 2 ⁇ 35S or rice Actin promoter, with poly A Or the NOS terminator ends to form a complete CRISPR Pol II expression cassette, which is then ligated into the FnCpfl expression cassette and placed in the LB and RB sequences of pCambia1300 ( Figure 18).
  • the constructed vector was transformed into Agrobacterium tumefaciens EHA105, and then the callus of the rice variety Nipponbare was infested by the strain.
  • the transformed callus was transferred to a screening culture containing hygromycin, and after 15 days of culture, the transfer was carried out.
  • DNA was randomly sampled from the regenerated plant population and amplified and sequenced at each target site.
  • the results showed that the long DR-guide array driven by the Pol II type promoter can be used for multi-site knockout of rice, with a mutation rate of up to 36%-48.3% in the 8 sites tested (Table 7, Table 8). ). However, some sites have lower mutation rates, such as LOC_Os01g43530, LOC_Os05g50710, LOC_Os06g21910, etc.
  • the 5' end cap structure and 3' end of the mRNA that may be transcribed by the Pol II type promoter may be attached.
  • the effect of the poly A tail can be solved by adding an RNA-cleaving function such as a transfer RNA (tRNA) sequence, a ribozyme sequence or a Csy4 recognition sequence to both ends of the DR-guide array.
  • tRNA transfer RNA
  • the PAM-guide sequence targeting these target sites is not efficient.
  • the 3' end of the PAM sequence is T (such as LOC_Os01g43530 and LOC_Os05g50710), or the 5' end of the guide sequence.
  • the first base if T (such as LOC_Os01g43530) may result in inefficient FnCpf1 targeted binding cleavage, which can be improved by altering or optimizing the PAM-guide sequence targeting the above sites.
  • Promoter with two other pol II types such as the ZmUbi promoter from maize or the promoter derived from cestrum yellow leaf curling virus (CmYLCV) instead of 2 ⁇ 35S or rice Actin promoter , got a similar result.
  • CmYLCV cestrum yellow leaf curling virus
  • Example 9 CRISPR-Cpf1 utilizes double DR sequence (DR-guide-DR) to improve single site knockout efficiency in rice
  • Examples 2-4 the inventors used AsCpf1, FnCpf1 and LbCpf1 to perform single-point knockout of rice endogenous genes, respectively, in which FnCpf1 and LbCpf1 can achieve high efficiency knockout at most sites, but there are also individual site efficiencies. Lower, such as the OsPDS-2 locus.
  • the PAM-guide sequence targeting this site has been optimized. For example, the 3' end of the PAM sequence is not T, the first base at the 5' end of the guide sequence is not T, the GC content of the guide sequence is moderate, and the four bases are The distribution of the base is also relatively balanced and so on.
  • the inventors added a DR sequence to U6/U3-DR-guide-DR-poly T in this framework, and used the RNA cleavage activity of Cpf1 itself to excise the poly U tail in the CRISPR transcript to become a clean DR-
  • the guide targets the sequence to increase knockout efficiency.
  • the inventors used LbCpf1 to target the OsPDS-2 site as an example, and turned the original crRNA expression cassette into U6-DR-guide-DR-poly T, except that a DR sequence was added after the guide.
  • the invariant the inventor called the dual DR system ( Figure 19).
  • the inventors also constructed a crRNA expression cassette driven by the Pol II type promoter Actin, using two ribozymes Hammerhead ribozyme (HH) and Hepatitis deltavirus ribozyme (HDV) with RNA self-shearing activity.
  • the post-transcriptional DR-guide targeting sequence was isolated: Actin-HH-DR-guide-HDV-poly T, which the inventors referred to as the H-HDV system ( Figure 20).
  • the above two crRNA expression cassettes were then cloned into the pCambia expression vector with the original LbCpfl expression cassette (Fig. 19, 20).
  • the constructed vector was transformed into Agrobacterium tumefaciens EHA105, and the callus of rice cultivar Nipponbare was infested by the strain. After 3 days of co-cultivation, the transformed callus was transferred to a screening culture containing hygromycin, and after 15 days of culture, it was transferred to contain Regenerated plants in the differentiation medium of hygromycin.
  • the regenerated plant population produced by each transformation was randomly sampled to extract DNA and the target site OsPDS-2 was amplified and sequenced.
  • the results are shown in Table 9. Both the double DR system and the H-HDV system can effectively improve the knockout efficiency of the OsPDS-2 site, and the knockout efficiency of the same site in the original example 4 was only 2.8%.
  • the improvement also shows that the dual DR system is as efficient as the H-HDV system, and the dual DR system does not need to be configured with complex Hammerhead ribozyme and Hepatitis deltavirus ribozyme ribozyme sequences, which is simpler in structure and more economical in vector construction. practical.
  • mutants produced chimeric mutants accounted for a large part, which may be due to the low expression of Cpf1, by replacing stronger promoters or ovaries, embryos. Specific promoters such as meristems can solve this problem.
  • Table 9 Identification results of the PAM-sg sequence and the T0 generation plants in which the CRISPR/LbCpf1 knocked out the rice OsPDS-2 site using the double DR system and the H-HDV system.

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Abstract

L'invention concerne un procédé d'édition dirigée sur un site pour un génome végétal sur la base de Cpf1. En particulier, l'invention concerne une construction d'acide nucléique, un vecteur ou une combinaison de vecteurs pour une édition dirigée sur un site génomique végétal sur la base de Cpf1 (AsCpf1, FnCpf1, LbCpf1) et un procédé d'édition dirigée sur un site pour un génome végétal. En particulier, la construction d'acide nucléique comprend une première cassette d'expression et une deuxième cassette d'expression facultative. La première cassette d'expression est une cassette d'expression de protéine de fusion Cpf1-NLS. La deuxième cassette d'expression est une cassette d'expression d'ARNcr. Avec le procédé, l'inactivation de gène unique, l'inactivation de multiples gènes ou la recombinaison homologue et l'insertion directionnelle de fragments étrangers peuvent être réalisées de manière simple et efficace au niveau d'un site génomique végétal prédéterminé.
PCT/CN2017/114259 2016-12-01 2017-12-01 Procédé d'édition dirigée sur un site pour génome végétal sur la base de cpf1 Ceased WO2018099475A1 (fr)

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WO2019238772A1 (fr) 2018-06-13 2019-12-19 Stichting Wageningen Research Constructions de polynucléotide et procédés d'édition génétique par cpf1
CN110129366A (zh) * 2019-05-24 2019-08-16 青岛农业大学 一种载体组合及其应用
CN110129366B (zh) * 2019-05-24 2020-09-01 青岛农业大学 一种载体组合及其应用
CN112280799A (zh) * 2020-10-22 2021-01-29 中国热带农业科学院橡胶研究所 利用CRISPR/Cas9系统对橡胶草或蒲公英基因定点突变的方法
CN112280799B (zh) * 2020-10-22 2023-02-17 中国热带农业科学院橡胶研究所 利用CRISPR/Cas9系统对橡胶草或蒲公英基因定点突变的方法
CN115820691A (zh) * 2022-07-25 2023-03-21 安徽农业大学 一种基于LbCpf1变体的水稻碱基编辑系统和应用
CN115820691B (zh) * 2022-07-25 2023-08-22 安徽农业大学 一种基于LbCpf1变体的水稻碱基编辑系统和应用

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