WO2019205939A1 - 一种重复片段介导的植物定点重组方法 - Google Patents

一种重复片段介导的植物定点重组方法 Download PDF

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WO2019205939A1
WO2019205939A1 PCT/CN2019/082095 CN2019082095W WO2019205939A1 WO 2019205939 A1 WO2019205939 A1 WO 2019205939A1 CN 2019082095 W CN2019082095 W CN 2019082095W WO 2019205939 A1 WO2019205939 A1 WO 2019205939A1
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sequence
nucleic acid
acid construct
site
plant cell
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French (fr)
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朱健康
陆钰明
田益夫
沈润东
王木桂
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中国科学院上海生命科学研究院
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Definitions

  • the present invention relates to the field of biotechnology, and in particular to a repeated fragment-mediated plant site-directed recombination method.
  • Genomic editing techniques include Zinc finger nuclease (ZFN), transcription activator-like (TAL) effector nucleases (Talen) and CRISPR/Cas technology. All three techniques can generate double-strand breaks (DSBs) in a specific site-specific cleavage DNA in an organism's genome, thereby performing site-directed editing using the characteristics of non-homologous end joining or homologous recombination possessed by the cells themselves.
  • ZFN and Talen technologies use specific proteins to guide genome cleavage, which is relatively complex to construct and has low editing efficiency.
  • a first aspect of the invention provides a nucleic acid construct having the structure of formula I from 5' to 3':
  • Y1 is a no or nucleotide sequence
  • Z1 is the first DSB sequence
  • Z2 is the first homologous sequence
  • Z3 is the DNA sequence of interest
  • Z4 is a second homologous sequence
  • Z5 is a second DSB sequence
  • Y2 is a no or nucleotide sequence
  • each "-" is independently a bond or nucleotide linkage sequence.
  • the nucleotide linking sequence comprises a sequence of m nucleotides in length, wherein m is from 1 to 30, preferably from 1 to 20, more preferably from 1 to 10 (e.g., 1) , 2, 3, 4, 5, 6, 7, 8, 9, 10).
  • each "-" is a bond.
  • the first and second DSB sequences are located (recognized) and cleaved with the participation of gRNA.
  • each of the DSB sequences is capable of being recognized and cleaved by a site-cutting nuclease.
  • each of the DSB sequences is independently: (a) itself contains a cleavage site, or (b) a cleavage site formed when the nucleic acid construct is integrated, such as by a target site, by NHEJ. .
  • the first DSB sequence may be outside the 5' end of the first homologous sequence.
  • the first DSB sequence partially overlaps the first homologous sequence.
  • the second DSB sequence may be outside the 3' end of the second homologous sequence.
  • the second DSB sequence partially overlaps the second homologous sequence.
  • the first DSB sequence is the same as or different from the second DSB sequence.
  • the first DSB sequence and the second DSB sequence are identical or different from the DSB sequence ("target site DSB sequence") of the cleavage site of the genomic target site.
  • the first DSB sequence, the second DSB sequence, and the target site DSB sequence are identical.
  • the site-cutting nuclease is selected from the group consisting of ZFN, Talen, and CRISPR/Cas9, or a combination thereof.
  • the site-cutting nuclease is CRISPR/Cas9.
  • the target DNA sequence is capable of being recognized and cleaved by an enzyme selected from the group consisting of Cas9, Cpf1, C2C1, C2C2, C2C3, and the like, a CRISPR-related enzyme.
  • the target DNA sequence is capable of being recognized and cleaved by an enzyme selected from the group consisting of Fok I.
  • the first DSB sequence is 10-50 bp, preferably 15-30 bp.
  • the second DSB sequence is 10-50 bp, preferably 15-30 bp.
  • the first homologous sequence is from 20 bp to 10 kb, preferably from 30 bp to 5 kb.
  • the second homologous sequence is from 20 bp to 10 kb, preferably from 30 bp to 5 kb.
  • the DNA sequence of interest is a sequence to be knocked in and/or replaced.
  • the DNA sequence of interest is from 1 bp to 10 kb, preferably from 5 bp to 5 kb.
  • the Y1 and Y2 are protected bases.
  • the lengths of Y1 and Y2 are respectively 1 to 50 bp, preferably 4 to 20 bp.
  • sequence homology H1 of the first homologous sequence to the DNA sequence of the target site side (eg, the upstream side or the left side) of the genome of the eukaryotic cell is ⁇ 90%, more preferably ⁇ 95%.
  • the first homologous sequence, the second homologous sequence and the DNA sequences flanking the target site of the eukaryotic genome respectively constitute a homologous repeat sequence (ie, the first homologous sequence and the The DNA sequence on one side of the target site (such as the upstream side or the left side) constitutes a homologous repeat, and the second homologous sequence is identical to the DNA sequence on the other side of the target site (such as the downstream side or the right side). To the repeat sequence; vice versa).
  • the eukaryotic cell comprises a plant cell.
  • the plant comprises an angiosperm and a gymnosperm.
  • the gymnosperm plant is selected from the group consisting of Cycadaceae, Podocarpaceae, Araucariaceae, Pinaceae, Cedaraceae, Cypress, and three-pointed Cedaraceae, Taxaceae, Ephedra, Matroaceae, Monocytogenes, Centennial Orchidaceae, or a combination thereof.
  • the plant comprises a monocot and a dicot.
  • the plant comprises a herbaceous plant and a woody plant.
  • the herbaceous plant is selected from the group consisting of Solanaceae, Gramineae, Leguminous, or a combination thereof.
  • the woody plant is selected from the group consisting of kiwifruit, Rosaceae, Moraceae, or a combination thereof.
  • the plant is selected from the group consisting of: cruciferous plants, gramineous plants, legumes, Solanaceae, kiwifruit, Malvaceae, Paeonia, Rosaceae, Liliaceae, or combinations thereof .
  • the plant is selected from the group consisting of rice, cabbage, soybean, tomato, corn, tobacco, wheat, sorghum, or a combination thereof.
  • the nucleic acid construct is a single-stranded DNA sequence or a double-stranded DNA sequence, preferably a double-stranded DNA sequence.
  • one or both of the nucleic acid constructs are phosphorylated with the 5' end of the single strand of the DNA.
  • the two 5' ends of the two DNA single strands of the nucleic acid construct are phosphorylated.
  • the diester bond between one or more (eg, 2, 3, 4, or 5) bases at the 5' and/or 3' end of the nucleic acid construct is sulfur Generation modification.
  • the nucleic acid construct does not carry a screening tag.
  • a second aspect of the invention provides a reagent combination for gene editing, comprising:
  • P1 is the first promoter
  • A1 is a coding sequence encoding a Cas9 protein
  • A2 is a terminator
  • a donor DNA element comprising: the nucleic acid construct of the first aspect of the invention, or a vector for expressing the nucleic acid construct.
  • the donor DNA comprises: a second nucleic acid construct, or a second vector comprising the second nucleic acid construct.
  • the second nucleic acid construct has the structure shown in Formula II from 5'-3':
  • P2 is a second promoter
  • A3 is the coding sequence of gRNA
  • A4 is a no- or transcription termination sequence
  • A5 is an expression cassette for the nucleic acid construct of claim 1;
  • the nucleotide linkage sequence is 1-60 nt.
  • nucleotide linking sequence does not affect the normal transcription and translation of each element.
  • the first promoter comprises a Pol class II promoter.
  • the first promoter is selected from the group consisting of a 35S promoter, a UBQ promoter, an Actin promoter, a UBI promoter, or a combination thereof.
  • the second promoter comprises a Pol class II promoter.
  • the second promoter is selected from the group consisting of a tRNA promoter, a 35S promoter, a UBQ promoter, an Actin promoter, a UBI promoter, or a combination thereof.
  • the tRNA promoter is selected from the group consisting of a U6 promoter, a U3 promoter, or a combination thereof.
  • the Cas9 protein is selected from the group consisting of Cas9, Cas9n, or a combination thereof.
  • the source of the Cas9 protein is selected from the group consisting of Streptococcus pyogenes, Staphylococcus aureus, or a combination thereof.
  • the terminator is selected from the group consisting of a NOS terminator, a UBQ terminator, or a combination thereof.
  • the transcription termination sequence is selected from the group consisting of PolyA, PolyT, NOS terminator, UBQ terminator, or a combination thereof.
  • the polyT sequence is Poly(T) n , wherein n is 5-30.
  • the polyA sequence is Poly(A) n , wherein n is 5-30.
  • the first carrier and the second carrier are different carriers.
  • first nucleic acid construct and the second nucleic acid construct are on different vectors.
  • the vector is a binary expression vector that can be transfected or transformed into a plant cell.
  • the vector is a plant expression vector.
  • the vector is a pCambia vector.
  • the plant expression vector is selected from the group consisting of pCambia 1300, pCambia 3301, pCambia 2300, or a combination thereof.
  • the carrier is an Agrobacterium Ti carrier.
  • the carrier is cyclic or linear.
  • the plant comprises an angiosperm and a gymnosperm.
  • the gymnosperm plant is selected from the group consisting of Cycadaceae, Podocarpaceae, Araucariaceae, Pinaceae, Cedaraceae, Cypress, and three-pointed Cedaraceae, Taxaceae, Ephedra, Matroaceae, Monocytogenes, Centennial Orchidaceae, or a combination thereof.
  • the plant comprises a monocot and a dicot.
  • the plant comprises a herbaceous plant and a woody plant.
  • the herbaceous plant is selected from the group consisting of Solanaceae, Gramineae, Leguminous, or a combination thereof.
  • the woody plant is selected from the group consisting of kiwifruit, Rosaceae, Moraceae, or a combination thereof.
  • the plant is selected from the group consisting of: cruciferous plants, gramineous plants, legumes, Solanaceae, kiwifruit, Malvaceae, Paeonia, Rosaceae, Liliaceae, or combinations thereof .
  • the plant is selected from the group consisting of rice, cabbage, soybean, tomato, corn, tobacco, wheat, sorghum, or a combination thereof.
  • the gene is edited as a gene-spotted knock-in and/or substitution.
  • a third aspect of the invention provides a kit comprising the reagent combination of the second aspect of the invention.
  • the kit further contains a label or instructions.
  • a fourth aspect of the invention provides a method for genetically editing a plant or plant cell, comprising: integrating the donor DNA into a target site of a plant cell genome by NHEJ in the presence of donor DNA, and subsequently The sequence from the donor DNA integrated into the target site is subjected to DSB cleavage to perform homologous sequence-based homologous recombination (HDR), thereby introducing a DNA sequence of interest from the donor DNA at a target site.
  • HDR homologous sequence-based homologous recombination
  • the DNA sequence of interest comprises a single base, a plurality of bases, a nucleic acid fragment, or a single gene, or a plurality of genes.
  • the homologous recombination is based on a homologous sequence between the first homologous sequence of the DNA sequence of interest on the donor DNA and the upstream (or left) side of the target site. Homology, and homology between the second homologous sequence of the DNA sequence of interest on the donor DNA and the homologous sequence on the downstream side (or right side) of the target site.
  • the method comprises the steps of:
  • Y1 is a no or nucleotide sequence
  • Z1 is the first DSB sequence
  • Z2 is the first homologous sequence
  • Z3 is the DNA sequence of interest
  • Z4 is a second homologous sequence
  • Z5 is a second DSB sequence
  • Y2 is a no or nucleotide sequence
  • each "-" is independently a bond or a nucleotide linkage sequence
  • step (b) after NHEJ and HDR, the homologous sequences flanking the target site of the plant cell are homologous sequences in the donor DNA.
  • a fifth aspect of the invention provides a method of genetically editing a plant or plant cell, comprising the steps of:
  • the first nucleic acid construct has a structure of formula I from 5' to 3':
  • P1 is the first promoter
  • A1 is a coding sequence encoding a Cas9 protein
  • A2 is a terminator
  • the donor DNA comprises: a second nucleic acid construct, or a second vector comprising the second nucleic acid construct.
  • the second construct has the structure shown in formula II from 5'-3':
  • P2 is a second promoter
  • A3 is the coding sequence of gRNA
  • A4 is a no- or transcription termination sequence
  • A5 is an expression cassette for the nucleic acid construct of claim 1;
  • the first carrier and the second carrier are introduced simultaneously or sequentially.
  • the introduction is introduced by Agrobacterium.
  • the introduction is by a gene gun.
  • the gene editing is a fixed point knock-in and/or replacement.
  • the target gene contains a site recognized and cleaved by a site-cutting nuclease.
  • the method is performed once Genetic transformation is achieved.
  • the method may It can be achieved by two genetic transformations, or by one genetic transformation.
  • 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 cell prepared by the method of the sixth aspect of the invention or the seventh aspect of the invention.
  • a tenth aspect of the invention provides a transgenic plant prepared by the method of the eighth aspect of the invention.
  • Figure 1 shows a schematic representation of a repeated fragment-mediated genomic spotting method.
  • the donor DNA fragment used as a site-directed knock-in/replacement comprises a sequence to be replaced/knock-in (101), which is 5' phosphorylated (102) at both ends, having a homologous region to the genomic sequence to be knocked into. (104), and the site has a site-directed cleavage site outside the homologous region, or a site-specific insertion site (103).
  • This fragment can generally be amplified by synthesis or by PCR using the corresponding primer (106).
  • Figure 2 shows a schematic diagram of a new formation of a cleavage site.
  • a cleavage site (201) is newly formed after the donor DNA fragment is spotted into the genome.
  • FIG. 3 shows a schematic representation of base substitution of the rice SLR1 gene.
  • A Donor DNA fragment sequence and its structure. Wherein 301 is the sequence to be replaced; 302 is a partial sequence of the newly formed CRISPR/Cas9 target gRNA-1 after site insertion into the genome; and 303 is a sequence homologous to the genomic DNA (underlined).
  • B Base replacement process. 304 is the wild-type SLR1 target position DNA sequence; 305 is the target sequence to be replaced; 306 is the CRISPR/Cas9 target gRNA-1; 307 is the donor DNA fragment (double strand); 308 is the donor DNA site-specific insertion into the genome The newly formed CRISPR/Cas9 target gRNA-1.
  • the red partial sequence forms a continuous repeat sequence with the green partial sequence.
  • Figure 4 shows the results of base substitution test of rice SLR1 gene.
  • A Schematic diagram of rice SLR1 gene; B.T0 generation plant PCR detection results; C.T0 generation plant #30 Sanger sequencing results; D.SLR1 gene fixed-point replacement efficiency statistics table; E.T0 generation plant phenotype, From left to right, the plant phenotypes of Indel mutation, site-replacement and wild type occurred.
  • Figure 5 shows the results of site-specific integration of GFP in rice ACT1 and GST1.
  • the present inventors have extensively and intensively studied to screen a donor DNA having a specific repeat sequence structure by screening, and cleavage of a specific site of a target gene by site-cutting nuclease to integrate the donor DNA fragment. Enter the cutting site.
  • the donor DNA of the present invention carries a sequence homologous to the sequence of the genomic target gene, and the region outside the homologous sequence of the donor DNA or overlapping with the homologous sequence has one or more site-directed cleavage sites, or is inserted into the genome at a fixed point. A site-directed cleavage site is then formed.
  • the donor fragment contains a sequence homologous to the target site, a homologous repeat sequence can be formed, and a fixed-point cleavage site existing or formed between the repeat sequences can generate a DSB, thereby generating Very efficient HDR for further efficient site-specific knock-in and/or replacement, and further experiments have shown that modification of donor DNA fragments can efficiently integrate donor DNA fragments into the genome of recipient plants, efficiency ⁇ 12%, the recombination efficiency is more than 6 times higher than the traditional method (only using NHEJ or HDR). On the basis of this, the inventors completed the present invention.
  • 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.
  • 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, Staphylococcus aureus.
  • the term "coding sequence of a Cas protein” refers to a nucleotide sequence that encodes a Cas protein having cleavage activity. In the case where the inserted polynucleotide sequence is transcribed and translated to produce a functional 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.
  • Codons protein The coding sequence is specifically covered. Furthermore, 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.
  • knock-in refers to the substitution of a large fragment, especially when the sequence is completely different from the original gene.
  • substitution refers to the replacement of small fragments, several amino acids, and several bases.
  • "expression cassette&quot refers to a stretch of polynucleotide sequences comprising a gene to be expressed and a sequence component that expresses the desired element.
  • the components required for expression include a promoter and a polyadenylation signal sequence.
  • the expression cassettes of the invention may or may not contain other sequences including, but not limited to, enhancers, secretion signal peptide sequences, and the like.
  • primary genetic transformation refers to the conventional conversion of transformants by one exogenous DNA transformation and tissue culture.
  • two genetic transformations refers to a transformant or explant that is first targeted for knock-in (NHEJ pathway) by one genetic transformation; then the transformant or explant obtained by the first genetic transformation is The receptor, through a second genetic transformation, introduces a targeted cutting element to cleave the repeat sequence and achieve precise editing via the HDR pathway.
  • Non-homologous end joining directly pulls the ends of the double-stranded breaks apart without the help of any template, and then breaks with the help of DNA ligase The two strands rejoined.
  • HDR Homology directed repair
  • Targeting knock-in/replacement of a sequence at a given site in the plant genome ie targeted knock-in/replacement techniques, has been a pressing need for plant research and breeding, but existing methods are very inefficient.
  • the present invention employs a donor DNA fragment having a specific repeat sequence structure, and successively passes NHEJ and HDR to achieve efficient targeted knock-in/replacement in plants.
  • the present invention is directed to a method for efficient targeted knock-in/replacement of plants. As shown in Figure 1, the implementation steps are briefly summarized as follows:
  • the site-directed cleavage site may be external to the homologous sequence or partially overlap with the homologous sequence, and when the site-directed cleavage site partially overlaps with the homologous sequence, it is also regarded as a fixed-point cleavage site. The outside of the homologous sequence.
  • the donor DNA contains a base or fragment to be knocked in/replaced (Fig. 1A, 101), one or both of which are homologous fragments for HDR, greater than 15 bp in length, preferably 20 bp to 10 kb, more preferably 30 bp- 5kb, Figure 1, 104).
  • the region overlapping the homologous region outside the homologous region has one or more site-directed cleavage sites, which may be the same as the cleavage site of the genomic target, or may be different from the cleavage site of the genomic target;
  • the cleavage site may be completely contained within the donor DNA or may be formed after the donor DNA has been spotted into the genome (Fig. 2, 201).
  • the donor DNA fragment is preferably subjected to a 5'-terminal phosphorylation modification (Fig. 1, 102).
  • a 5'-terminal phosphorylation modification Fig. 1, 102
  • the terminal bases of the donor DNA fragment can be thiolated.
  • the preparation of the donor DNA fragment of the invention can be carried out by the following method:
  • the modified oligonucleotide single strand can be directly synthesized and directly annealed to generate double-stranded donor DNA;
  • Both ZFN, Talen, and CRISPR/Cas9 technologies can produce site-directed cleavage on the plant genome to produce double-stranded DNA breaks (DSBs). Therefore, DNA elements expressing these three site-cutting nucleases can be used in the present invention.
  • the DNA element can be a plasmid or a linear fragment. Since the CRISPR/Cas9 technology is relatively simple and efficient, the present invention preferably produces CRISPR/Cas9 for point-cutting on the plant genome.
  • the invention provides a reagent combination for gene editing, the reagent combination comprising (i) a first nucleic acid construct, or a first vector comprising the first nucleic acid construct, the first nucleic acid construct Has a structure of formula I from 5'-3':
  • P1 is the first promoter (including a Pol class II promoter, such as a 35S promoter, a UBQ promoter, an Actin promoter, a UBI promoter, etc.);
  • a Pol class II promoter such as a 35S promoter, a UBQ promoter, an Actin promoter, a UBI promoter, etc.
  • A1 is a coding sequence encoding a Cas9 protein
  • A2 is a terminator
  • P2 is a second promoter (including a PolII promoter, such as a tRNA promoter, a 35S promoter, a UBQ promoter, an Actin promoter, a UBI promoter, etc.);
  • a PolII promoter such as a tRNA promoter, a 35S promoter, a UBQ promoter, an Actin promoter, a UBI promoter, etc.
  • A3 is the coding sequence of gRNA
  • A4 is a transcription termination sequence (such as PolyA, PolyT, NOS terminator, UBQ terminator);
  • A5 is the nucleic acid construct of the first aspect of the invention.
  • the various elements used in the constructs of the present invention can be obtained by conventional methods, such as PCR methods, full artificial chemical synthesis, enzymatic cleavage methods, and then joined together by well-known DNA ligation techniques to form the constructs of the present invention. .
  • 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 nucleic acid construct constructed by the present invention can be introduced into a plant cell by a conventional plant recombination technique (for example, Agrobacterium transformation 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 transformation technology
  • the main feature of this vector is to drive the expression of Cas protein in CRISPR/Cas system by strong promoter such as 35S, Actin or UBI, and guide it to the target position in the genome by guide RNA, and cut the target by Cas protein. Plant targeted knock-in or replacement by NHEJ and HDR mechanisms.
  • proteins are usually linked by some flexible short peptides, namely Linker (linker peptide sequence).
  • Linker linker peptide sequence
  • the Linker can use XTEN.
  • the present invention selects specific promoters suitable for plant cells, such as 35S, Actin or UBI promoters and the like.
  • the expression cassette of the guide RNA suitable for plant cells is selected and constructed in a different vector from the open expression cassette (ORF) of the above proteins.
  • the vector is not particularly limited, and any binary vector may be, not limited to, a pCambia vector, and is not limited to these two kinds of resistance, as long as a carrier satisfying the following requirements can be used in the present invention: (1) It can be transformed into plants by Agrobacterium-mediated transformation; (2) allowing normal transcription of RNA; (3) allowing plants to acquire new resistance.
  • the vector is selected from the group consisting of pCambia 1300, pCambia 3301, pCambia 2300, or a combination thereof.
  • the modified donor DNA fragment and the DNA fragment donor expressing the site-cutting nuclease are introduced into a plant recipient.
  • 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.
  • site-recombinant cells are obtained by conventional 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 donor DNA fragment with a specific repeat sequence provided by the present invention can efficiently perform site-directed recombination (knock-in and/or replace) and knock, compared to the conventional direct-point precision knock-in/replace method by HDR.
  • the donor DNA of the present invention does not need to contain a screening tag.
  • the plant gene editing method of the present invention is simple and easy to popularize and apply.
  • the modified DNA fragment synthesized in vitro was used as a donor DNA fragment, and the CRISPR/Cas9 technology was used to accurately replace and delete multiple bases in the rice SLR1 gene.
  • the specific operation process is as follows.
  • the target gRNA-1 (SEQ ID NO.: 1) was designed for the region of the rice SLR1 gene to be replaced, and the gRNA-1 leader sequence was constructed into the rice CRISPR/Cas9 vector, wherein the OSU6-gRNA-1 sequence is shown in the sequence listing (SEQ. ID NO.: 2).
  • the donor DNA ends can be phosphorylated and thio-modified (5'P represents a 5'-terminal phosphorylation modification, and * indicates a base-to-base thio modification) to promote NHEJ; 82 bp is homologous to the sequence of the SLR1 target site; after site-integration of the fragment, an additional 5 bases (CCTCGG) at the end and a CRISPR/Cas9 target gRNA-1 re-formed on the genome sequence to facilitate HDR.
  • 5'P represents a 5'-terminal phosphorylation modification, and * indicates a base-to-base thio modification
  • the synthesized single-stranded oligonucleotide fragment was dissolved in water to 100 ⁇ M, diluted to 10 ⁇ M with annealing buffer (10 mM Tris-Cl, 0.1 mM EDTA, 50 mM NaCl, pH 8.0), and the PCR instrument was annealed to form a double-stranded donor.
  • annealing buffer 10 mM Tris-Cl, 0.1 mM EDTA, 50 mM NaCl, pH 8.0
  • PCR instrument was annealed to form a double-stranded donor.
  • DNA Dn-SLR1
  • SLR1-HR is a single-stranded donor DNA, which is used as a control group in a conventional HDR experiment.
  • the CRISPR/Cas9 plasmid, donor DNA and gold powder were mixed according to the following system, and rice calli were pretreated with hypertonic medium for 4 hours according to the operation manual of Bole PDS-1000 benchtop gene gun.
  • hygromycin as a screening marker, a positive resistant callus was obtained after screening by conventional tissue culture, and further differentiated to obtain a stably transformed plant.
  • the experimental group and the control group were subjected to tissue culture and screened for resistance callus, and further differentiated to obtain stable transformed plants.
  • 47 strains and 81 strains of T0 plants were obtained, and genomic DNA was extracted one by one for detection.
  • Primers were designed upstream and downstream of the target for PCR amplification. The primer sequences are shown in the following table:
  • Fig. 4A in which SLR1-F1 and SLR1-R1 amplify a genomic fragment, which serves as an internal reference; SLR1-F1 and SLR1-R1 specifically amplify a recombinant fragment, and detect site-directed recombination efficiency.
  • Fig. 4B The results of electrophoresis after PCR amplification are shown in Fig. 4B. Three samples in the experimental group were able to detect specific amplified fragments, but none of the control groups were detected. Further sequencing results showed that 2 of the 3 positive samples had the expected site-directed replacement (Fig. 4C) with a recombination efficiency of 4.2% (Fig. 4D).
  • plants with precise substitution at the SLR1 locus can produce a semi-dwarf phenotype, and the recombinant plants actually obtained in this example can indeed observe a significant semi-dwarf phenotype (Fig. 4E).
  • the experimental method based on the present invention succeeded in obtaining a rice plant which was site-recombined, confirming the practical application value of the present invention.
  • the DNA fragment was amplified by PCR and used as a donor DNA fragment.
  • the GFP gene was knocked into the 3' end of the rice high expression genes ACT1 and GST1 to form a fusion protein.
  • the specific procedure is as follows.
  • Target gRNA-2 and gRNA-3 (SEQ ID NO.: 9, 10) were designed for the 3' ends of rice ACT1 and GST1 genes, respectively, and these two guide sequences were constructed into rice CRISPR/Cas9 vector, of which OSU6-gRNA- 2 and OSU6-gRNA-3 sequences are shown in the Sequence Listing (SEQ ID NO.: 11, 12).
  • the donor DNA was amplified by PCR, and the primers used for amplification were shown in the following table. Among them, ACT1 knock-in donor DNA fragment (sequence 9, 1528 bp) was amplified from primers ACT1-F1 and NOS-R1; GST1 knock-in donor DNA fragment was amplified from primers GST1-F1 and NOS-R1 (sequence 10) , 1412bp).
  • the ends of the PCR amplification primers can be phosphorylated and thiolated (5'P represents 5'-terminal phosphorylation, and * represents thiol modification between bases) to promote NHEJ; about 400 bp of sequence and target in the fragment
  • the sequence of the position is homologous; after the spot is integrated, the sequence of the genome forms a repeat structure; the target sequence of the additional gRNA-2 or gRNA-3 at the end can be cleaved again, so that HDR occurs between the repeats, and GFP is realized.
  • the CRISPR/Cas9 plasmid, donor DNA and gold powder were mixed according to the following system, and rice calli were pretreated with hypertonic medium for 4 hours according to the operation manual of Bole PDS-1000 benchtop gene gun.
  • hygromycin as a screening marker, a positive resistant callus was obtained after screening by conventional tissue culture, and further differentiated to obtain a stably transformed plant.
  • the two groups of experiments were subjected to tissue culture and screened for resistance callus, and further differentiated to obtain stable transformed plants.
  • ACT1 and GST1 21 strains and 64 strains of T0 plants were obtained, and genomic DNA was extracted one by one for detection.
  • Primers were designed upstream and downstream of the target for PCR amplification. The primer sequences are shown in the following table:
  • the present invention combines NHEJ and HDR to achieve high-efficiency targeted knock-in/replacement, is simple to implement, and has low difficulty, and can be a routine experimental method.

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Abstract

提供一种具有特定重复序列的供体DNA，以及用于基因编辑的试剂组合。还提供了通过所述重复片段介导的植物定点重组方法，包括采用具有特定结构的供体DNA，借助定点切割核酸酶对目标基因的特定位点进行切割，并借助供体DNA中的同源臂将该供体DNA片段整合入切割位点。

Description

一种重复片段介导的植物定点重组方法 技术领域
本发明涉及生物技术领域，具体地，涉及一种重复片段介导的植物定点重组方法。
背景技术
基因组编辑技术包括锌指核酸技术(Zinc finger nuclease,ZFN)、转录激活因子样效应物核酸酶技术(transcription activator-like(TAL)effector nucleases,Talen)和CRISPR/Cas技术。这三种技术都可以在生物体基因组指定位点特异性的切割DNA产生双链断裂(DSB)，从而利用细胞自身具有的非同源末端连接或同源重组的特性，进行定点编辑。ZFN和Talen技术采用特异性的蛋白来引导进行基因组切割，其构建相对较复杂，编辑效率很低。
通常认为，在植物细胞内的DNA断裂修复机制以NHEJ为主导，HDR的概率相对非常低。因此，在进行基因组编辑时，往往以NHEJ修复后的结果为主。虽然通过NHEJ途径也可以进行定点的敲入或者替换，但是其编辑结果表现为靶点位置的Indel。并且通过NHEJ在水稻中的重组效率很低，最高只有2％左右。目前，通过HDR实现精准的敲入，在植物领域一直是一项难题。除非敲入或者替换序列为筛选标签，否则其实现效率非常低。到目前为止，在植物领域一直缺乏高效的基因组精准敲入/替换的方法。
综述所述，为了植物研究和育种的需要，本领域迫切需要开发一种高效的基因组精准敲入/替换技术。
发明内容
本发明的目的在于提供一种高效的基因组精准敲入/替换技术。
本发明第一方面提供了一种核酸构建物，所述核酸构建物具有从5’-3’的式I所示的结构：
Y1-Z1-Z2-Z3-Z4-Z5-Y2  (I)
其中，Y1为无或核苷酸序列；
Z1为第一DSB序列；
Z2为第一同源序列；
Z3为目的DNA序列；
Z4为第二同源序列；
Z5为第二DSB序列；
Y2为无或核苷酸序列；
并且，各“-”独立地为键或核苷酸连接序列。
在另一优选例中，所述的核苷酸连接序列包括长度为m个核苷酸的序列，其中，m为1-30，较佳地1-20，更佳地1-10(如1、2、3、4、5、6、7、8、9、10)。
在另一优选例中，各“-”均为键。
在另一优选例中，所述第一和第二DSB序列在gRNA的参与下被定位(识别)并切割。
在另一优选例中，所述各DSB序列能够被定点切割核酸酶识别并切割。
在另一优选例中，所述各DSB序列各自独立地：(a)自身含有切割位点，或(b)当所述核酸构建物通过NHEJ方式整合如靶位点后，形成的切割位点。
在另一优选例中，所述第一DSB序列可在第一同源序列5’端的外侧。
在另一优选例中，所述第一DSB序列与第一同源序列部分重叠。
在另一优选例中，所述第二DSB序列可在第二同源序列3’端的外侧。
在另一优选例中，所述第二DSB序列与第二同源序列部分重叠。
在另一优选例中，所述第一DSB序列与第二DSB序列是相同的或不同的。
在另一优选例中，所述的第一DSB序列与第二DSB序列与基因组靶位点的切割位点的DSB序列(“靶位点DSB序列”)是相同或不同的。
在另一优选例中，所述的第一DSB序列、第二DSB序列和所述靶位点DSB序列是相同的。
在另一优选例中，所述定点切割核酸酶选自下组：ZFN、Talen和CRISPR/Cas9、或其组合。
在另一优选例中，所述定点切割核酸酶为CRISPR/Cas9。
在另一优选例中，所述靶DNA序列能够被选自下组的酶识别并切割：Cas9、Cpf1、C2C1、C2C2、C2C3等CRISPR相关酶。
在另一优选例中，所述靶DNA序列能够被选自下组的酶识别并切割：Fok I。
在另一优选例中，所述第一DSB序列为10-50bp，优选15-30bp。
在另一优选例中，所述第二DSB序列为10-50bp，优选15-30bp。
在另一优选例中，所述第一同源序列为20bp-10kb，较佳地，30bp-5kb。
在另一优选例中，所述第二同源序列为20bp-10kb，较佳地，30bp-5kb。
在另一优选例中，所述目的DNA序列为待敲入和/或替换的序列。
在另一优选例中，所述目的DNA序列为1bp-10kb，较佳地，5bp-5kb。
在另一优选例中，所述Y1、Y2为保护碱基。
在另一优选例中，所述Y1、Y2的长度分别为1-50bp，较佳地，4-20bp。
在另一优选例中，所述第一同源序列与真核细胞基因组的靶位点一侧(如上游侧或左侧)的DNA序列的序列同源性H1，所述第二同源序列分别与真核细胞基因组的靶位点另一侧(如下游侧或右侧)的DNA序列的同源性H2，均≥90％，更佳地，≥95％。
在另一优选例中，所述第一同源序列、第二同源序列与真核细胞基因组的靶位点两侧的DNA序列分别构成同向重复序列(即第一同源序列与所述靶位点一侧(如上游侧或左侧)的DNA序列构成同向重复序列，而第二同源序列与所述靶位点另一侧(如下游侧或右侧)的DNA序列构成同向重复序列；反之亦然)。
在另一优选例中，所述真核细胞包括植物细胞。
在另一优选例中，所述植物包括被子植物和裸子植物。
在另一优选例中，所述裸子植物选自下组：苏铁科(Cycadaceae)、罗汉松科(Podocarpaceae)、南洋杉科(Araucariaceae)、松科(Pinaceae)、杉科、柏科、三尖杉科、红豆杉科、麻黄科、买麻藤科、单型科、百岁兰科、或其组合。
在另一优选例中，所述植物包括单子叶植物和双子叶植物。
在另一优选例中，所述植物包括草本植物和木本植物。
在另一优选例中，所述草本植物选自下组：茄科、禾本科植物、豆科植物、或其组合。
在另一优选例中，所述木本植物选自下组：猕猴桃科、蔷薇科、桑科、或其组合。
在另一优选例中，所述植物选自下组：十字花科植物、禾本科植物、豆科植物、茄科、猕猴桃科、锦葵科、芍药科、蔷薇科、百合科、或其组合。
在另一优选例中，所述的植物选自下组：水稻、白菜、大豆、番茄、玉米、烟草、小麦、高粱或其组合。
在另一优选例中，所述核酸构建物为单链DNA序列或双链DNA序列，优选为双链DNA序列。
在另一优选例中，所述核酸构建物的一条和/或两条DNA单链的5’末端进 行磷酸化修饰。
在另一优选例中，所述核酸构建物的两条DNA单链两个5’末端均进行磷酸化修饰。
在另一优选例中，所述核酸构建物的5’和/或3’端的最末端的一个或多个(如2、3、4、或5个)碱基间的磷酸二脂键进行硫代修饰。
在另一优选例中，所述核酸构建物上不带有筛选标签。
本发明第二方面提供了一种用于基因编辑的试剂组合，包括：
(i)第一核酸构建物，或含有所述第一核酸构建物的第一载体，所述第一核酸构建物具有从5’-3’的式I结构：
P1-A1-A2  (I)
其中，P1为第一启动子；
A1为编码Cas9蛋白编码序列；
A2为终止子；
并且，“-”为键或核苷酸连接序列；和
(ii)供体DNA元件，所述的供体DNA元件包括：本发明第一方面所述的核酸构建物，或用于表达所述核酸构建物的载体。
在另一优选例中，所述的供体DNA包括：第二核酸构建物，或含有所述第二核酸构建物的第二载体。
在另一优选例中，所述第二核酸构建物具有从5’-3’的式II所示的结构：
P2-A3-A4-A5  (II)
其中，P2为第二启动子；
A3为gRNA的编码序列；
A4为无或转录终止序列；
A5为权利要求1所述的核酸构建物的表达盒；
并且，“-”为键或核苷酸连接序列。
在另一优选例中，所述核苷酸连接序列为1-60nt。
在另一优选例中，所述核苷酸连接序列不影响各元件的正常转录和翻译。
在另一优选例中，所述第一启动子包括PolII类启动子。
在另一优选例中，所述第一启动子选自下组：35S启动子、UBQ启动子、Actin启动子、UBI启动子、或其组合。
在另一优选例中，所述第二启动子包括PolII类启动子。
在另一优选例中，所述第二启动子选自下组：tRNA启动子、35S启动子、UBQ启动子、Actin启动子、UBI启动子、或其组合。
在另一优选例中，所述tRNA启动子选自下组：U6启动子、U3启动子、或其组合。
在另一优选例中，所述Cas9蛋白选自下组：Cas9、Cas9n、或其组合。
在另一优选例中，所述Cas9蛋白的来源选自下组：酿脓链球菌(Streptococcus pyogenes)、葡萄球菌(Staphylococcus aureus)、或其组合。
在另一优选例中，所述终止子选自下组：NOS终止子、UBQ终止子、或其组合。
在另一优选例中，所述转录终止序列选自下组：PolyA、PolyT、NOS终止子、UBQ终止子、或其组合。
在另一优选例中，所述polyT序列为Poly(T) n，其中n为5-30。
在另一优选例中，所述polyA序列为Poly(A) n，其中n为5-30。
在另一优选例中，所述第一载体和所述第二载体为不同的载体。
在另一优选例中，所述第一核酸构建物和所述第二核酸构建物位于不同的载体上。
在另一优选例中，所述的载体为可转染或转化植物细胞的双元表达载体。
在另一优选例中，所述的载体为植物表达载体。
在另一优选例中，所述的载体为pCambia载体。
在另一优选例中，所述的植物表达载体选自下组：pCambia1300、pCambia3301、pCambia2300、或其组合。
在另一优选例中，所述的载体为农杆菌Ti载体。
在另一优选例中，所述载体是环状的或者是线性的。
在另一优选例中，所述植物包括被子植物和裸子植物。
在另一优选例中，所述裸子植物选自下组：苏铁科(Cycadaceae)、罗汉松科(Podocarpaceae)、南洋杉科(Araucariaceae)、松科(Pinaceae)、杉科、柏科、三尖杉科、红豆杉科、麻黄科、买麻藤科、单型科、百岁兰科、或其组合。
在另一优选例中，所述植物包括单子叶植物和双子叶植物。
在另一优选例中，所述植物包括草本植物和木本植物。
在另一优选例中，所述草本植物选自下组：茄科、禾本科植物、豆科植物、或其组合。
在另一优选例中，所述木本植物选自下组：猕猴桃科、蔷薇科、桑科、或其组合。
在另一优选例中，所述植物选自下组：十字花科植物、禾本科植物、豆科植物、茄科、猕猴桃科、锦葵科、芍药科、蔷薇科、百合科、或其组合。
在另一优选例中，所述的植物选自下组：水稻、白菜、大豆、番茄、玉米、烟草、小麦、高粱、或其组合。
在另一优选例中，所述基因编辑为基因定点敲入和/或替换。
本发明第三方面提供了一种试剂盒，所述试剂盒含有本发明第二方面所述的试剂组合。
在另一优选例中，所述试剂盒还含有标签或说明书。
本发明第四方面提供了一种对植物或植物细胞进行基因编辑的方法，包括：在供体DNA存在下，将所述供体DNA通过NHEJ方式整合入植物细胞基因组的靶位点，并随后对整合入靶位点的来自供体DNA的序列进行DSB切割，从而进行基于同源序列的同源重组(HDR)，由此在靶位点定点引入来自所述供体DNA的目的DNA序列。
在另一优选例中，所述的目的DNA序列包括单碱基、多个碱基、一个核酸片段、或单个基因、或多个基因。
在另一优选例中，所述的同源重组是基于所述供体DNA上的位于目的DNA序列的第一同源序列与靶位点上游侧(或左侧)的同源序列之间的同源性，以及所述供体DNA上的位于目的DNA序列的第二同源序列与靶位点下游侧(或右侧)的同源序列之间的同源性。
在另一优选例中，所述的方法包括步骤：
(a)提供一供体DNA和待编辑植物，其中所述供体DNA具有从5’-3’的式I所示的结构：
Y1-Z1-Z2-Z3-Z4-Z5-Y2  (I)
其中，Y1为无或核苷酸序列；
Z1为第一DSB序列；
Z2为第一同源序列；
Z3为目的DNA序列；
Z4为第二同源序列；
Z5为第二DSB序列；
Y2为无或核苷酸序列；
并且，各“-”独立地为键或核苷酸连接序列；
(b)在所述供体DNA存在下，对所述待编辑植物先后进行NHEJ和HDR，从而实现对所述植物细胞的靶基因进行编辑。
在另一优选例中，在步骤(b)中，经过NHEJ和HDR后，所述植物细胞的靶位点两侧的同源序列为所述供体DNA中的同源序列。
本发明第五方面提供了一种对植物或植物细胞进行基因编辑的方法，包括步骤：
(i)提供待编辑植物或植物细胞；
(ii)将第一核酸构建物或含所述第一核酸构建物的第一载体、以及供体DNA元件，所述的供体DNA元件包括：本发明第一方面所述的核酸构建物，或用于表达所述核酸构建物的载体导入所述待编辑植物的植物细胞，从而实现对所述植物或植物细胞的靶基因进行编辑；
其中，所述第一核酸构建物具有从5’-3’的式I结构：
P1-A1-A2  (I)
其中，P1为第一启动子；
A1为编码Cas9蛋白编码序列；
A2为终止子；
并且，“-”为键或核苷酸连接序列。
在另一优选例中，所述的供体DNA包括：第二核酸构建物，或含有所述第二核酸构建物的第二载体。
在另一优选例中，所述第二构建物具有从5’-3’的式II所示的结构：
P2-A3-A4-A5  (II)
其中，P2为第二启动子；
A3为gRNA的编码序列；
A4为无或转录终止序列；
A5为权利要求1所述的核酸构建物的表达盒；
并且，“-”为键或核苷酸连接序列。
在另一优选例中，所述第一载体、第二载体同时、或先后导入。
在另一优选例中，所述导入为通过农杆菌导入。
在另一优选例中，所述导入为通过基因枪导入。
在另一优选例中，所述的基因编辑为定点敲入和/或替换。
在另一优选例中，所述靶基因含有定点切割核酸酶识别并切割的位点。
在另一优选例中，当A5元件中的第一DSB序列和第二DSB序列与基因组靶位点的切割位点的DSB序列(“靶位点DSB序列”)相同时，所述方法通过一次遗传转化实现。
在另一优选例中，当A5元件中的第一DSB序列和第二DSB序列与基因组靶位点的切割位点的DSB序列(“靶位点DSB序列”)不相同时，所述方法可以通过两次遗传转化实现，也可通过一次遗传转化实现。
本发明第六方面提供了一种制备转基因植物细胞的方法，包括步骤：
(i)将本发明第一方面所述的核酸构建物或本发明第二方面所述的试剂组合导入或转染植物细胞，使得本发明第一方面所述的核酸构建物或本发明第二方面所述试剂组合中的所述构建物与所述植物细胞中的染色体发生定点敲入和/或替换，从而制得所述转基因植物细胞。
在另一优选例中，所述的转染采用农杆菌转化法或基因枪轰击法。
本发明第七方面提供了一种制备转基因植物细胞的方法，包括步骤：
(i)将本发明第一方面所述的核酸构建物或本发明第二方面所述的试剂组合导入或转染植物细胞，使得所述植物细胞含有本发明第一方面所述的核酸构建物或本发明第二方面所述试剂组合中的所述构建物，从而制得所述转基因植物细胞。
本发明第八方面提供了一种制备转基因植物的方法，包括步骤：
本发明第六方面或本发明第七方面所述方法制备的所述转基因植物细胞再生为植物体，从而获得所述转基因植物。
本发明第九方面提供了一种转基因植物细胞，所述的植物细胞是用本发明第六方面或本发明第七方面所述的方法制备的。
本发明第十方面提供了一种转基因植物，所述的植物是用本发明第八方面所述的方法制备的。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了重复片段介导的基因组定点敲入方法示意图。(A)用作定点敲入/替换的供体DNA片段包含待替换/敲入序列(101)，其两端5’磷酸化修饰(102)，与待敲入位置的基因组序列具有同源区域(104)，并且该同源区外侧具有一个定点切割位点，或定点插入基因组后可以形成一个定点切割位点(103)。该片段一般可以通过合成或者采用相应的引物(106)PCR扩增得到。(B)由核酸酶靶向切割基因组位点造成DSB，(C)使供体DNA片段通过NHEJ整合至目标位点；由于供体片段含有与靶位点同源的序列，可以形成同向的重复序列，其重复序列间存在或者形成的定点切割位点可以产生DSB；由于重复序列间的DSB可以产生非常高效的HDR(105)，(D)借助细胞的这一特性实现定点精准敲入/替换。
图2显示了新形成一个切割位点示意图。供体DNA片段定点插入基因组后新形成一个切割位点(201)。
图3显示了水稻SLR1基因碱基替换示意图。A.供体DNA片段序列及其结构。其中301为待替换的序列；302为定点插入基因组后新形成的CRISPR/Cas9靶点gRNA-1的部分序列；303为与基因组DNA同源的序列(下划线)。B.碱基替换个过程。304为野生型SLR1靶点位置DNA序列；305为需要替换的目标序列；306为CRISPR/Cas9靶点gRNA-1；307为供体DNA片段(双链)；308为供体DNA定点插入基因组后新形成的CRISPR/Cas9靶点gRNA-1。其中红色部分序列与绿色部分序列形成连续的重复序列。
图4显示了水稻SLR1基因碱基替换检测结果。A.水稻SLR1基因示意图；B.T0代植株定点替换的PCR检测结果；C.T0代植株#30的Sanger测序结果；D.SLR1基因定点替换效率统计表；E.T0代植株的表型，从左到右分别为发生Indel突变、定点替换和野生型的植株表型。
图5显示了水稻ACT1和GST1定点整合GFP的结果。A.GFP整合至ACT1和GST1的3’端的示意图；B.T0代植株PCR检测结果，其中上部为特异性扩增结果，下部为内参对照；C.T0代植株ACT#01和GST#03的Sanger测序结果；E.GFP靶向敲入结果统计表。
具体实施方式
本发明人经过广泛而深入地研究，通过大量筛选，筛选出一种具有特定重复序列结构的供体DNA，借助定点切割核酸酶对目标基因的特定位点进行切割， 将该供体DNA片段整合入切割位点。
本发明的供体DNA带有与基因组靶基因序列同源的序列，并且该供体DNA的同源序列外侧或与同源序列重叠的区域具有一个或多个定点切割位点，或定点插入基因组后会形成一个定点切割位点，由于该供体片段含有与靶位点同源的序列，可以形成同向的重复序列，其重复序列间存在或者形成的定点切割位点可以产生DSB，从而产生非常高效的HDR，进一步实现高效的定点敲入和/或替换，并且进一步的实验表明，对供体DNA片段进行修饰，可高效的将供体DNA片段定点整合至受体植物的基因组中，效率≥12％，比传统方法(仅用NHEJ或HDR)的重组效率提高了6倍以上。在此基础上，本发明人完成了本发明。
术语
如本文所用，术语“植物启动子”指能够在植物细胞中启动核酸转录的核酸序列。该植物启动子可以是来源于植物、微生物(如细菌、病毒)或动物等，或者是人工合成或改造过的启动子。
如本文所用，术语“Cas蛋白”指一种核酸酶。一种优选的Cas蛋白是Cas9蛋白。典型的Cas9蛋白包括(但并不限于)：来源于酿脓链球菌(Streptococcus pyogenes)、葡萄球菌(Staphylococcus aureus)的Cas9。如本文所用，术语“Cas蛋白的编码序列”指编码具有切割活性的Cas蛋白的核苷酸序列。在插入的多聚核苷酸序列被转录和翻译从而产生功能性Cas蛋白的情况下，技术人员会认识到，因为密码子的简并性，有大量多聚核苷酸序列可以编码相同的多肽。另外，技术人员也会认识到不同物种对于密码子具有一定的偏好性，可能会根据在不同物种中表达的需要，会对Cas蛋白的密码子进行优化，这些变异体都被术语“Cas蛋白的编码序列”所具体涵盖。此外，术语特定地包括了全长的、与Cas基因序列基本相同的序列，以及编码出保留Cas蛋白功能的蛋白质的序列。
如本文所用，术语“植物”包括全植株、植物器官(如叶、茎、根等)、种子和植物细胞以及它们的子代。可用于本发明方法的植物的种类没有特别限制，一般包括任何可进行转化技术的高等植物类型，包括单子叶、双子叶植物和裸子植物。
如本文所用，术语“敲入”指大片段的置换，尤其是当置换上的是和原基因完全不同的序列时。
如本文所用，术语“替换”指小片段、几个氨基酸、几个碱基的置换。
如本文所用，术语“表达盒”是指含有待表达基因以及表达所需元件的序 列组件的一段多聚核苷酸序列。表达所需的组件包括启动子和聚腺苷酸化信号序列。此外，本发明的表达盒还可以含有或不含有其他序列，包括(但并不限于)：增强子、分泌信号肽序列等。
如本文所用，术语“一次遗传转化”指：常规的通过一次外源DNA转化和组织培养得到转化子。
如本文所用，术语“两次遗传转化”指：首先通过一次遗传转化靶向敲入(NHEJ途径)的转化子或外植体；然后以第一次遗传转化得到的转化子或外植体为受体，通过第二次遗传转化导入靶向切割元件切割重复序列，经HDR途径实现精准编辑。
NHEJ
非同源性末端连接(Non-homologous end joining,NHEJ)：不需要任何模版的帮助，直接将双链裂断的末端彼此拉近，再藉由DNA连接酶(ligase)的帮助下，将断裂的两股重新接合。
HDR
同源重组(Homology directed repair,HDR)：一种细胞内依赖于同源DNA片段进行双链裂断修复的机制。
靶向敲入/替换
在植物基因组的指定位点靶向敲入/替换一段序列，即靶向敲入/替换技术，一直是植物研究和育种迫切需要的技术，但现有的方法效率非常低。本发明采用具有特定重复序列结构的供体DNA片段，先后经过NHEJ和HDR，在植物中实现了高效的靶向敲入/替换。
本发明人经过广泛而深入的研究和实验，发现具有特定重复序列结构的DNA供体先后经过NHEJ和HDR确实能够极大的提高植物基因组编辑中靶向敲入/替换的效率。因此，本发明旨在提供一种适用于植物的高效的靶向敲入/替换的方法。如图1所示，其实施步骤简要概括如下：
a)体外制备DNA片段用作定点敲入/替换，其特征为1)该片段的两端或一端与待敲入位置的基因组序列具有同源区域；2)该同源区外侧具有一个定点切割位点，或定点插入基因组后可以形成一个定点切割位点，以便转化细胞后产生DSB；和
b)体外制备表达定点切割核酸酶的DNA片段；和
c)将上述两个DNA片段转化植物受体，并在合适的条件下，使转化的植物细胞中的DNA表达核酸酶，定点切割目标位点造成双链断裂，使供体DNA片段通过NHEJ整合至目标位点；由于供体片段含有与靶位点同源的序列，可以形成同向的重复序列，其重复序列间存在或者形成的定点切割位点可以产生DSB；由于重复序列间的DSB可以产生非常高效的HDR，借助细胞的这一特性实现定点精准敲入/替换。
在本发明中，所述定点切割位点可在同源序列的外侧，也可与同源序列部分重叠，当所述定点切割位点与同源序列部分重叠时，也被视为定点切割位于同源序列的外侧。
供体DNA片段制备
供体DNA内包含有待敲入/替换的碱基或片段(图1A，101)，其一侧或两侧为用于HDR的同源片段，长度大于15bp，优选20bp-10kb，更优选30bp-5kb，图1，104)。该同源区外侧与同源区重叠的区域具有一个或多个定点切割位点，该切割位点可以与基因组靶点的切割位点一样，也可以与基因组靶点的切割位点不同；该切割位点可以完整的包含在供体DNA内，也可以是待该供体DNA定点插入基因组后可以形成的(图2，201)。为了增加NHEJ的效率，优选地将供体DNA片段进行5’端磷酸化修饰(图1，102)。为了阻止植物细胞核酸外切酶的降解，供体DNA片段的末端碱基间可进行硫基化修饰。
在一优选实施方式中，本发明的供体DNA片段的制备可以通过以下方法进行：
1)对于较短的供体DNA的制备(通常120bp以内)，可以直接合成修饰的寡核苷酸单链后直接退火生成双链的供体DNA；
2)对与较长的供体DNA的制备，可以采用硫基化修饰的引物通过PCR扩增获得(图1)；
3)或者直接通过酶切质粒等外源DNA制备。
定点切割核酸酶DNA构建物的制备
ZFN、Talen和CRISPR/Cas9技术都可以在植物基因组上制造定点切割产生双链DNA断裂(DSB)。因此，表达这3种定点切割核酸酶的DNA元件都可以用 于本发明。该DNA元件可以是质粒，也可以是线性片段。由于CRISPR/Cas9技术相对简单高效，本发明优选CRISPR/Cas9在植物基因组上制造定点切割。
用于基因编辑的试剂组合
本发明提供了一种签用于基因编辑的试剂组合，所述试剂组合包括(i)第一核酸构建物，或含有所述第一核酸构建物的第一载体，所述第一核酸构建物具有从5’-3’的式I结构：
P1-A1-A2  (I)
其中，P1为第一启动子(包括PolII类启动子，如35S启动子、UBQ启动子、Actin启动子、UBI启动子等)；
A1为编码Cas9蛋白编码序列；
A2为终止子；
并且，“-”为键或核苷酸连接序列；和
(ii)第二核酸构建物，或含有所述第二核酸构建物的第二载体，所述第二核酸构建物具有从5’-3’的式II所示的结构：
P2-A3-A4-A5  (II)
其中，P2为第二启动子(包括PolII类启动子，如tRNA启动子、35S启动子、UBQ启动子、Actin启动子、UBI启动子等)；
A3为gRNA的编码序列；
A4为转录终止序列(如PolyA、PolyT、NOS终止子、UBQ终止子)；
A5为本发明第一方面所述的核酸构建物；
并且，“-”为键或核苷酸连接序列。
本发明的构建物中所用的各种元件可以用常规方法，如PCR方法、全人工化学合成法、酶切方法获得，然后通过熟知的DNA连接技术连接在一起，就形成了本发明的构建物。
将本发明的载体转化植物细胞从而介导本发明的载体对植物细胞染色体进行整合，制得转基因植物细胞。
将本发明的转基因植物细胞再生为植物体，从而获得转基因植物。
将本发明构建好的上述核酸构建物，通过常规的植物重组技术(例如农杆菌转化技术)，可以导入植物细胞，从而获得携带所述核酸构建物(或带有所述核酸构建物的载体)的植物细胞，或获得基因组中整合有所述核酸构建物的植物细胞。
载体构建
该载体的主要特征是利用35S、Actin或UBI等强启动子驱动CRISPR/Cas系统中的Cas蛋白的表达，并由guide RNA引导至基因组中的靶点位置，由Cas蛋白切割靶点，并先后通过NHEJ和HDR机制进行植物靶向敲入或替换。
一般的，为了增加蛋白的活性，蛋白间一般通过一些柔性短肽连接，即Linker(连接肽序列)。优选的，该Linker可以选用XTEN。
为了增加敲入和/或替换效率，本发明选择特定的适用于植物细胞的启动子，比如35S、Actin或UBI启动子等。选择适用于植物细胞的guide RNA的表达框，并将其与上述蛋白的开放表达框(ORF)构建在不同的载体。
在本发明中，所述载体没有特别限制，任何双元载体都可以，不限于pCambia载体，也不限于这两种抗性，只要满足如下要求的载体都可以用在本发明中：(1)能通过农杆菌介导，转化进入植物中；(2)让RNA正常转录；(3)让植物获得新的抗性。
在一优选实施方式中，所述载体选自下组：pCambia1300、pCambia3301、pCambia2300、或其组合。
遗传转化
在本发明的一优选实施方式中，将修饰的供体DNA片段与表达定点切割核酸酶的DNA片段供体导入到植物受体中。导入方法包括但不局限于：基因枪法、显微注射法、电击法、超声波法和聚乙二醇(PEG)介导法等。受体植物包括但不限于水稻、大豆、番茄、玉米、烟草、小麦、高粱等。上述两种DNA片段导入植物细胞后，推测通过一下几步实现精确整合：
1)核酸酶切割靶位点产生DSB；
2)供体DNA片段通过NHEJ整合至目标位点：供体DNA片段5’磷酸化可以促进NHEJ；末端碱基间进行硫基化修饰可以阻止细胞内核酸外切酶的降解；
3)供体DNA片段定点整合后，由于含有与靶位点同源的序列，因此可以形成同向的重复序列结构；
4)核酸酶切割重复序列间存在或者新形成的切割位点产生DSB；
5)由于重复序列间的DSB可以产生非常高效的HDR，借助细胞的这一特性实现定点精准敲入/替换。
最后，定点重组的细胞通过常规的组织培养获得植株。
应用
本发明可以用于植物基因工程领域，用于植物研究和育种，尤其是具有经济价值的农作物和林业作物的遗传改良。
本发明的主要优点包括：
(1)相比传统的直接通过HDR实现定点精准敲入/替换方法，本发明提供的具有特定重复序列的供体DNA片段，可以高效的实现定点重组(敲入和/或替换)，并且敲入和/或替换效率≥12％(与传统方法(仅用NHEJ或HDR)相比，提高了6倍以上)，可以广泛的用于植物研究和育种。
(2)本发明的供体DNA不需含有筛选标签。
(3)本发明的植物基因编辑方法简便，容易推广应用。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。本发明中所涉及的实验材料和试剂如无特殊说明均可从市售渠道获得。
实施例1水稻SLR1基因碱基替换
利用体外合成的修饰后的DNA片段作为供体DNA片段，结合CRISPR/Cas9技术，对水稻SLR1基因内的多个碱基进行了精准的替换和删除。具体操作流程如下。
CRISPR/Cas9载体制备
针对水稻SLR1基因的待替换区域设计靶点gRNA-1(SEQ ID NO.:1)，将该gRNA-1引导序列构建至水稻CRISPR/Cas9载体，其中OSU6-gRNA-1序列见序列表(SEQ ID NO.:2)。
供体片段设计和体外制备
如图3A和3B所示，该供体DNA末端可进行磷酸化和硫代修饰(5’P代表 5’端磷酸化修饰，*号代表碱基间硫代修饰)，以促进NHEJ；片段中82bp与SLR1靶点位置的序列同源；待片段定点整合后，末端额外的5个碱基(CCTCGG)与基因组上的序列重新形成的CRISPR/Cas9靶点gRNA-1，以便于HDR。
在体外合成修饰或未修饰的单链寡核苷酸片段：
Figure PCTCN2019082095-appb-000001
合成后的单链寡核苷酸片段用水溶解至100μM后，用退火缓冲液(10mM Tris-Cl,0.1mM EDTA,50mM NaCl,pH8.0)稀释至10μM，PCR仪退火结合成双链供体DNA(Dn-SLR1)。其中，SLR1-HR为单链的供体DNA，用作常规HDR实验，为对照组。
基因枪转化水稻愈伤组织
将CRISPR/Cas9质粒、供体DNA与金粉按下列体系混合，按照伯乐PDS-1000台式基因枪的操作手册，转化用高渗培养基预处理4小时的水稻愈伤组织。利用潮霉素作为筛选标签，经常规组织培养筛选后获得阳性的抗性愈伤，进一步分化获得稳定转化植株。
Figure PCTCN2019082095-appb-000002
Figure PCTCN2019082095-appb-000003
靶向敲入效率检测
将实验组和对照组经组织培养筛选后的抗性愈伤，进一步分化获得稳定转化植株。实验组和对照组共计分别获得47株和81株T0代植株，逐一提取基因组DNA进行检测。在靶点上下游设计引物进行PCR扩增检测，引物序列见下表：
Figure PCTCN2019082095-appb-000004
如图4A所示，其中，SLR1-F1和SLR1-R1扩增基因组片段，用作内参；SLR1-F1和SLR1-R1特异性地扩增重组片段，检测定点重组效率。PCR扩增后电泳检测结果如图4B所示，实验组有3个样品能够检测到特异性的扩增片段，而对照组全部都未检测到。进一步的测序结果显示，这3个阳性样品中有2个发生了预期的定点替换(图4C)，其重组效率为4.2％(图4D)。根据实验设计，SLR1位点发生精准替换的植株可以产生半矮表型，本实施例实际获得的重组植株确实可以观察到显著的半矮表型(图4E)。
与传统的HDR实验(对照组)相比，基于本发明的实验方法成功获得了定点重组的水稻植株，证实了本发明的实际应用价值。
实施例2 GFP的定点敲入实验
利用PCR扩增得到DNA片段作为供体DNA片段，结合CRISPR/Cas9技术，在水稻高表达基因ACT1和GST1的3’端定点敲入GFP基因，使之形成融合蛋白，具体操作流程如下。
CRISPR/Cas9载体制备
针对水稻ACT1和GST1基因3’端分别设计靶点gRNA-2和gRNA-3(SEQ ID NO.:9、10)，将这两个引导序列构建至水稻CRISPR/Cas9载体，其中OSU6-gRNA-2和OSU6-gRNA-3序列见序列表(SEQ ID NO.:11、12)。
供体DNA片段的设计与制备
如图5A所示，该供体DNA通过PCR扩增得到，扩增所用引物见下表。其中，由引物ACT1-F1和NOS-R1扩增ACT1敲入的供体DNA片段(序列9，1528bp)；由引物GST1-F1和NOS-R1扩增GST1敲入的供体DNA片段(序列10，1412bp)。PCR扩增引物的末端可进行磷酸化和硫代修饰(5’P代表5’端磷酸化修饰，*号代表碱基间硫代修饰)，以促进NHEJ；片段中约400bp的序列与靶点位置的序列同源；待片段定点整合后，与基因组的序列形成重复结构；其末端额外的gRNA-2或gRNA-3的靶序列可以再次产生切割，从而使重复序列间发生HDR，使GFP实现精准的靶向敲入。
Figure PCTCN2019082095-appb-000005
基因枪转化水稻愈伤组织
将CRISPR/Cas9质粒、供体DNA与金粉按下列体系混合，按照伯乐PDS-1000台式基因枪的操作手册，转化用高渗培养基预处理4小时的水稻愈伤组织。利用潮霉素作为筛选标签，经常规组织培养筛选后获得阳性的抗性愈伤，进一步分化获得稳定转化植株。
Figure PCTCN2019082095-appb-000006
靶向敲入效率检测
将两组实验经组织培养筛选后的抗性愈伤，进一步分化获得稳定转化植株。ACT1和GST1的实验共计分别获得21株和64株T0代植株，逐一提取基因组DNA进行检测。在靶点上下游设计引物进行PCR扩增检测，引物序列见下表：
Figure PCTCN2019082095-appb-000007
Figure PCTCN2019082095-appb-000008
由于ACT1和GST1在水稻中具有较高的表达量，融合GFP后可以通过荧光判断其是否整合。荧光镜检显示，分别有3株ACT1和8株GST1的植株具有显著的GFP荧光信号(图5D)。采用ACT1-F1+GFP-R1和GST1-F1+GFP-R1分别检测这两个位点的GFP整合结果，发现这几株具有GFP荧光的植株都能扩增出目标条带(图5B)。进一步的测序结果显示，这些植株确实发生了预期的靶向敲入(图5C)，其重组效率分别为14.3％和12.5％(与仅用NHEJ或HDR的传统方法相比，分别提高了至少7倍和6倍)(图5E)。以上实验表明，基于本发明的实验方法成功获得了定点重组的水稻植株，进一步证实了本发明的实际应用价值。
如图4D所示，仅用HDR时，重组效率为0，无法得到靶向敲入/替换的植株，其他位点(包括水稻NRT1.1b基因和EPSPS基因)的单独HDR实验的重组效率也为0，并且也未成功实现靶向碱基替换。
因此，本发明结合NHEJ和HDR，实现了高效率的靶向敲入/替换，实施简易，难度低，可以成为一种常规的实验方法。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (22)

  1. 一种核酸构建物，其特征在于，所述核酸构建物具有从5’-3’的式I所示的结构：
    Y1-Z1-Z2-Z3-Z4-Z5-Y2      (I)
    其中，Y1为无或核苷酸序列；
    Z1为第一DSB序列；
    Z2为第一同源序列；
    Z3为目的DNA序列；
    Z4为第二同源序列；
    Z5为第二DSB序列；
    Y2为无或核苷酸序列；
    并且，各“-”独立地为键或核苷酸连接序列。
  2. 如权利要求1所述的核酸构建物，其特征在于，所述第一和第二DSB序列在gRNA的参与下被定位并切割。
  3. 如权利要求1所述的核酸构建物，其特征在于，所述各DSB序列能够被定点切割核酸酶识别并切割。
  4. 如权利要求1所述的核酸构建物，其特征在于，所述各DSB序列各自独立地：(a)自身含有切割位点，或(b)当所述核酸构建物通过NHEJ方式整合如靶位点后，形成的切割位点。
  5. 如权利要求3所述的核酸构建物，其特征在于，所述定点切割核酸酶选自下组：ZFN、Talen和CRISPR/Cas9、或其组合。
  6. 如权利要求1所述的核酸构建物，其特征在于，所述第一DSB序列、第二DSB序列能够被选自下组的酶识别并切割：Cas9、Cpf1、C2C1、C2C2、C2C3等CRISPR相关酶。
  7. 如权利要求1所述的核酸构建物，其特征在于，所述第一DSB序列、第二DSB序列能够被选自下组的酶识别并切割：Fok I。
  8. 如权利要求1所述的核酸构建物，其特征在于，所述目的DNA序列为待敲入和/或替换的序列。
  9. 如权利要求1所述的核酸构建物，其特征在于，所述核酸构建物为单链DNA序列或双链DNA序列，优选为双链DNA序列。
  10. 如权利要求1所述的核酸构建物，其特征在于，所述核酸构建物的一条和/或两条DNA单链的5’末端进行磷酸化修饰。
  11. 如权利要求1所述的核酸构建物，其特征在于，所述核酸构建物的5’和/或3’端的最末端的一个或多个(如2、3、4、或5个)碱基间的磷酸二脂键进行硫代修饰。
  12. 一种用于基因编辑的试剂组合，其特征在于，包括：
    (i)第一核酸构建物，或含有所述第一核酸构建物的第一载体，所述第一核酸构建物具有从5’-3’的式I结构：
    P1-A1-A2     (I)
    其中，P1为第一启动子；
    A1为编码Cas9蛋白编码序列；
    A2为终止子；
    并且，“-”为键或核苷酸连接序列；和
    (ii)供体DNA元件，所述的供体DNA元件包括：权利要求1所述的核酸构建物，或用于表达所述核酸构建物的载体。
  13. 如权利要求12所述的试剂组合，其特征在于，所述的供体DNA元件包括：第二核酸构建物，或含有所述第二核酸构建物的第二载体。
  14. 如权利要求13所述的试剂组合，其特征在于，所述第二核酸构建物具有从5’-3’的式II所示的结构：
    P2-A3-A4-A5      (II)
    其中，P2为第二启动子；
    A3为gRNA的编码序列；
    A4为无或转录终止序列；
    A5为权利要求1所述的核酸构建物的表达盒；
    并且，“-”为键或核苷酸连接序列。
  15. 如权利要求12所述的试剂组合，其特征在于，所述基因编辑为基因定点敲入和/或替换。
  16. 一种试剂盒，其特征在于，所述试剂盒含有权利要求12所述的试剂组合。
  17. 一种对植物或植物细胞进行基因编辑的方法，其特征在于，包括：在供体DNA存在下，将所述供体DNA通过NHEJ方式整合入植物细胞基因组的靶位点，并随后对整合入靶位点的来自供体DNA的序列进行DSB切割，从而进行基于同源序 列的同源重组(HDR)，由此在靶位点定点引入来自所述供体DNA的目的DNA序列。
  18. 一种对植物或植物细胞进行基因编辑的方法，其特征在于，包括步骤：
    (i)提供待编辑植物或植物细胞；
    (ii)将第一核酸构建物或含所述第一核酸构建物的第一载体、以及供体DNA元件，所述的供体DNA元件包括：权利要求1所述的核酸构建物，或用于表达所述核酸构建物的载体导入所述待编辑植物的植物细胞，从而实现对所述植物细胞的靶基因进行编辑；
    其中，所述第一核酸构建物具有从5’-3’的式I结构：
    P1-A1-A2     (I)
    其中，P1为第一启动子；
    A1为编码Cas9蛋白编码序列；
    A2为终止子；
    并且，“-”为键或核苷酸连接序列。
  19. 一种制备转基因植物细胞的方法，其特征在于，包括步骤：
    (i)将权利要求1所述的核酸构建物或权利要求12所述的试剂组合导入或转染植物细胞，使得权利要求1所述的核酸构建物或权利要求12所述试剂组合中的所述构建物与所述植物细胞中的染色体发生定点敲入和/或替换，从而制得所述转基因植物细胞。
  20. 一种制备转基因植物细胞的方法，其特征在于，包括步骤：
    (i)将权利要求1所述的核酸构建物或权利要求12所述的试剂组合导入或转染植物细胞，使得所述植物细胞含有权利要求1所述的核酸构建物或权利要求12所述试剂组合中的所述构建物，从而制得所述转基因植物细胞。
  21. 一种制备转基因植物的方法，其特征在于，包括步骤：
    权利要求19或权利要求20所述方法制备的所述转基因植物细胞再生为植物体，从而获得所述转基因植物。
  22. 一种转基因植物细胞，其特征在于，所述的植物细胞是用权利要求19或20所述的方法制备的。
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