Method for site-specific replacement or insertion of DNA fragments in plant genome

Abstract

The invention discloses a method for site-specific replacement or insertion of DNA fragments in a plant genome, and belongs to the technical field of biology. The method disclosed by the invention comprises the following steps: editing a genome of a plant protoplast by adopting a CRISPR / Cas9 system and taking a double-stranded DNA repair template as a donor template to obtain an edited protoplast; and carrying out culture regeneration on the edited protoplast by utilizing a plant protoplast transformation method to obtain a site-directed mutagenesis plant in the genome so as to realize site-directed mutagenesis of the target plant genome. The method disclosed by the invention can be used for accurately replacing DNA in a plant genome at a fixed point. According to the invention, a recognition sequence of recombinase can be accurately inserted into a genome at a fixed point through homologous recombination, and then accurate fixed-point replacement of a gene large fragment is realized by virtue of a specific recombinase system.

Description

Technical Field

[0001] The present invention relates to a method for site-directed replacement or insertion of DNA fragments in the genome of plants in the field of biotechnology. Background Art

[0002] Gene targeting (GT) is a technology for site-directed modification or replacement of genomic sequences and plays an important role in basic research and biotechnology. Gene targeting in plants is usually achieved by homologous recombination (HR) between the genome and exogenous DNA fragments. However, the frequency of homologous recombination in plant cells is very low, which hinders the widespread application of this technology. The CRISPR / Cas9 (Clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9) system can cleave DNA at specific sites in the organism genome and generate double-strand breaks. The double-strand breaks of DNA are mainly repaired by the non-homologous end-joining (NHEJ) pathway, and homology-directed repair (HDR) occurs with an extremely low probability.

[0003] The gene targeting technology mediated by homology-directed repair (HDR) can achieve the insertion and replacement of any sequence. Although CRISPR / Cas9 has been used to improve the efficiency of homologous repair in plants, most of the technologies rely on Agrobacterium-mediated genetic transformation. The T-DNA provided by Agrobacterium transformation cannot provide a large number of repair templates, and the number of repair templates in cells is an important factor determining the efficiency of homologous repair. Therefore, the efficiency of gene targeting relying on homologous repair in plants is still very low at present. Efficient site-directed insertion of DNA fragments in the plant genome remains a difficult problem. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to site-directed replace or insert DNA fragments in the plant genome.

[0005] To solve the above technical problem, the present invention first provides a method for site-directed mutagenesis in the plant genome, including: using the CRISPR / Cas9 system and taking a double-stranded DNA repair template as a donor template to edit the genome of plant protoplasts to obtain edited protoplasts; using the plant protoplast transformation method to culture and regenerate the edited protoplasts to obtain a plant with site-directed mutagenesis in the genome, thereby achieving site-directed mutagenesis of the genome of the target plant; The double-stranded DNA repair template sequentially contains an upstream homologous arm identical to the plant genomic sequence, a target DNA fragment, and a downstream homologous arm identical to the plant genomic sequence; The target DNA fragment contains (or is) a fragment after site-directed mutagenesis, and the fragment after site-directed mutagenesis is located between the upstream homologous arm and the downstream homologous arm; The ends of the double-stranded DNA repair template are modified with biotin and / or phosphorothioate linkages.

[0006] In the double-stranded DNA repair template, the upstream homologous arm, the target DNA fragment, and the downstream homologous arm are sequentially connected.

[0007] In the above method, the biotin modification can be to modify the 5'-terminal nucleotides of the two strands of the double-stranded DNA repair template with biotin.

[0008] In the above method, the phosphorothioate linkage modification can be the phosphorothioate linkage modification between 2-5 consecutive nucleotide residues at the 5'-ends of the two strands of the double-stranded DNA repair template.

[0009] The 2-5 consecutive nucleotide residues can be 2, 3, 4, or 5 consecutive nucleotide residues.

[0010] In an embodiment of the present invention, the phosphorothioate linkage modification is two phosphorothioate linkage modifications between 3 consecutive nucleotides at the 5'-ends of the two strands of the double-stranded DNA repair template.

[0011] In the above method, two mutant target DNA fragments respectively connected to the upstream homologous arm and the downstream homologous arm are further contained at both ends of the double-stranded DNA repair template, and there are 1-5 different nucleotides between the mutant target DNA fragment and the target DNA fragment of the CRISPR / Cas9 system; The mutant target DNA fragments are located at one end of the upstream homologous arm far from the target DNA fragment and one end of the downstream homologous arm far from the target DNA fragment.

[0012] In an embodiment of the present invention, the target DNA fragment of the CRISPR / Cas9 system is 5'-N 20 PAM-3', and the N 20 is 20 Ns, and the PAM (protospacer adjacent motif) is NGG; the N is A, G, C, or T.

[0013] The mutant target DNA fragment can be obtained by mutating 1-5 nucleotides on the basis of the target DNA fragment of the CRISPR / Cas9 system.

[0014] The PAM sequence of the mutant target DNA fragment is directly connected to the upstream homologous arm and the downstream homologous arm.

[0015] Specifically, in the double-stranded DNA repair template, the mutant target DNA fragment, the upstream homologous arm, the target DNA fragment, the downstream homologous arm, and the mutant target DNA fragment are connected in sequence. In the same strand, the sequences of the mutant target DNA fragments connected to the upstream homologous arm and the downstream homologous arm are reverse complementary.

[0016] The "presence of 1-5 different nucleotides" may mean the presence of 1, 2, 3, 4, or 5 different nucleotides.

[0017] In one embodiment of the present invention, there are 4 different nucleotides between the mutant target DNA fragment and the target DNA fragment.

[0018] Both ends of the double-stranded DNA repair template further contain boundary sequences, which are located at one end of the mutant target DNA fragment far from the target DNA fragment and are connected to the mutant target DNA fragment. The biotin modification and the phosphorothioate bond modification are both located at the 5′ ends of the two strands of the boundary sequence.

[0019] In the above method, the fragments in the double-stranded DNA repair template are connected end to end in sequence through 3′,5′-phosphodiester bonds.

[0020] In the above method, the length of the fragment after site-directed mutagenesis can be 1-100 bp. Specifically, the length of the fragment after site-directed mutagenesis can be 1-10 bp, 11-20 bp, 21-30 bp, 31-40 bp, 41-50 bp, 51-60 bp, 61-70 bp, 81-90 bp, or 91-100 bp.

[0021] In the above method, the site-directed mutagenesis can be substitution, insertion, and / or deletion of DNA at the target position in the plant genome.

[0022] In the above method, Cas9 in the CRISPR / Cas9 system can be SpCas9, and SpCas9 is a protein containing positions 41-1047 of SEQ ID No.1 or a fusion protein containing positions 41-1047 and 1424-1537 of SEQ ID No.1.

[0023] In one embodiment of the present invention, the sequence of the fusion protein is as shown in SEQ ID No.1.

[0024] In the above method, "editing the genome of plant protoplasts using the CRISPR / Cas9 system with a double-stranded DNA repair template as the donor template" can transform the Cas9 expression vector, the sgRNA expression vector, and the double-stranded DNA repair template into plant protoplasts to achieve editing of the genome of plant protoplasts.

[0025] The Cas9 expression vector contains the coding gene of Cas9.

[0026] The sgRNA expression vector can transcribe sgRNA targeting the target DNA fragment in the plant genome.

[0027] The present invention also provides a method for site-specific insertion or replacement of large DNA fragments in the plant genome. The length of the large DNA fragment is 100 bp - 20 kb. The method includes: 1) Using the recognition fragment of the recombinase as the fragment after the site-specific insertion or replacement, and using the method for site-specific mutagenesis in the plant genome to insert or replace the recognition fragment of the recombinase at the target position in the plant genome to obtain a recombinase recognition sequence insertion or replacement strain; 2) Transforming the expression vector of the recombinase and the donor vector containing the target large DNA fragment into the protoplasts of the recombinase recognition sequence insertion or replacement strain to achieve site-specific insertion of the large DNA fragment in the plant genome; both ends of the target large DNA fragment contain a recombinase recognition site.

[0028] The recognition fragment of the recombinase can be the recognition site (Recombinase site, RS) of recombinases such as Cre (Cre - loxP system), FLP (FLP - FRT system), or Bxb1 recombinase (Bxb1 - att system).

[0029] The target large DNA fragment can be a fluorescent protein coding gene such as GFP, RFP, or mCherry, or an expression element of a foreign gene.

[0030] The method may further include culturing and regenerating the protoplasts after the transformation in step 2) to obtain a plant with site-specific insertion of a large DNA fragment in the genome.

[0031] The length of the large DNA fragment can be 100 bp - 20 kb, 100 bp - 500 bp, 500 bp - 1 kb, 1 kb - 5 kb, 5 kb - 10 kb, 10 kb - 15 kb, or 15 kb - 20 kb.

[0032] The present invention also provides a product containing the CRISPR / Cas9 system and the double-stranded DNA repair template.

[0033] The CRISPR / Cas9 system may be the Cas9 protein and sgRNA, or may also be a Cas9 expression vector and an sgRNA expression vector.

[0034] The product may also contain an expression vector of the recombinase and a donor vector containing the target large DNA fragment.

[0035] The product can be used for site-directed mutagenesis in the plant genome, and can also be used for site-directed insertion or replacement of large DNA fragments in the plant genome.

[0036] In the present invention, the plant may be a dicotyledonous plant or a monocotyledonous plant that can be subjected to protoplast transformation and regeneration.

[0037] The method for precisely site-directed replacement of DNA in the plant genome by homologous recombination repair in the present invention. First, the inventors transferred the CRISPR / Cas9 plasmid and the double-stranded DNA repair template into protoplasts, and precisely site-directed inserted loxP sequences in the genome through homologous recombination, and then, with the aid of a specific recombinase system, achieved precise site-directed replacement of gene fragments. This method can be used for small fragment insertion at any site and site-directed insertion or replacement of large fragment DNA. Compared with the prior art, the present invention has the following characteristics: 1. This technology can achieve precise insertion and replacement of any DNA sequence at any site, and does not rely on resistance genes to obtain marker-free site-directed insertion and replacement plant lines; 2. The gene targeting system based on transient protoplast transformation can quickly obtain site-directed replacement plant lines without transgenes and any redundant sequences, avoiding multi-generation backcrossing or self-crossing to remove transgenic sequences.

[0038] The following further describes the present invention in detail in conjunction with specific embodiments. The given embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 、Flow chart of PEKT based on protoplast regeneration.

[0040] Figure 2 、Comparison of HDR efficiency in protoplasts 3 days after transformation and cell clusters after 30 days of regeneration culture in Example 1. (A) Flow chart of protoplast transformation and regeneration. (B) Structure diagram of plasmid for small fragment site-directed insertion mediated by HDR. Plasmid 1 is a SpCas9 expression plasmid, and plasmid 2 is an LFY-sgRNA expression plasmid or a RAX1-sgRNA expression plasmid. (C) LFY 5' end of the coding regionlox66 Sequence site-directed insertion. (D) RAX1 5' end of the coding region lox66 Sequence site-directed insertion. (E) LFY Comparison of HDR efficiency in protoplasts and cell clusters at the site. (F) RAX1 Comparison of HDR efficiency in protoplasts and cell clusters at the site. (G) Comparison of NHEJ efficiency in protoplasts and cell clusters

[0041] Figure 3 In Example 2 RAX1 Results of gene targeting at the site. (A) PCR identification of positive callus. (B) Sanger sequencing results of positive single plants. The orange font is the lox66 sequence, and the red font is random nucleotide insertions and deletions. (C) Statistical table of positive rates in callus

[0042] Figure 4 In Example 3 LFY Results of small fragment site-directed insertion at the site. (A) Structure diagrams of plasmids and repair templates. Plasmid 1 is the SpCas9 expression plasmid, and plasmid 2 is the LFY-NPTII-sgRNA expression plasmid. (B) Process of resistance screening and regeneration culture of transformed protoplasts. (C) Schematic diagram of primers for PCR positive identification of regenerated seedlings. (D) Sanger sequencing results of positive single plants. The orange font is the lox66 sequence, and the red font is random nucleotide insertions and deletions. (E) Phenotypes of the progeny of Col wild type and homozygous GT lines. (F) Statistical table of GT positive rates of regenerated seedlings

[0043] Figure 5 In Example 4 GL2 Results of small fragment site-directed insertion at the site. (A) GL2 5' end of the coding region lox66 Sequence site-directed insertion. (B) Sanger sequencing results of positive single plants. The orange font is the lox66 sequence, and the red font is random nucleotide insertions and deletions. (C) Phenotypes of the progeny of Col wild type and homozygous GT lines. (D) Statistical table of GT positive rates of regenerated seedlings

[0044] Figure 6 In Example 5 TOR Results of small fragment site-directed insertion at the site. (A) TOR 5' end of the coding region lox66 Sequence site-directed insertion. (B) Sanger sequencing results of positive single plants. The orange font is the lox66 sequence, and the red font is random nucleotide insertions and deletions. (C) Statistical table of GT positive rates of regenerated seedlings

[0045] Figure 7, Results of small fragment site-directed insertion at the GL1 locus mediated by SpCas9-mSA in Example 6. (A) GL1 5' end of the coding region lox66 Sequence site-directed insertion. (B) Structure diagram of the SpCas9-mSA expression module. (C) Sanger sequencing results of GT-positive single plants. Orange font is lox66 sequence, and red font is random nucleotide insertions and deletions. (D) Statistical table of GT positive rates of regenerated seedlings.

[0046] Figure 8 , Results of Example 7. (A) Schematic diagram of gene fragment site-directed integration. (B) Schematic diagram of the plasmid structure for gene fragment site-directed knock-in. Plasmid 1 is the Cre enzyme expression plasmid, and plasmid 2 is the donor plasmid. (C) Schematic diagram of the site-directed integration of the mCherry coding sequence. (D) PCR detection of positive site-directed integration events. (E) Statistical table of the efficiency of gene fragment site-directed integration in regenerated seedlings. Detailed implementation manners

[0047] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial channels unless otherwise specified. For the quantitative tests in the following examples, at least three repeated experiments are set, and the results are averaged.

[0048] Based on the CRISPR / Cas9 system and combined with polyethylene glycol (PEG)-mediated protoplast transformation and regeneration, the present invention has developed a method for precise site-directed knock-in of plant genomes (Protoplast eccDNA kink-in mediated Targeting, hereinafter referred to as PEKT), and the flow chart is as Figure 1 shown, and the specific steps are as follows: 1. Put 20-30 mg of Arabidopsis thaliana seeds in each 2 ml centrifuge tube. After sterilization, germinate them on SCA medium (3.2 g / L Gamborg’s B-5 medium with vitamins, 1.14 g / L MgSO 4 ·7H 2 O, 20 g / L sucrose, 3 g / L gelzan, pH 5.8), and perform a low-temperature dark treatment at 4 °C for 2 days, then place them in a greenhouse at 22 °C for long-day cultivation (16 hours of light, 8 hours of darkness).

[0049] 2. Subject 7-day-old Arabidopsis thaliana seedlings to a low-temperature dark treatment at 4 °C for 24 hours, cut off the cotyledons and place them in MMC solution (1.952 g / L MES·H2 O, 1.47 g / L CaCl 2 ·2H 2 O, 85 g / L mannitol, pH 5.8) for 1 hour and incubated overnight at 25°C for enzymatic digestion to obtain protoplasts. Each transformation reaction requires the SpCas9 expression plasmid (about 40 μg, 4 pmol), the sgRNA expression plasmid without the resistance screening module or the sgRNA expression plasmid with the resistance screening module (about 10 μg, 4 pmol), and the dsDNA repair template (about 10 μg, 8 pmol), dissolved in 300 μL of MMG solution (4 mM MES, 0.4 M mannitol, 15 mM MgCl 2 , pH 5.7), and mixed with 1×10 6 protoplasts. An equal volume of 40% PEG solution (40% (mass percentage) PEG4000, 0.2 M mannitol, 100 mM CaCl 2 ) was added, inverted and mixed well, incubated at room temperature for 10 minutes, and 10 mL of W5 solution (2 mM MES, 154 mM NaCl, 125 mM CaCl 2 , 5 mM KCl, pH 5.7) was added to terminate the reaction. The transformed protoplasts were washed twice with W5 solution, and then washed twice with MMM solution (1.952 g / L MES, 1.02 g / L MgCl 2 ·6H 2 O, 1.25 g / L MgSO 4 ·7H 2 O, 85 g / L mannitol, pH 5.8).

[0050] 3. Embed the transformed protoplasts in alginate, and the obtained alginate blocks (protoplast-alginate hydrogel) were placed in PIM liquid medium (505 mg / L KNO 3 , 160 mg / L NH 4 NO 3 , 440 mg / L CaCl 2 ·2H 2 O, 370 mg / L MgSO 4 ·7H 2 O, 170 mg / L KH 2 PO 4 , 30 mg / L FeCitrateNH 4 , 0.01 mg / L KI, 1 mg / L H 3 BO 3, 0.1 mg / L Mn 4 SO 4 ·4H 2 O, 1 mg / L ZnSO 4 ·7H 2 O, 0.03 mg / LCuSO 4 ·5H 2 O, 0.03 mg / L AlCl 3 , 0.03 mg / L NiCl 2 ·6H 2 O, 100 mg / L inositol, 1 mg / L calcium pantothenate, 0.01 mg / L biotin, 1 mg / L niacin, 1 mg / L vitamin B6, 1 mg / L vitamin B1, 0.2 mg / L folic acid, 40000 mg / L glucose, 60000 mg / L mannitol, 1 mg / L 2,4-D, 0.022 mg / L thidiazuron, 700 mg / L MES, 8 mg / L bromocresol purple, pH 5.6), cultured in the dark in an incubator at 25 °C for 3 days.

[0051] 4. After culturing in PIM liquid medium for 3 days, transfer the obtained protoplast cells to PCA liquid medium containing 20 mg / L kanamycin (3.2 g / L Gamborg’s B-5 medium with vitamins, 0.746 g / LMgSO 4 ·7H 2 O, 0.45 g / L CaCl 2 ·2H 2 O, 0.05 g / L L-glutamine, 20 mL / L coconut water, 80 g / L glucose, 0.5 mg / L NAA, 0.1 mg / L 2-IP, pH 5.8) in an incubator at 25 °C, under long-day conditions (16 h light, 8 h dark), and weak light (5 - 10 μmol / m 2 / s) for resistance screening; after 10 days, transfer the obtained cell clusters to PCA liquid medium without antibiotics, and continue to culture them in an incubator at 25 °C, under long-day conditions (16 h light, 8 h dark) and weak light (5 - 10 μmol / m 2 / s) for another 10 - 15 days to obtain cell clusters.

[0052] 5. Transfer the obtained cell clusters onto SRA solid medium (4.3 g / L MS Medium with vitamins, 30 g / L sucrose, 0.9 mg / L 2-IP, 0.1 mg / L NAA, 6 g / L agar, pH 5.8), and culture them in an incubator at 25°C under long-day conditions (16 hours of light and 8 hours of darkness) with weak light (5-10 μmol / m 2 / s) for 7 days to induce callus.

[0053] 6. Subsequently, transfer the callus onto SIM solid medium (4.3 g / L MS Medium with vitamins, 30 g / L sucrose, 0.47 g / L MES·H 2 O, 0.1576 mg / L IAA, 0.501 mg / L 2-IP, 8 g / L agar, pH 5.8), and perform shoot induction for 2-3 weeks in an incubator at 25°C under long-day conditions (16 hours of light and 8 hours of darkness) with weak light (5-10 μmol / m 2 / s). Then, induce root formation on RM solid medium (2.15 g / L MS Medium with vitamins, 10 g / L sucrose, 0.47 g / L MES·H 2 O, 1 mg / L IBA, 8 g / L agar, pH 5.8) to finally obtain regenerated plants with foreign sequences inserted at specific sites in the genome.

[0054] Among them, the SpCas9 expression plasmid and the sgRNA expression plasmid without a resistance screening module are both described in the literature (Yan, L. H. et al. High-efficiency genome editing in Arabidopsis using YAO promoter-driven CRISPR / Cas9 system. Mol. Plant 8, 1820-1823 (2015)), and their names in the literature are 35S-Cas9-SK and AtU6-26-sgRNA-SK, respectively.

[0055] The sgRNA expression plasmid with a resistance screening module was prepared as follows: 1) The sgRNA expression plasmid (Yan, L. H. et al. High-efficiency genome editing in Arabidopsis using YAOpromoter-driven CRISPR / Cas9 system. Mol. Plant 8, 1820-1823 (2015)) was digested with SpeI to obtain the SpeI-digested sgRNA expression plasmid; 2) Using plasmid pART27 (Gleave, A.P. A versatilebinary vector system with a T-DNA organisational structure conducive toefficient integration of cloned DNA into the plant genome. Plant Mol Biol 20,1203–1207 (1992)) as a template, PCR amplification was carried out using Kan-1F and Kan-1R, and the resulting PCR product contained a kanamycin resistance gene expression module; 3) The resulting PCR product was subjected to homologous recombination with the SpeI-digested sgRNA expression plasmid through a homologous recombinase (pEASY -Basic Seamless Cloning andAssembly Kit, CU201, TransGen Biotech), and the recombinant plasmid with the correct sequence obtained was the sgRNA expression plasmid with a resistance screening module. The amplification primers used were as follows: Kan-1F: GTTAGTCTCTCTGCATCTGATCA CAACGTCGTGACTGGGAAAAC (the underlined part is the sequence on the sgRNA expression plasmid); Kan-1R: GGGAACAAAAGCTGGAGCTCA TGGAATTAATTCGATCTAGTAACATAG (the underlined part is the sequence on the sgRNA expression plasmid).

[0056] The sequence of the kanamycin resistance gene expression module is as follows (the underlined part is the NPTII coding sequence): CAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGAAATTGTAAACGTTAATGGGTTTCTGGAGTTTAATGAGCTAAGCACATACGTCAGAAACCATTATTGCGCGTTCAAAAGTCGCCTAAGGTCACTATCAGCTAGCAAATATTTCTTGTCAAAAATGCTCCACTGACGTTCCATAAATTCCCCTCGGTATCCAATTAGAGTCTCATATTCACTCTCAATCCAAATAATCTGCA ATGGCAATTACCTTATCCGCAACTTCTTTA CCTATTTCCGCCCGGATCCGGGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAAC AGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGA CCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGC GCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCC TGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCC GGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTC GATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGC CCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTC TGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCT GAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCG CCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCGATCCAACACTTACGTTTGCAACGTCCAAGAGCAAATAGACCACGAACGCCGGAAGGTTGCCGCAGCGTGTGGATTGCGTCTCAATTCTCTCTTGCAGGAATGCAATGATGAATATGATACTGACTATGAAACTTTGAGGGAATACTGCCTAGCACCGTCACCTCATAACGTGCATCATGCATGCCCTGACAACATGGAACATCGCTATTTTTCTGAAGAATTATGCTCGTTGGAGGATGTCGCGGCAATTGCAGCTATTGCCAACATCGAACTACCCCTCACGCATGCATTCATCAATATTATTCATGCGGGGAAAGGCAAGATTAATCCAACTGGCAAATCATCCAGCGTGATTGGTAACTTCAGTTCCAGCGACTTGATTCGTTTTGGTGCTACCCACGTTTTCAATAAGGACGAGATGGTGGAGTAAAGAAGGAGTGCGTCGAAGCAGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGAATTAATTCCA。

[0057] Both 5'-ends of the two strands of the dsDNA repair template used are biotinylated, modified with two phosphorothioate bonds, and the mutant sgRNA target sequences are carried at both ends of the dsDNA repair template.

[0058] The left and right homologous arms in the following embodiments, namely the left homologous arm and the right homologous arm, that is, the upstream homologous arm and the downstream homologous arm.

[0059] Example 1: Detection of small fragment insertion efficiency during protoplast regeneration To verify whether HDR can be enriched during protoplast regeneration, in this example, the inventors used 7-day-old Col-0 wild-type Arabidopsis seedlings as materials, enzymatically digested the cotyledons to obtain protoplasts, and transiently transformed the SpCas9 expression plasmid, the sgRNA expression plasmid without a resistance screening module, and the double-stranded DNA repair template into Arabidopsis cotyledon protoplasts by PEG transformation ( Figure 2 in B), respectively, at RAX1 and LFY Precisely insert after the start codon of the gene lox66 sequence.

[0060] Respectively use RAX1 ( REGULATOR OF AXILLARY MERISTEMS1 ) and LFY(LEAFY) as the target genes, and design sgRNA target sites near the start codon (ATG) ( Figure 2 in C and D). LFY The target sequence of the LFY gene is AGAGAGAGAAAAATAGATTA, and its DNA fragment is inserted upstream of the DNA fragment of sgRNA in the sgRNA expression plasmid without a resistance screening module. The resulting recombinant plasmid is denoted as the LFY-sgRNA expression plasmid, which can transcribe the sgRNA targeting the RAX1 gene, and its transcription is driven by the AtU6 promoter. RAX1 The target sequence of the

[0061] LFY gene is CAATTCACTAACGAGAGAAA, and its DNA fragment is inserted upstream of the DNA fragment of sgRNA in the sgRNA expression plasmid without a resistance screening module. The resulting recombinant plasmid is denoted as the RAX1-sgRNA expression plasmid, which can transcribe the sgRNA targeting the lox66 gene, and its transcription is driven by the AtU6 promoter. LFY The repair template used for inserting the lox66 sequence into the RAX1 gene ( lox66 repair template) carries the lox66 sequence, and the lengths of the left and right homologous arms are 562 bp and 547 bp respectively. The repair template used for inserting the RAX1 sequence into the Figure 2 gene ( LFY The repair template is double-stranded DNA, and one strand is as follows (the part with a gray background is the boundary sequence; the boxed part is the Cas9 target sequence with mutations, which is 4 nucleotides different from the sgRNA target; the underlined part is the PAM sequence): 5′-Biotin-T*G*GCGGGACTAGTGGCCCGTAGAGAAAAATAGATTA TGG TGATCGTGCTCTCATGGTACAAACAATGAGACCCCCCAAAAAAAAAAAACCCCCGAGGATGAAATTAAAACAAATAACACAGCTTGAACAAAACATCAAAAACAGAGGATCATCACAGTAAATATTAATACTTGAACAAAACATCAAAAAGATAGGATCATCACAGTAAATATTAATATGCATAAAGAAGAGAATCACCACAGTGAAAACCCTAATCATTTTATATTCCTACGTGTCAAATTATGAATGGTCATAATCAGAGTAGAGAAAAAGAATGTCGATGAAAGAAAAAGTTTGACTTGTTAAGTCCCAACTGTCAATTTCCCAGCAAGACACATATCTTCTTTTACATCACATTACATCTACACATAAATGCTTTATTGCAAAAATAGCGATATAAAAAAAGAATATACAAAAACCTAGTATTTATTTTCTAGATTCTTTACTGTTGTATATATAGAAGTTGCTCGAGTGGTCATTTTTACAATAAAGCAATCTGCTCAAAAGAGTAAAGAAAGAGAGAAAAAGAGAGTGATAGAGAGAGAGAGAAAAATAGATTA TG ATAACTTCGTATAGCATACATTATACGAACGGTA GATCCTGAAGGTTTCACGAGTGGCTTATTCCGGTGGAACCCAACGAGAGCATTGGTTCAAGCACCACCTCCGGTTCCACCTCCGCTGCAGCAACAGCCGGTGACACCGCAGACGGCTGCTTTTGGGATGCGACTTGGTGGTTTAGAGGGACTATTCGGTCCGTACGGTATACGTTTCTACACGGCGGCGAAGATAGCGGAGTTAGGTTTTACGGCGAGCACGCTTGTGGGTATGAAGGACGAGGAGCTTGAAGAGATGATGAATAGTCTCTCTCATATCTTTCGTTGGGAGCTTCTTGTTGGTGAACGGTACGGTATCAAAGCTGCCGTTAGAGCTGAACGGAGACGATTGCAAGAAGAGGAGGAAGAGGAATCTTCTAGACGCCGTCATTTGCTACTCTCCGCCGCTGGTGATTCCGGTACTCATCACGCTCTTGATGCTCTCTCCCAAGAAGGTACAATAATTATGAATACATATAGAAATATCAAATATCGCACGTTTTATAAATATGTGTATATATTGTTCTCAACATTTCTATGCCCGACTT CCA TAATCTATTTTTCTCTACGGGCCACTAGTCCCGC*C*A-Biotin-3′。

[0062] The RAX1 repair template is double-stranded DNA. One of the strands is as follows (the gray background part is the boundary sequence; the boxed part is the Cas9 target sequence with mutations, which has 4 nucleotides different from the sgRNA target; the underlined part is the PAM sequence): 5′-Biotin-T*G*GCGGGACTAGTGGCTGGCTCACTAACGAGAGAAA TGGGATCAAATCAAGAAACCTGCTGATAATTTTAGTTAATGACTTTTTTTTTTGTCAACTCATTACAAAGTTCTGGATTTTAAATATTTGCATTTCTCTTGATATTAATGATTCAATTTTGATTGTTTGGAGTTTGGACCATGCGTATGCTGACAACCAAGTTACTCTGGAATTACTCTGGATTTTGGTAGGGATATTCCAGACTAACCCCAAGTTATGAAATAAAAGACATTGAAATCCTCATAAAGAAGGGATAAATAAAGTTTATCTTTTGAACAGTTAATTTGTTTTAACGTAATTTTATCAAACGAAGCGCGAGCTAAAATAAAATGAATAATTGGAAATCTAATTTTTTCAAACACGAACAATAAATATGCATGCATGTCAATCATGTTGACTTCTCCATACCCATAGCTTTAGAGACAAAATGAAATAAATAATTGGTTCTCCAGATAATAATATATAATAATAATTGATAGGGTGTGTCCAAATCCAAGTCTCTTATTAAAAGGGCACATATTCAACTACTCCTCCATAAACACAAAAAGTCCATCCTAAAAAAAGCAAGTAAGAGATAACAATTTCTCTCTTTTTCTTTTCTCTTCTTCAAACTTCAATTCACTAACGAGAGAAA TG ATAACTTCGTATAGCATACATTATACGAACGGTA GGAAGAGCTCCGTGTTGCGACAAGACAAAAGTGAAGCGAGGGCCTTGGTCGCCTGAAGAAGACTCTAAACTTAGAGATTACATTGAAAAGTATGGTAATGGTGGAAATTGGATCTCTTTCCCCCTCAAAGCCGGTAAGTTCATACACATGTTCTTAATCCTAGATAACCTTTTCGAGCTTCTAAGCCCAAATAAGAGCTTATGCCATTCTTTGTTGTGGTTTAAGGTTTGAGGAGATGTGGGAAGAGTTGTAGACTGAGGTGGCTAAACTATTTGAGACCAAACATAAAGCATGGTGACTTCTCTGAGGAAGAAGACAGGATCATTTTTAGTCTCTTCGCTGCCATAGGAAGCAGGTTCTCTAATTAATTCCTTTTGAAATCATCTTTAATCATATAAGATCCTTACAAAAAATGTAGAAATGAAATCATTTTTTCGCCCACTTGCCTGTGAAGGCCCTTTCATAGTTATCTGCTTAGATAGGTTTAAAGAAAATATTAATTCACACTCTCCTTCAATTAATCTTATAATAGTAACAAAATTTCTCCTAAATCTAATTTTGAATTTATGTGTTATTTTGAAGTAATGATCAAAATATTTCCAAACGTGCTTAATTTCTTCATAAATCTAGTCAAAATCACGAGCTACATTGATTCCAT CCA TTTCTCTCGTTAGTGAGCCAGCCACTAGTCCCGC*C*A-Biotin-3′。

[0063] According to steps 2-4 of the above PEKT method, replace the "sgRNA expression plasmid with a resistance screening module" with the "LFY-sgRNA expression plasmid", and use LFY the repair template as the dsDNA repair template for the experiment. Collect the protoplasts 3 days after transformation and the cell clusters after 30 days of regeneration culture after transformation respectively ( Figure 2 A in). Extract genomic DNA from the protoplasts 3 days after transformation and the cell clusters 30 days after transformation respectively, design primers on both sides of the target site, and perform two rounds of PCR. The PCR products are subjected to next-generation sequencing.

[0064] According to Steps 2-4 of the above-mentioned PEKT method, replace the "sgRNA expression plasmid with a resistance screening module" with the "RAX1-sgRNA expression plasmid", and use RAX1 the repair template as the dsDNA repair template for the experiment. Protoplasts collected 3 days after transformation and cell clusters after 30 days of regeneration culture after transformation were respectively collected ( Figure 2 in A). Genomic DNA was extracted from the protoplasts 3 days after transformation and the cell clusters 30 days after transformation. Primers were designed on both sides of the target site. After two rounds of PCR, the PCR products were subjected to next-generation sequencing. The first-round PCR primers for the LFY gene were LFY-O1F + LFY-O1R, and the second-round PCR primers were LFY-IF + LFY-IR; the first-round PCR primers for the RAX1 locus were RAX1-O1F + RAX1-O1R, and the second-round PCR primers were RAX1-IF + RAX1-IR. The specific primers used for PCR amplification are as follows: LFY-O1F: GCGTCCATGCCAATACAGTTA; LFY-O1R: GACTGCATACTCTATACACACA; LFY-IF: TAGAAGTTGCTCGAGTGGTCAT; LFY-IR: GGTGCTTGAACCAATGCTCTC; RAX1-O1F: TCTGCATGCGTAAATCAAACACATG; RAX1-O1R: CTAGTAGGAGGCTACCACTGC; RAX1-IF: CTCCATAAACACAAAAAGTCCATCC; RAX1-IR: CAATGTAATCTCTAAGTTTAGAG.

[0065] The sequencing data was analyzed using the CRISPResso2 software to detect the accurate HDR (Accurate HDR) efficiency, imperfect HDR (Imperfect HDR) efficiency, and NHEJ efficiency in each sample. Among them, accurate HDR is lox66 the precise insertion of the sequence, and the accurate HDR efficiency is lox66 the proportion of the number of reads with precise sequence insertion to the total number of sequencing reads; imperfect HDR is the inserted lox66 sequence with random base mutations or base insertions and deletions inside or on both sides, and the imperfect HDR efficiency is the proportion of the number of reads with imprecise insertion to the total number of sequencing reads; the NHEJ efficiency is the proportion of the number of reads with random base mutations or base insertions and deletions at 3 bp upstream of the PAM sequence within the sgRNA target site to the total number of sequencing reads.

[0066] Sequencing data analysis results showed that in the LFY gene, the precise HDR efficiency (0.167 ± 0.068%) in cell clusters after 30 days of regeneration culture was approximately 4.8 times higher than that in protoplasts 3 days after transformation (0.035 ± 0.008%) ( Figure 2 in E), and the proportion of inaccurate HDR also increased significantly. In the RAX1 gene, the precise HDR efficiency (0.401 ± 0.268%) in cell clusters after 30 days of regeneration culture was approximately 8.9 times higher than that in protoplasts 3 days after transformation (0.045 ± 0.016%) ( Figure 2 in F), and the proportion of inaccurate HDR also increased significantly. The NHEJ efficiency in cell clusters also increased significantly ( Figure 2 in G). This indicates that HDR can be enriched during protoplast regeneration.

[0067] Example 2. Using PEKT to RAX1 Precisely insert a small fragment at the 5' end of the coding region In this example, Arabidopsis RAX1 was used as the target gene, and a lox66 sequence was precisely inserted after the start codon (ATG).

[0068] According to steps 2 - 6 of the PEKT method above, the "RAX1-sgRNA expression plasmid of Example 1" was used, and the RAX1 repair template of Example 1 was used as the dsDNA repair template for the experiment. Among them, after protoplast transformation in step 4, there was no 10-day resistance screening. The cells were cultured in PCA liquid medium without kanamycin for 20 days, and then through callus induction and bud induction, regenerated seedlings were obtained, and other steps remained unchanged.

[0069] During the experiment, callus samples were taken and genomic DNA was extracted. Two rounds of PCR were used for positive identification. The first round of PCR amplification was performed with primers outside the left and right homologous arms (RAX1-O2F + RAX1-O2R), and then the second round of amplification was performed with primers specific to the inserted sequence (RAX1-O2F + RAX1-TR and RAX1-TF + RAX1-O2R) ( Figure 3 in A). Among 636 calli, 8 positive calli were identified ( Figure 3 in C). The calli with positive bands amplified were subjected to bud induction to obtain regenerated seedlings. The long fragment was amplified with primers outside the homologous arms (RAX1-O2F + RAX1-O2R), and genotype identification was performed by Sanger sequencing. Finally, 7 positive single plants were obtained by Sanger sequencing. The primers used are as follows: RAX1-O2F: GTTCACTGGTCCGTCCAATGC; RAX1-O2R: GCTGCTATTATTGACCACCTAC; RAX1-TF: CGAGAGAAATGATAACTTCGTATAGCATAC; RAX1-TR: GCTCTTCCTACCGTTCGTATAATGTATGC.

[0070] According to the sequencing results, among the 7 positive single strains (# RAX1-GT-133 to # RAX1-GT-138, # RAX1-GT-142), 5 strains were chimeras and 2 strains had heterozygous genotypes ( Figure 3 in B).

[0071] Example 3. Using PEKT to LFY site-specifically insert a small fragment at the 5' end of the coding region In this example, Arabidopsis LFY was used as the target gene, and an sgRNA target site was designed near the start codon. A sequence was precisely inserted after the start codon (ATG) ( lox66 sequence ( Figure 2 in C).

[0072] LFY The target sequence of the gene was AGAGAGAGAAAAATAGATTA, and its DNA fragment was inserted upstream of the DNA fragment of sgRNA in the sgRNA expression plasmid with a resistance screening module. The resulting recombinant plasmid was designated as the LFY-NPTII-sgRNA expression plasmid. This plasmid could transcribe an sgRNA targeting the LFY gene, and its transcription was driven by the AtU6 promoter. Moreover, this plasmid contained a kanamycin resistance screening gene expression module ( Figure 4 in A).

[0073] According to steps 2-6 of the above PEKT method, replace the "sgRNA expression plasmid with a resistance screening module" with the "LFY-NPTII-sgRNA expression plasmid", and use the LFY repair template of Example 1 as the dsDNA repair template for the experiment to obtain regenerated seedlings ( Figure 4 in B).

[0074] Samples were taken from the regenerated seedlings and genomic DNA was extracted. Two rounds of PCR were used for positive identification. The first round of PCR amplification was performed with primers outside the left and right homologous arms (LFY-O2F + LFY-O2R), and then the second round of amplification was performed with primers near the target region (LFY-IF + LFY-IR) ( Figure 4 in C). Single strains with positive bands amplified were selected, and long fragments were amplified with primers outside the homologous arms (LFY-O2F + LFY-O2R), and genotype identification was performed by Sanger sequencing. The primers used were as follows: LFY-O2F: TAGTAACTTATCGGGCTTCTGC; LFY-O2R: GACTGCATACTCTATACACACA; LFY-IF: TAGAAGTTGCTCGAGTGGTCAT; LFY-IR: GGTGCTTGAACCAATGCTCTC.

[0075] According to the sequencing results, among 65 transgenic regenerated seedlings, 2 plants precisely inserted lox66 the sequence, and the GT efficiency (i.e., the efficiency of precisely inserting the target sequence at the target position) was 3.0% ( Figure 4 in D, F). The sequencing results showed that in one GT single plant (#LFY-GF-9), both alleles were edited. In addition to the precise insertion, there was a 1-bp insertion before the ATG in the other allele, indicating that this GT single plant was of the bi-allele genotype. In another GT line (#LFY-GF-61), in addition to the precise insertion, there were two 1-bp insertions, indicating that this GT single plant was chimeric. Due to LFY frameshift mutations in the gene expression cassette in the GT lines, flower development was abnormal, which was consistent with the expectation ( Figure 4 in E).

[0076] Example 4. Using PEKT to GL2 precisely insert a small fragment at the 5' end of the coding region In this example, GL2 was used as the target gene, and sgRNA target sites were designed near the start codon, and precisely inserted lox66 the sequence ( Figure 5 in A) after the start codon (ATG).

[0077] GL2 The target sequence of the gene was AGAAAAATGAAGTCGATCGA, and its DNA fragment was inserted upstream of the DNA fragment of sgRNA in the sgRNA expression plasmid with a resistance screening module. The resulting recombinant plasmid was denoted as the GL2-NPTII-sgRNA expression plasmid, and this plasmid could transcribe sgRNA targeting GL2 the gene, and its transcription was driven by the AtU6 promoter.

[0078] GL2 The repair template used for inserting the gene into the lox66 sequence ( GL2 repair template) carried a 34-bp lox66 sequence, the left homologous arm was 520 bp in length, and the right homologous arm was 695 bp in length ( Figure 5 in A). The repair template was as follows: GL2The repair template is double-stranded DNA, and one of the strands is as follows (the gray-background part is the boundary sequence; the boxed part is the Cas9 target sequence with mutations, which has 4 nucleotides different from the sgRNA target; the underlined part is the PAM sequence): 5′-Biotin-T*G*GCGGGACTAGTGGCCCGTAAATGAAGTCGATCGA TGG GGAGGGAAGAAGAAAGCAGAAAATGCGGTTGGAGAATTAGGTGCTAAAAGTTAGTTGAGTCCATCTCAGTATCTAACGGTCAACTCTCTCTCTCTCTAGAGAAAACAATTAAGAAATCTGACATACACATATGTCTCTCTCTCTCTCTCTCTCTAGTCTATACACACAATTCAATTAAAGAAGAGACAGAGAAGTTCGTCTTTTTTGTTTTTATACCCTTAAATCAATCATGCAATTGTAACCCTTCCTTCTTATTCTCATTCCTTCCCCCCCTGTCTACAGTAATCTATAGCAACGCCATTATGTACTACTTTTAACGGATAATTTGCTCATGTTTCAATATGGCTTCATTGTATATATGTTCAAGTTCTTCTCAATCCTTTATATCATTCCAACATAATTCATATTAAAGTTAGTAGCTGAAATTGGAAGGCTGATATATTTTCCATAATTCAAATTTGAATTTTGCTCATCATATATATATGTATATATTAAAAATCGAATATTAAGAAGAAAAATGATAACTTCGTATAGCATACATTATACGAACGGTAAAGTCGATCGA TGGCTGCCAATGCTGTAGCTGGCCATGTTTTAAACTACTCAATTGTCGGATTGAAGTATAGCCAAAATATATAAAACCGTAAAAGGACTAAATATAATAATATAATAGGTATTAATTAATTAAAACTAATTAATTATAAAAGAAGCACCTAAAAGTCAAGAGCAGTAGAGAAATGGAAGAAATATCTGAAAAACGACCGCTTATATATATATGTATCATTGGAATTGAAGAGGCTATATATATATATATATATATATATATCGATCTTAGCTTATATATTAATTGAAAGTACATTTTGGTGTATAAGTAATTAAAGAAGAAAGAAAAAAAGAGAGATAATATATAAGGAAGAAGGAGTGCGAGGAGAAGAGGGAAGAGATCATAATTAAGCAAAGAAGCTAGCTAGGGACAGGATTTGTATGTCAATGGCCGTCGACATGTCTTCCAAACAACCCACCAAAGACTTTTTCTCCTCTCCAGCCCTCTCTCTATCTCTCGTATACTACTCTCTCTTTTCTACATACATGTACTTGCTAGCTCTCACATAAACATATATTATATATATGTCTTGATTTTAATGGAGATAGGAATATATACAGGCTGGGATATTCCGGAATGCATCCTCCGGCAGCACCAACCCTGAGGAGGATTTCCTGGGCAGAAGAGTAGTTGACGATGAGGATC CCA TCGATCGACTTCATTTACGGGCCACTAGTCCCGC*C*A-Biotin-3′。

[0079] According to steps 2-6 of the above PEKT method, replace the "sgRNA expression plasmid with a resistance screening module" with the "GL2-NPTII-sgRNA expression plasmid", and use GL2 the repair template as the dsDNA repair template for the experiment to obtain regenerated seedlings.

[0080] Samples of the regenerated seedlings were taken and genomic DNA was extracted. Two rounds of PCR were used for positive identification. The first round of PCR amplification was carried out with primers outside the left and right homologous arms (GL2-OF + GL2-OR), and then the second round of amplification was carried out with primers near the target region (GL2-IF + GL2-IR). Single plants with positive bands amplified were selected, and long fragments were amplified with primers outside the homologous arms (GL2-OF + GL2-OR), and genotype identification was carried out by Sanger sequencing. The primers used are as follows: GL2-OF: CACAAGTCATAATAAGAGTAATGAGAGG; GL2-OR: TGAGTTCTCGCTGCTCATCTC; GL2-IF: GCTGAAATTGGAAGGCTGATAT; GL2-IR: TGGCTATACTTCAATCCGACAA.

[0081] After sequencing analysis, among 100 transgenic regenerated seedlings, 3 plants had precise knock-ins at the target site lox66 sequence, and the GT efficiency was 3.0% ( Figure 5 B, D in). The three GT single plants were all bi-allele genotypes. In one single plant, in addition to the precise knock-in allele, a random insertion of 152 bp occurred in the other allele; in another GT single plant, in addition to the precise knock-in genotype, a deletion of 399 bp and an insertion of 184 bp occurred in the other allele. The phenotypes of the leaves were examined. In the GT lines GL2 the coding sequence had a frameshift mutation, resulting in the phenotype of hairless epidermis in homozygous offspring, which was consistent with the expectation ( Figure 5 C in).

[0082] Example 5. Using PEKT to TOR precisely knock in a small fragment at the 3' end of the coding region In this example, TOR was used as the target gene, and an sgRNA target site was designed near the stop codon (TGA), and a 36-bp lox66 sequence was precisely knocked in before the stop codon ( Figure 6 A in).

[0083] TOR The target sequence of the gene was GGTGCCCTTTCTGGTGAGAT, and its DNA fragment was inserted upstream of the DNA fragment of sgRNA in the sgRNA expression plasmid with a resistance screening module. The resulting recombinant plasmid was denoted as the TOR-NPTII-sgRNA expression plasmid, and this plasmid could transcribe an sgRNA targeting TOR gene, and its transcription was driven by the AtU6 promoter.

[0084] TORGene insertion lox66 The repair template used for the sequence ( TOR repair template) carries a 36bp lox66 sequence, with a left homologous arm length of 656bp and a right homologous arm length of 573bp. The repair template is as follows: TOR The repair template is double-stranded DNA. One strand is as follows (the gray background part is the boundary sequence; the boxed part is the Cas9 target sequence with mutations, which is 4 nucleotides different from the sgRNA target; the underlined part is the PAM sequence): 5′-Biotin-T*G*GCGGGACTAGTGGCAAGTCCCTTTCTGGTGAGAT GGG AATGGCAATGATGGAAGCGTTTGTACATGATCCTTTAATCAATTGGCGTCTTTTCAATTTCAATGAAGTCCCCCAATTAGCACTGCTCGGTAACAACAACCCCAATGCTCCTGCTGATGTTGAGCCTGACGAAGAAGATGAAGATCCCGCTGATATAGATCTTCCTCAGCCTCAAAGGAGTACTCGAGAGAAGGAGATTCTTCAGGTTTCTTTTGAAAATCTTATTATGTTTTTTTCTTCTCAAAGGGGGATTTTTTTATATTTAAATTTGATTTTTGGATTATTTCAGGCTGTAAATATGCTTGGAGATGCTAATGAAGTTTTAAATGAGCGTGCCGTAGTTGTTATGGCACGTATGAGTCATAAGCTTACAGGGCGTGATTTTTCTTCGTCTGCAATTCCGAGCAATCCCATTGCTGATCATAATAACTTGCTCGGAGGAGATTCTCATGAAGTCGAACATGGTTTGTCTGTGAAAGTTCAGGTTCAAAAACTAATCAATCAAGCCACTTCCCATGAGAATCTCTGTCAAAACTATGTTGGGTATGTTCCTCTACACTTCTACACTCCTAAACCCCGATTGCTTTAGTTCATTTAAAATTGAACATGCATATTTCTTGTGTTTGTTCTTTTATTACAGGTGGTGCCCTTTCTGGATAACTTCGTATAGCATACATTATACGAACGGTAGGTGAGAT GGGTCATCTGTACATTTTTAATGTAAATAGGCTTTTTAATTTTCAACTAATTTGCATTTATGTTTGACTAATTCGATAATAAACTATGTTCTTTTTAATGTAAATAATTTATTAGTTTCATAGTCATGTATTAATACATGATGTAGCGTGGATGAATAAATGGGCAGGTCACCTTTTTTACTTTTCTGAAATATACATTCTCATATCCTCAAGGTATTTTAATGGAATTGCGACAAAAGAAGAAGAAGAAGAAGATGGTGTACAATGTACATGTTTTATGAACTATATAAAGCAATTGGAATATGCATGTTCTTTTTAATGAAACATGTTTCCGGAGTTTAAGATGGATTTCATAAAAATCAACATAATTTTGTTAATAGTCCAAATAATATAATTTTTTTATTTTTTTTTTGTTAAAGCAAATTTGTCACATTTTTTTATATAAAATTTGTGATGTCAAAAGAGAAGACAAATGTAAAATTGTGTTGTGGGTTGTTACAGTATGTAGTTGGTATCTTTATTAAACCTACCAAAACGAGACAATTTGTAACATTGAGGGAGCATGTG CCC ATCTCACCAGAAAGGGACTTGCCACTAGTCCCGC*C*A-Biotin-3′。

[0085] According to steps 2-6 of the above PEKT method, replace the "sgRNA expression plasmid with a resistance screening module" with the "TOR-NPTII-sgRNA expression plasmid", and use GL2 the repair template as the dsDNA repair template for the experiment to obtain regenerated seedlings.

[0086] Sample the regenerated seedlings and extract genomic DNA, and perform positive identification by two rounds of PCR. Use the primers outside the left and right homologous arms (TOR -OF + TOR -OR) for the first round of PCR amplification, and then use the primers near the target region (TOR -IF + TOR -IR) for the second round of amplification. Select the single plants with positive bands amplified, use the primers outside the homologous arms to amplify the long fragment (TOR -OF + TOR -OR), and perform genotype identification by Sanger sequencing. The primers used are as follows: TOR-OF: TCCGCCTGACAAGAATGCT; TOR-OR: CAAGTGGTGCCGAGACAAC; TOR-IF: TTTATTACAGGTGGTGCCCTT; TOR-IR: ATTCATCCACGCTACATCATGT.

[0087] After sequencing analysis, among 144 transgenic regenerated seedlings, 4 plants precisely knocked in the lox66 sequence, and the GT efficiency was 2.8% ( Figure 6 in B and C below). Among them, 2 GT single plants were of bi-allele genotype, and 2 GT single plants were chimeric.

[0088] Example 6, Test of precise knock-in of small fragments at the 5' end of the GL1 coding region In this example, monomeric streptavidin (mSA) was fused and expressed at the C-terminus of the SpCas9 protein (denoted as SpCas9-mSA fusion protein), and GL1 using lox66 as the target gene, an sgRNA target site was designed near the start codon, and precisely knocked in Figure 7 sequence (

[0089] The SpCas9-mSA fusion gene was synthesized, and the DNA fragment between the SwaI and BamHI recognition sequences of the SpCas9 expression plasmid was replaced with the SpCas9-mSA fusion gene to obtain a recombinant plasmid, namely the SpCas9-mSA expression plasmid. This plasmid can express the SpCas9-mSA fusion protein, and the expression of the SpCas9-mSA fusion gene is driven by the 35S promoter.

[0090] The SpCas9-mSA fusion gene is as follows (the gray background part is the FLAG tag coding sequence; the boxed part is the SV40NLS and Nucleoplasmin NLS; the bold part is the Linker sequence):

[0091] The SpCas9-mSA fusion protein is as follows (the amino acid sequence of the FLAG tag is in the gray background; the boxed parts are the SV40 NLS and Nucleoplasmin NLS; the underlined part is the sequence of SpCas9; the bold part is the Linker sequence): MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAA DKKYSIGLDIGTNSVGWAVITDEYKVPS KKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDV DKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFD LAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLT LLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKG ASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLK TYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV SGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIK ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRG KSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNT KYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYD VRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN IVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIM ERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGSETPGTSESATPESAEAGITGTWYNQSGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKKRPAATKKAGQAKKKK (SEQ ID No.1).

[0092] GL1 The target sequence of the gene is ATAAAAAAAGAATGAGAATA, and its DNA fragment is inserted upstream of the DNA fragment of sgRNA in the sgRNA expression plasmid with a resistance screening module. The resulting recombinant plasmid is denoted as the GL1-NPTII-sgRNA expression plasmid, and this plasmid can transcribe the sgRNA targeting GL1 the gene, and its transcription is driven by the AtU6 promoter.

[0093] GL1 The gene is inserted into lox66 the repair template used for the GL1 repair template) with a 34bp lox66 sequence, the left homologous arm is 656bp in length, and the right homologous arm is 774bp in length. The repair template is as follows: GL1The repair template is double-stranded DNA, and one of the strands is as follows (the part with the gray background is the boundary sequence; the boxed part is the Cas9 target sequence with mutations, which differs from the sgRNA target by 4 nucleotides; the underlined part is the PAM sequence): 5′-Biotin-T*G*GCGGGACTAGTGGCCCGTAAAAAGAATGAGAATA AGG GGAGAACCATTTCATTGATCCATCAAAGTTTCTTTTTTCATGTTTTATCTTCTGCAAAAAAACTGTTTTAGGGTGAACTCTGTGGTACTTTTAAATAGGTTGTTCCGATCCCACTTTGTTGTATAAATAACTAAAAATAATATGTAGTTCCAATGAAAAAGATCATTATCAAATATTAGTAGAATGTAAGTGTATACAGTGCATAAACAAACTGGTTAACGTGTCAAATAAATTACAAATCAGTTAAATCATACAATATAGAACATTTCGCAGTAATCTGCCCCATATTTAGTAGATTTGTTTTTGTAAATTTCATGTCGCAAGTATAGTATTTAGATATTGCCAGAGGAGAAAAAACAATTTTTTTATAAATTTTTTGGCCATAGATACAATTAAACCAACTGCCAAATTTTAGCTTTTAATACGTAACGTAGGGTAACAAAGGATAACGCTATAGTCGCTATCTATACATTATTAGGTACATATTTATGCTTATAGCCATGATTACACAAAGATACGTATTGATGTGAGTGTATATATAAGCACGTGTCACGAAAACCCATCATAAGTTCACTACTCATAATCTCTTTCTCTCTCTCACACACACACACAGACACACACAAACATAGAAGGAATCAGAGAATAAAAAAAGAATGATAACTTCGTATAGCATACATTATACGAACGGTAAGAATA AGGAGAAGAGATGAAAAAGAGAATCAAGAATACAAGAAAGGTTTATGGACAGTTGAAGAAGACAACATCCTTATGGACTATGTTCTTAATCATGGCACTGGCCAATGGAACCGCATCGTCAGAAAAACTGGTACTCTTTCTCTCTCTCGCGCGTTTTGTGATTATATATATTAACTTTAGTCTCTAGCTAGTTCATATATAAATATATCTCAGTTAGACTAAATTTGATTCGTTGATAGGGCTAAAGAGATGTGGGAAAAGTTGTAGACTGAGATGGATGAATTATTTGAGCCCTAATGTGAACAAAGGCAATTTCACTGAACAAGAAGAAGACCTCATTATTCGTCTCCACAAGCTCCTCGGCAATAGGTACATAACTTTATGTTACTACAATATTATACACCATGTCTTTTTATTTAATCGATCGTTAACTGATTTTTTCTTTCGTCAAATATTTGAATTGTTGTTTTAAACAAAGATTTGGCCGGTTAAGTTGATTTTTAAAAATCTAATTGAAGGATTTTCTTACTTGCAAATGTAAAAATAAAGAGAGTTGACAAGTAAAGACTGTTTTATTTCAATTAAACTATGTGTTCGTGGATTATTTCACATTTCAAAAGGTGTTTTCTTCACAAAATAAATCTTAAAAATAATCCTTTGAACTTAAAATATGTTCAAAATTTTCAAGTTGATATATTTTTCTGAAGAAGAAAAGAGTTGCACAAGATAAAAGTTTGTCTTCTCTTCAATGAAACTCAACCGATGTGT CCT TATTCTCATTCTTTTTACGGGCCACTAGTCCCGC*C*A-Biotin-3′。

[0094] According to steps 2-6 of the above PEKT method, replace "SpCas9 expression plasmid" with "SpCas9-mSA expression plasmid", replace "sgRNA expression plasmid with a resistance screening module" with "GL1-NPTII-sgRNA expression plasmid", and use GL1 the repair template as the dsDNA repair template for the experiment to obtain regenerated seedlings.

[0095] Samples were taken from the regenerated seedlings and genomic DNA was extracted. Two rounds of PCR were used for positive identification. The first round of PCR amplification was carried out with primers outside the left and right homologous arms (GL1-OF + GL1-OR), and then the second round of amplification was carried out with primers near the target region (GL1-IF + GL1-IR). Single plants with positive bands amplified were selected, and long fragments were amplified with primers outside the homologous arms (GL1-OF + GL1-OR), and genotype identification was carried out by Sanger sequencing. The amplification primers are as follows: GL1-OF: GCCGTTAAAGCTCTTGGCTATATTCTCG; GL1-OR: GAAGGTGGTAACTGGTAAGTAGGGAG; GL1-IF: AGAACATAGTCCATAAGGATGTTGTC; GL1-IR: ACCCATCATAAGTTCACTACTCATAATC.

[0096] After sequencing analysis, among the 13 transgenic regenerated seedlings, 1 plant had precise knock-in at the target site lox66 sequence, and the GT efficiency was 7.7% ( Figure 7 C and D in). The GT single plant was chimeric.

[0097] Example 7, Precise insertion of gene fragments by combining PEKT and sequence-specific recombinase To achieve site-directed knock-in of larger DNA fragments, the inventors used RAX1 the strain with precise knock-in at the site lox66 (#RAX1-GT-138 obtained in Example 2) for the second round of protoplast transformation and regeneration, and site-directed knock-in of gene fragments was achieved through the specific recombinase system ( Figure 7 A, C in).

[0098] The inventors constructed a donor plasmid containing the mCherry open reading frame, with two recombinase recognition sites on both sides of the open reading frame (this fragment was denoted as loxP-mCherry-lox71), and also constructed a Cre enzyme expression plasmid driven by UBQ10 the promoter ( Figure 7 B in).

[0099] The construction steps of the donor plasmid containing the mCherry open reading frame are as follows: The mCherry coding sequence was obtained from transgenic plants pCLV3::mCherry-N7(Amplified from (Xu M, Du Q, Tian C, Wang Y, Jiao Y. Stochastic gene expression drives mesophyll protoplast regeneration. Sci Adv. 2021 Aug 11;7(33):eabg8466)), loxP The sequence and the linker sequence were added to the amplification primers. The amplified blunt-ended product was directly ligated to pEASY Blunt ( pEASY -Blunt Cloning Kit, CB101, TransGen Biotech) vector, and the resulting recombinant vector containing loxP-mCherry-lox71 with the correct sequence was the donor plasmid containing the mCherry reading frame. The amplification primers and the sequences of loxP-mCherry-lox71 used are as follows: Lox-mCh-F: ATAACTTCGTATAGCATACATTATACGAAGTTATGGATGGTGAGCAAGGGCGAGGAG; Lox-mCh-R: ATAACTTCGTATAATGTATGCTATACGAACGGTACCGGCGCGTCCAGGAGCAGCAGAAGGACCCTTGTACAGCTCGTCCATGCC.

[0100] LoxP- mCherry -linker-lox71 sequence: ATAACTTCGTATAGCATACATTATACGAAGTTAT GGATGGTGAGCAAGGGCGAGGAGGATAACATGGC CATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGC GAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCG CCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTA CTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTG ACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACG GCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAA GGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCC AAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACA CCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAG GGTCCTTCTGCTGCTCCTGGACGCGCCGGTACCGTTCGTATAGCATACATTATACGAAGTTAT.

[0101] The steps for constructing the Cre expression plasmid are as follows: Amplify the UBQ10 promoter (denoted as UBQ10-p) from the Col wild-type Arabidopsis genome. The amplification primers are UBQ10-F and UBQ10-R. Homologously recombine the amplification product with the BJ36 vector digested with XhoI (Cao X, Du Q, Guo Y, Wang Y, Jiao Y. Condensation of STM is critical for shoot meristem maintenance and salt tolerance in Arabidopsis. Mol Plant. 2023 Sep 4;16(9):1445-1459.). The correctly sequenced recombinant vector obtained is denoted as BJ36-pUBQ10. Amplify the Cre enzyme-encoding gene using the primer pair Cre-F and Cre-R. Homologously recombine the amplification product with the BJ36-pUBQ10 vector digested with EcoRI. The correctly sequenced recombinant vector obtained is denoted as BJ36-pUBQ10-Cre. Using plasmid pART27 as a template, amplify using the primer pair Kan-2F and Kan-2R. The amplification product obtained contains the kanamycin resistance gene expression module (see above). Homologously recombine the amplification product with the BJ36-pUBQ10-Cre vector digested with NdeI. The correctly sequenced recombinant vector is the Cre enzyme expression plasmid.

[0102] UBQ10-F: CATATGTCGACCTGCAGACGCGTC TACCCGACGAGTCAGTAATAAACGG (the underlined sequence is the sequence on the BJ36 vector); UBQ10-R: CGAACCCGGGGTACCGAATTCC AGTGTTAATCAGAAAAACTCAGATTAATC (the underlined sequence is the sequence on the BJ36 vector); Cre-F: CTGAGTTTTTCTGATTAACACTGG ATGTCCAATTTACTGACCGTACAC (the underlined sequence is the sequence on the BJ36-pUBQ10 vector); Cre-R: TCGATTTCGAACCCGGGGTACCCG CTAATCGCCATCTTCCAGCAGG (the underlined sequence is the sequence on the BJ36-pUBQ10 vector); Kan-2F: GGGAGCTCGCGGCCGCATGCA CAACGTCGTGACTGGGAAAAC (the underlined sequence is the sequence on the BJ36-pUBQ10-Cre vector); Kan-2R: GACGCGTCTGCAGGTCGACATGGAATTAATTCGATCTAGTAACATAG (The underlined sequence is on the BJ36-pUBQ10-Cre vector).

[0103] pUBQ10 sequence: TACCCGACGAGTCAGTAATAAACGGCGTCAAAGTGGTTGCAGCCGGCACACACGAGTCGTGTTTATCAACTCAAAGCACAAATACTTTTCCTCAACCTAAAAATAAGGCAATTAGCCAAAAACAACTTTGCGTGTAAACAACGCTCAATACACGTGTCATTTTATTATTAGCTATTGCTTCACCGCCTTAGCTTTCTCGTGACCTAGTCGTCCTCGTCTTTTCTTCTTCTTCTTCTATAAAACAATACCCAAAGAGCTCTTCTTCTTCACAATTCAGATTTCAATTTCTCAAAATCTTAAAAACTTTCTCTCAATTCTCTCTACCGTGATCAAGGTAAATTTCTGTGTTCCTTATTCTCTCAAAATCTTCGATTTTGTTTTCGTTCGATCCCAATTTCGTATATGTTCTTTGGTTTAGATTCTGTTAATCTTAGATCGAAGACGATTTTCTGGGTTTGATCGTTAGATATCATCTTAATTCTCGATTAGGGTTTCATAGATATCATCCGATTTGTTCAAATAATTTGAGTTTTGTCGAATAATTACTCTTCGATTTGTGATTTCTATCTAGATCTGGTGTTAGTTTCTAGTTTGTGCGATCGAATTTGTAGATTAATCTGAGTTTTTCTGATTAACACT。

[0104] Cre enzyme coding sequence:

[0105] According to Steps 1-6 of the above PEKT method, replace "Arabidopsis thaliana" with "#RAX1-GT-138" obtained in Example 2, and replace "SpCas9 expression plasmid (about 40 μg, 4 pmol), sgRNA expression plasmid (about 10 μg, 4 pmol) and dsDNA repair template (about 10 μg, 8 pmol)" with "Cre enzyme expression plasmid (20 μg) and donor plasmid (20 μg)", and keep other steps unchanged to obtain regenerated seedlings.

[0106] After resistance screening, screen for regenerated plants in which the mCherry coding sequence is site-specifically integrated into RAX1 the 5' end of the coding region. After PCR positive identification and Sanger sequencing, 9 positive single plants were identified from 30 regenerated seedlings, and the gene fragment insertion efficiency was 30% ( Figure 8 D, F in). The primers used are as follows: R-F: GTTCACTGGTCCGTCCAATGC; R-R: GCTGCTATTATTGACCACCTAC; m-F: ACATCAAGTTGGACATCACCTC; m-R: CTTGGAGCCGTACATGAACTGAG.

[0107] In vivo observation of the apical meristem of one of the positive plants also found red fluorescence signals in the nucleus ( Figure 8 E in).

[0108] The above details the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any changes, uses or improvements of the present invention, including those that depart from the scope disclosed in this application and are made by conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. A method for site-directed mutagenesis in a plant genome, comprising: The CRISPR / Cas9 system is used to edit the genome of plant protoplasts using a double-stranded DNA repair template as a donor template to obtain edited protoplasts; Using the plant protoplast transformation method, the edited protoplasts are cultured and regenerated to obtain plants with site-directed mutations in the genome, thereby achieving site-directed mutations in the genome of the target plant; The double-stranded DNA repair template sequentially contains an upstream homology arm identical to the plant genome sequence, a target DNA fragment, and a downstream homology arm identical to the plant genome sequence; The target DNA fragment contains a fragment after site-directed mutation, and the fragment after site-directed mutation is located between the upstream homology arm and the downstream homology arm; The ends of the double-stranded DNA repair template are modified with biotin and / or phosphorothioate bonds.

2. The method according to claim 1, characterized in that: The biotin modification is to modify the 5′ terminal nucleotides of the two strands of the double-stranded DNA repair template using biotin.

3. The method according to claim 1 or 2, characterized in that: The phosphorothioate bond modification is a phosphorothioate bond modification between 2-5 nucleotide residues at the 5′ ends of the two strands of the double-stranded DNA repair template.

4. The method according to any one of claims 1 to 3, characterized in that: The double-stranded DNA repair template also contains mutant target DNA fragments at both ends, and the mutant target DNA fragments are 1-5 different nucleotides from the target DNA fragments of the CRISPR / Cas9 system; The mutant target DNA fragment is located at one end of the upstream homology arm away from the target DNA fragment and one end of the downstream homology arm away from the target DNA fragment.

5. The method according to any one of claims 1 to 4, characterized in that: The fragment after site-directed mutation is 1-100 bp.

6. The method according to any one of claims 1 to 5, characterized in that: The site-directed mutagenesis is the replacement, insertion and / or deletion of DNA at a target position in the plant genome.

7. The method according to any one of claims 1 to 6, characterized in that: In the CRISPR / Cas9 system, Cas9 is SpCas9, which is a protein containing positions 41-1047 of SEQ ID No.1 or a fusion protein containing positions 41-1047 and positions 1424-1537 of SEQ ID No.

1.

8. A method for inserting or replacing a large DNA fragment at a specific site in a plant genome, wherein the large DNA fragment has a length of 100 bp-20 kb, the method comprising: 1) Using the recognition fragment of a recombinase as the fragment after site-specific insertion or replacement, inserting or replacing the recognition fragment of a recombinase at a target position in a plant genome using the method described in any one of claims 1 to 7 to obtain a strain with insertion or replacement of the recognition sequence of the recombinase; 2) transforming the expression vector of the recombinase and the donor vector containing the target large DNA fragment into the protoplasts of the recombinase recognition sequence insertion or replacement strain to achieve site-specific insertion of the large DNA fragment in the plant genome; the two ends of the target large DNA fragment each contain a recombinase recognition site.

9. The method according to any one of claims 1 to 8, characterized in that: The plant is a dicotyledon or monocotyledon that can undergo protoplast transformation and regeneration.

10. A product comprising a CRISPR / Cas9 system and the double-stranded DNA repair template described in claims 1-7.

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