WO2017092201A1 - 利用CRISPR-Cas9系统定点修饰ALS基因获得抗除草剂水稻的系统及其应用 - Google Patents

利用CRISPR-Cas9系统定点修饰ALS基因获得抗除草剂水稻的系统及其应用 Download PDF

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WO2017092201A1
WO2017092201A1 PCT/CN2016/077337 CN2016077337W WO2017092201A1 WO 2017092201 A1 WO2017092201 A1 WO 2017092201A1 CN 2016077337 W CN2016077337 W CN 2016077337W WO 2017092201 A1 WO2017092201 A1 WO 2017092201A1
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plant
target
sequence
expression cassette
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夏兰琴
孙永伟
赵云德
马有志
吴传银
张欣
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中国农业科学院作物科学研究所
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C12N15/8278—Sulfonylurea
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the invention belongs to the field of genetic engineering, and particularly relates to a system for obtaining herbicide resistant rice by using a CRISPR-Cas9 system (CRISPR/Cas9 system) to modify the ALS gene and its application.
  • CRISPR-Cas9 system CRISPR/Cas9 system
  • the CRISPR/Cas9 system is a new technology for genome-based editing after ZFNs and TALENs technology. Unlike ZFNs and TALENs, the recognition of target sites by the CRISPR/Cas9 system relies on base-pair pairing between nucleic acids, allowing editing of any 20-bp target sequence immediately following PAM (NGG), and its target The frequency of distribution in the genome is high, so it is easier to find a suitable target site for target genes that require site-specific editing. In addition, the CRISPR/Cas9 system can simultaneously edit the positions of different sites or multiple genes of the same gene to make it more flexible.
  • CRISPR/Cas9 system is simple and fast to operate, and only needs to replace the 20-30 bp nucleotide sequence on the original vector for each target, which is more suitable for large-scale, high-throughput operation.
  • CRISPR/Cas9 has broad development potential and application prospects, and is expected to become one of the most powerful tools for future gene-oriented editing.
  • CRISPR/Cas9 has been applied to fixed-point knockout studies of rice, wheat, Arabidopsis and N. benthamiana genomes, but there has not been any genetic improvement of agronomic traits of important crops through site-specific modification (amino acid substitution or site-specific integration). .
  • CRISPR/Cas9 system and exogenous modified fragments were simultaneously introduced into maize by gene gun or Agrobacterium method, and the proline at position 165 of ALS2 was successfully modified to serine (P165S). Plants are also resistant to sulfonylurea herbicides.
  • herbicide resistance gene drift may form a concern for the safety of genetically modified organisms such as super weeds;
  • the key enzymes in the synthesis of plant amino acids have always been important target enzymes in the development of new herbicides, and the endogenous genes of crops have been mutated by EMS to improve important agriculture.
  • Herbicide resistance of crops has been reported in wheat, barley and rice, but due to the random nature of EMS mutations, it is necessary to extensively screen large-scale mutants with herbicide resistance and other agronomic traits unchanged. Technology is difficult to get widely used.
  • acetolactate synthase (ALS) inhibitor herbicide bispyribac is one of the most active areas of development.
  • Acetolactate synthase (ALS) is present in plants and can catalyze pyruvic acid as acetolactate. It has high specificity and high catalytic efficiency, thus synthesizing three essential branched-chain amino acids in plants. Acid, leucine and isoleucine).
  • Sulfonylureas, imidazolinones, pyrimidine carboxylic acid herbicides inhibit ALS activity and destroy the synthesis of proline, leucine and isoleucine in plants, leading to plant death. Swanson et al.
  • Sulfonylureas, imidazolinones, and pyrimidinecarboxylic acid herbicides are widely used in production, and have the advantages of high biological activity, wide herbicidal spectrum, and safety to humans and animals (Endo et al., 2007).
  • the invention provides a system for obtaining herbicide resistant rice by site-directed modification of ALS gene by using CRISPR-Cas9 system and application thereof.
  • the system for site-directed modification of a plant genome comprises a vector for site-directed modification of a plant genome and a donor DNA A;
  • the vector for site-directed modification of a plant genome comprises a Cas9 protein expression cassette, a gRNA expression cassette and DNA B;
  • the gRNA expression cassette encodes two gRNAs, each of which targets two target sites of the target DNA of the plant of interest;
  • the target modified DNA in the target DNA is located at the two targets Between the sites; the target site located upstream of the two target sites is an upstream target site, and the target site located downstream is a downstream target site;
  • the donor DNA B contains the upstream target site, the downstream target site, and a site-directed modification fragment located between the upstream target site and the downstream target site; the site-directed modified fragment is to be replaced a DNA fragment of the modified fragment to be determined;
  • the donor DNA A is identical to the nucleotide sequence of the donor DNA B.
  • the Cas9 protein expression cassette, the gRNA expression cassette and the donor DNA B may be present on the same plasmid, or may be present on both plasmids in any combination, or may be present on one plasmid, respectively.
  • the fixed point modification may be amino acid substitution, site-directed integration of foreign genes or site-directed integration of foreign fragments.
  • the donor DNA B further comprises an upstream homology arm and a downstream homology arm for homologous recombination with the target DNA, the upstream homology arm being located at the upstream target site and the Between site-directed modifications, the downstream homology arm is positioned between the site-directed modification fragment and the downstream target site.
  • the plant or the plant of interest may be a monocot or a dicot.
  • the monocot may be a gramineous plant.
  • the gramineous plant may specifically be rice, such as Nipponbare rice.
  • the target DNA may be a gene encoding acetolactate synthase; the acetolactate synthase is a1 or a2:
  • A1 an amino acid sequence such as the protein shown in SEQ ID NO: 2 of the Sequence Listing;
  • A1-derived protein having acetolactate synthase activity obtained by performing substitution and/or deletion and/or addition of one or several amino acid residues in SEQ ID NO:2.
  • the nucleotide sequence of the target DNA is specifically shown as Sequence 3 of the Sequence Listing.
  • the upstream target site is specifically shown as nucleotides 7590-7609 from the 5' end of SEQ ID NO: 1 of the Sequence Listing.
  • the downstream target site is specifically shown in nucleotide sequence 1032 to 8051 from the 5' end of Sequence 1 of the Sequence Listing.
  • the site-directed modified fragment is specifically shown as nucleotides at positions 5716 to 7979 from the 5' end of SEQ ID NO: 1 of the Sequence Listing.
  • the gRNA expression cassette includes a gRNA expression cassette 1 and a gRNA expression cassette 2.
  • the gRNA expression cassette 1 encodes gRNA1 (such as gRNAW548L), which encodes gRNA2 (such as gRNAS627I).
  • gRNA1 is targeted to the upstream target site and the gRNA2 is targeted to the downstream target site.
  • the Cas9 protein expression cassette includes a promoter that initiates transcription of the Cas9 gene (such as the Ubiquitin promoter), a Cas9 gene, and a terminator that terminates transcription of the Cas9 gene (such as the NOS terminator).
  • the gRNA expression cassette 1 includes a promoter that initiates transcription of a gRNA1 encoding gene (eg, rice U3 initiation) The transcript), the gRNA1 encoding gene, and a terminator that terminates transcription of the gRNA1 encoding gene (such as the Poly-A terminator).
  • the gRNA expression cassette 2 includes a promoter that initiates transcription of a gRNA2 encoding gene (such as a rice U3 promoter), the gRNA2 encoding gene, and a terminator that terminates transcription of the gRNA2 encoding gene (such as a Poly-T terminator).
  • a promoter that initiates transcription of a gRNA2 encoding gene (such as a rice U3 promoter)
  • the gRNA2 encoding gene such as a rice U3 promoter
  • a terminator that terminates transcription of the gRNA2 encoding gene
  • the gRNA expression cassette 1 (e.g., gRNAW548L expression cassette) is specifically shown as nucleotides 261 to 747 from the 5' end of SEQ ID NO: 1 of the Sequence Listing.
  • the gRNA expression cassette 2 (e.g., gRNAS627I expression cassette) is specifically shown as nucleotides 8328-8814 from the 5' end of SEQ ID NO: 1 of the Sequence Listing.
  • the vector for site-directed modification of the plant genome may be the recombinant vector pCXUN-cas9-gRNA548-gRNA627-arm donor, as shown in SEQ ID NO:1 of the Sequence Listing.
  • nucleotides 900-7570 are the Cas9 protein expression cassette (nucleotides 5580-7570 are the Ubiquitin promoter that initiates transcription of the Cas9 protein gene, and the 1446-5576 nucleus
  • the nucleotide is the Cas9 protein gene
  • the nucleotides 900-1152 are the NOS terminator for terminating the transcription of the Cas9 protein gene
  • the nucleotides 261-747 are the gRNA expression cassette 1 (p.
  • the nucleotide 747 is the rice U3 promoter which initiates transcription of the gRNA1 encoding gene
  • the nucleotides 271-366 are the gRNA1 encoding genes
  • the nucleotides 261-270 terminate the transcription of the gRNA1 encoding gene.
  • nucleotides 8328-8814 are the gRNA expression cassette 2 (nucleotides 8328-8708 are the rice U3 promoter that initiates transcription of the gRNA2 encoding gene, 8709-8804 The nucleotide is the gRNA2 encoding gene, the nucleotides 8805-8814 are the Poly-T terminator that terminates the transcription of the gRNA2 encoding gene, and the nucleotides 7590-8051 are the donor DNA B.
  • nucleotides at positions 7590-7609 are the upstream target sites
  • nucleotides at 7616-7715 are the upstream homologous arms
  • nucleotides at positions 7716-7979 are Said point modified fragment of nucleotides 7980-8025 of the downstream homology arm, of 8032-8051 nucleotides downstream of the target site).
  • the donor DNA A can be represented by nucleotides 7590-8051 from the 5' end of SEQ ID NO: 1 of the Sequence Listing.
  • the above system may also include other reagents required for PCR amplification, reagents required for gel electrophoresis, a PCR instrument, an electrophoresis apparatus, a gel imaging system, and a camera.
  • the present invention also provides a method for site-directed modification of a plant genome.
  • the invention provides a method for fixed-point modification of plant genome, comprising the following steps
  • the vector for site-directed modification of the plant genome and the donor DNA A are introduced into the plant of interest to obtain a plant whose plant genome is site-modified.
  • the molar ratio of the vector for site-directed modification of the plant genome to the donor DNA A may be 1: (0-40), specifically 1:20.
  • the fixed point modification may be amino acid substitution, site-directed integration of foreign genes or site-directed integration of foreign fragments.
  • the site-specific modification may specifically mutate the 548th tryptophan (W) of acetolactate synthase (ALS) to leucine (L) and the serine (S) at position 627 to isoleucine (I). ).
  • the invention also provides the use of the above system for site-directed modification of a plant genome, which is any one of the following 1)-5):
  • the site-directed modification may be amino acid substitution, site-directed integration of foreign genes or site-directed integration of foreign fragments.
  • the site-modified ALS gene is the 548th tryptophan (W) in the acetolactate synthase shown by SEQ ID No. 2 (the codon of the tryptophan at this position in the wild type)
  • W tryptophan
  • L leucine
  • S serine
  • I isoleucine
  • ATT codon of the isoleucine at this position
  • the herbicide may be a pyrimidine carboxylic acid, a sulfonylurea or an imidazolinone herbicide, and specifically may be a pyrimidine carboxylic acid herbicide.
  • the pyrimidine carboxylic acid herbicide may specifically be Bismuth (BS).
  • the plant may be a monocot or a dicot.
  • the plant of interest may be a monocot or a dicot.
  • the monocotyledonous plant may be a gramineous plant, and the gramineous plant may specifically be rice, such as Nipponbare rice.
  • the experiment demonstrates that the in vitro construction of the expression cassette containing the Cas9 protein, the gRNA expression cassette of the two targets, and the recombination of the target sequence of two gRNA recognizable by the end of the arm donor (donor DNA) using the present invention.
  • the vector pCXUN-cas9-gRNA548-gRNA627-arm donor and the exogenous fragment arm donor co-transformed rice callus by gene gun method, and successfully mutated the 548th tryptophan (W) in acetolactate synthase (ALS).
  • the leucine (L) and the serine (S) at position 627 were mutated to isoleucine (I), and the fixed-point modification of the rice ALS gene was achieved, and homozygous homologous plants were obtained without off-target effects, and The homologously reconstituted plants have the properties of the herbicide resistant bispyribac.
  • Figure 1 shows the results of restriction enzyme digestion of PCR products of some T 0 generation modified rice.
  • M is DL2000 DNA molecular weight Marker.
  • Figure 2 shows the result of synonymous mutation of the nucleotide sequence near the 548th amino acid position and the 627th amino acid position of the ALS of the Cas9-arm donor group.
  • Figure 3 shows the sequencing results of homologous recombination types of plants in the Cas9-arm donor group; wherein WT ALS is the gene of ALS of wild type rice plants; Donor is the donor DNA genotype.
  • Figure 4 shows the identification of the relevant sequence of the recombinant vector pCXUN-cas9-gRNA548-gRNA627-arm donor in the Cas9-arm donor group; wherein A is the primer position map for detecting the Cas9-arm donor group; B is PCR detection of Cas9-arm donor group containing agarose gel electrophoresis map of Cas9 protein gene; C for PCR detection of Cas9-arm donor group containing gRNA expression cassette agarose gel electrophoresis map; D for PCR detection The foreign-integrated fragment of the recombinant vector pCXUN-cas9-gRNA548-gRNA627-arm donor in the Cas9-arm donor group is a complete agarose gel electrophoresis map; vector is the recombinant vector pCXUN-cas9-gRNA548-gRNA627 -arm donor, WT is the result of enzyme digestion of PCR products of wild type rice plants.
  • Figure 5 shows the growth of plants after 36 days of spraying Bispyribac-sodium; among them, 1, 2 and 3 are plants with successful double-site homologous recombination, and 4, 5 and 6 are wild type rice plants.
  • Nipponbare rice is used as a plant for site-directed modification of the genome
  • the acetolactate synthase gene of Nipponbare rice is used as a target DNA (as shown in the sequence 3 of the Sequence Listing) to construct an acetolactate synthase of Nipponbare rice.
  • the 548th tryptophan (W) (the codon of the tryptophan at this position in the wild type is TGG) is mutated to leucine (L) (the codon of the position leucine is TTG), the serine at position 627 (S) (The codon of the serine at this position in the wild type is AGT) is mutated to a fixed-point modified rice of isoleucine (I) (the codon of the isoleucine at this position is ATT).
  • the acetolactate synthase gene in Nipponbare rice also contains the EcoR V restriction recognition sequence, and the restriction enzyme recognition sequence in the arm donor is site-directed and mutated without changing the amino acid.
  • Nipponbare Rice Seeds is a product of the National Crop Germplasm Conservation Center of the Crop Science Institute of the Chinese Academy of Agricultural Sciences. Nipponbare rice is also known as wild type rice and is expressed by WT.
  • R1 solid medium (pH 5.8): 4.3 g / L MS & Vitamins salt + 30 g / L sucrose + 0.5 g / L MES + 300 mg / L casein amino acid + 2.8 g / L L-valine + 2 mg / L 2, 4-D+4g/L plant gel with the balance being water.
  • R4 solid medium (pH 5.8): 4.3 g/L MS & Vitamins salt + 30 g / L sucrose + 0.5 g / L MES + 2 g / L casein amino acid + 30 g / L sorbitol + 2 mg / L kinetin + 1 mg / L NAA +4 g/L plant gel with the balance being water.
  • R5 solid medium (pH 5.8): 2.15 g/L MS & Vitamins salt + 15 g / L sucrose + 0.5 g / L MES + 2 g / L plant gel, the balance is water.
  • Example 1 Construction of a vector for site-directed mutagenesis of acetolactate synthase (ALS) gene
  • Sequence 1 of the synthetic sequence listing was constructed from the double-stranded DNA molecule shown at position 5590-8051 at the 5' end, and was named arm donor (donor DNA).
  • the recombinant vector pCXUN-cas9-gRNA548-gRNA627-arm donor (loop plasmid) was synthesized.
  • the recombinant vector pCXUN-cas9-gRNA548-gRNA627-arm donor is shown in SEQ ID NO:1 of the Sequence Listing.
  • nucleotides 900-7570 constitute the Cas9 protein expression cassette (the Ubiquitin promoter is composed of nucleotides 5580-7570, and the Cas9 gene is composed of nucleotides 1446-5576, number 900-1152
  • the nucleotides constitute the NOS terminator
  • the nucleotides 261-747 constitute the gRNA expression cassette 1
  • the nucleotides 367-747 constitute the OsU3 promoter
  • nucleotides 271-366 constitute the gRNA1 encoding gene
  • the nucleotides 261-270 constitute the Poly-A terminator
  • the nucleotides 8328-8814 constitute the gRNA expression cassette 2 (the nucleotides 8328-8708 constitute the OsU3 promoter, and the nucleotides 8709-8804)
  • the acid constitutes the gRNA2 encoding gene
  • the nucleotides 8805-8814 constitute the Poly-T terminator
  • the Cas9 protein expression cassette expresses the Cas9 protein.
  • the gRNA expression cassette 1 designated as the gRNAW548L expression cassette, expresses gRNAW548L.
  • the gRNA expression cassette 2 designated as the gRNAS627I expression cassette, expresses gRNAS627I.
  • Example 2 Acquisition and verification of rice at 548 and 627 double-site amino acid fixed-point modification of ALS
  • step 2 the induced callus was hypertonic treated with R1 solid medium containing 0.3 M mannitol and 0.3 M sorbitol for 4-6 h to obtain callus after hypertonic treatment.
  • the recombinant carrier pCXUN-cas9-gRNA548-gRNA627-arm donor and arm-donor were mixed at 1:20, and then the hypertonic callus obtained by step 2 of the gene gun bombardment (using 0.6 ⁇ m of gold powder, bombardment) The bombardment was performed at a pressure of 900 psi to obtain a transformed callus.
  • the transformed callus was cultured on R1 solid medium containing 0.3 M mannitol and 0.3 M sorbitol for 16 h, and then transferred to R1 solid medium containing 50 mg/L hygromycin. The cells were incubated at 28 ° C for 2 weeks, then transferred to R1 solid medium containing 0.4 ⁇ M Bispyribac-sodium and incubated at 28 ° C for 2 weeks.
  • step 4 select the positive callus with good growth and tender yellow color, transfer it to R4 solid medium containing 0.4 ⁇ M Bispyribac-sodium with sterile forceps, and incubate at 28 °C until the differentiated seedling grows to 2 -5mm.
  • step 5 the seedlings are transferred to R5 solid medium, incubated at 28 °C for 2-3 weeks, then transplanted into soil and cultured in a greenhouse (temperature 28-30 ° C, 16 h light / 8 h dark) A total of 116 T 0 generation fixed-point modified rice were obtained, which was recorded as Cas9-arm donor group.
  • the hypertonic callus obtained in step 2 was cultured on R1 solid medium containing 0.3 M mannitol and 0.3 M sorbitol for 16 h, then transferred to R1 solid medium and incubated at 28 ° C for 2 weeks, then Transfer to R1 solid medium and incubate at 28 ° C for 2 weeks.
  • step 7 the callus which grows well and tender yellow is selected, transferred to R4 solid medium with sterile forceps, and cultured at 28 ° C until the differentiated seedlings grow to 2-5 mm.
  • step 8 the seedlings are transferred to R5 solid medium, incubated at 28 ° C for 2-3 weeks, then transplanted into soil and cultured in a greenhouse (temperature 28-30 ° C, 16 h light / 8 h dark) ), T 0 generation non-fixed-point modified rice was obtained and recorded as wild type group.
  • the 52 strains of the Cas9-arm donor group obtained in the first step and the wild type group obtained in the first step of the first step were randomly identified as follows:
  • RNA was extracted using a plant genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.).
  • the acetolactate synthase (ALS) gene was amplified by PCR using genomic DNA as a template and primers consisting of 753F and 753R, and then digested with restriction endonuclease EcoR V.
  • PCR reaction system 25 ⁇ L: 10 ⁇ PCR Buffer 2.5 ⁇ L, dNTP 2 ⁇ L, 753F 0.5 ⁇ L, 753R 0.5 ⁇ L, genomic DNA 1 ⁇ L, rTaq 0.2 ⁇ L, and ddH 2 O 18.3 ⁇ L.
  • PCR reaction conditions pre-denaturation at 94 ° C for 4 min; denaturation at 94 ° C for 40 s, annealing at 58 ° C for 40 s, extension at 72 ° C for 1 min, a total of 35 cycles; finally extending at 72 ° C for 10 min.
  • 753F and 753R are respectively set in the rice genome and correspond to arm-Donor. Upstream and downstream of the section.
  • the rice ALS gene contains the restriction endonuclease EcoR V restriction endonuclease recognition sequence (gatatc).
  • gatatc restriction endonuclease EcoR V restriction endonuclease recognition sequence
  • the plant to be tested is a successfully reconstituted plant.
  • the genomic DNA of the plant to be tested is a template, if the PCR amplification product can be cleaved by the restriction enzyme EcoR V (488 bp and 265 bp), the plant to be tested is a plant that has not been successfully recombined.
  • the agarose gel electrophoresis pattern of some digested products is shown in Figure 1.
  • 48 strains could not be cleaved by restriction endonuclease EcoR V, and 4 strains could be restricted by restriction endonuclease EcoR V Cut open. Wild type rice can be cut by restriction endonuclease EcoR V.
  • the 52 strains of Cas9-arm donor rice randomly taken in step 1 were extracted, and the genomic DNA was extracted.
  • the primer pair consisting of 753F and 753R was used for PCR amplification, and then the PCR amplification products were sequenced.
  • nucleotide sequence near the 548th amino acid position of the Cas9-arm donor group and the nucleotide sequence near the 627th amino acid position are shown in Figure 3 (B99-12 is heterozygous, B99-12-39, B99-12-17 is the sequencing result of the relevant interval in the two chromosomes).
  • the following primer pairs were designed: a pair of primers for detecting the Cas9 protein gene (Cas9-F/Cas9-R), which was amplified in plants containing the Cas9 protein gene.
  • the fragment length was 738 bp; the primer pair (U3F/U3R) of the gRNA expression cassette was detected, and the fragment amplified in the plant containing the gRNA expression cassette was 614 bp in length; the recombinant vector pCXUN-cas9-gRNA548-gRNA627 was randomly detected in the plant.
  • the length of the amplified fragment is 365 bp when all the arm donors in the plant are edited, and the arm donor in the plant is not edited.
  • the amplified fragment was 841 bp in length, and the arm donor portion of the plant was expanded to contain both 365 bp and 841 bp amplified fragments.
  • the 52 strains of Cas9-arm donor rice randomly selected in step 1 were extracted, and genomic DNA was extracted, and the primer pair of Cas9 protein gene was detected by PCR.
  • the results showed that except for plants B99-9, B99-10, B99-11 and B99-12, other plants contained the Cas9 protein gene, and the PCR amplification product contained an amplified fragment of 738 bp in length.
  • the plants containing the Cas9 protein gene have all been homologously recombined successfully and are homozygous strains, so gRNA cannot recognize the ALS gene after site-specific modification, and the absence of chimera occurs.
  • the 52 strains of Cas9-arm donor rice randomly taken in step 1 were extracted, and genomic DNA was extracted, and the primer pair of the gRNA expression cassette was used for PCR detection.
  • the results showed that all plants contained a gRNA expression cassette (C in Figure 4), and the PCR amplification products contained amplified fragments of 614 bp in length.
  • plant B99-9 was the only plant that still had a 841 bp long fragment PCR amplification product (D in Figure 4), and the sequencing results showed that the plant was at the amino acid codons at positions 548 and 627 of ALS. Non-homologous recombination occurred.
  • the possible off-target sites of gRNA548 and gRNA627 were predicted, and primer pairs were designed according to the flanking sequences that may exist at the off-target site: for the gRNAW548L expression cassette Primer pairs are OFF1F/OFF1R and OFF2F/OFF2R, and primer pairs for the gRNAS627I expression cassette are OFF3F/OFF3R, OFF4F/OFF4R, and OFF5F/OFF5R.
  • the amplified fragments of OFF5R on off-target plants were 492 bp, 606 bp, 597 bp, 388 bp and 382 bp, respectively.
  • the invention establishes a technical system for obtaining herbicide-resistant rice by site-directed modification of acetolactate synthase (ALS) gene by using CRISPR/Cas9 system, and at the same time, using the CRISPR/Cas9 system for fixed-point modification of genes in rice and other crops, Replacement and site-specific integration of exogenous genes provide the basis for improving agronomic traits of other important crops.
  • ALS acetolactate synthase

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Abstract

提供了利用CRISPR-Cas9系统定点修饰ALS基因获得抗除草剂水稻的系统及其应用。

Description

利用CRISPR-Cas9系统定点修饰ALS基因获得抗除草剂水稻的系统及其应用 技术领域
本发明属于基因工程领域，具体涉及利用CRISPR-Cas9系统(CRISPR/Cas9系统)定点修饰ALS基因获得抗除草剂水稻的系统及其应用。
背景技术
CRISPR/Cas9系统是继ZFNs和TALENs技术之后出现的基因组定点编辑新技术。与ZFNs和TALENs不同的是，CRISPR/Cas9系统对靶位点的识别依赖于核酸之间碱基互补配对，可对任何紧随PAM(NGG)的20bp的靶点序列进行编辑，且其靶点在基因组中的分布频率很高，因此对于需要定点编辑的靶基因，更容易找到合适的靶位点。另外CRISPR/Cas9系统可同时对同一基因的不同位点或多个基因的位点进行定向编辑，使其运用更加灵活。此外，CRISPR/Cas9系统操作简单快捷，每次打靶只需替换原有载体上20-30bp的核苷酸序列，更适宜规模化，高通量操作。CRISPR/Cas9作为一种新的靶向基因修饰技术，展现了广阔的发展潜力和应用前景，有望成为未来基因定向编辑的最强有力的工具之一。CRISPR/Cas9目前已经应用于水稻、小麦、拟南芥以及本生烟基因组的定点敲除研究中，但尚未有进行重要农作物目标农艺性状通过定点修饰(氨基酸替换或定点整合)进行遗传改良的研究。
利用CRISRP/Cas9系统在作物中对基因组编辑主要分为三种，基因的定点敲除方法获得突变体、对靶标基因的定点修饰及外源基因定点整合。因定点敲除操作方便，技术成熟等特点，在现有报道中最多。Shan等(2013)成功获得水稻OsBADH2、OsPDS基因突变体，突变概率为7.1％-9.4％。Wang等(2013)利用CRISPR技术成功定点敲除小麦TaMLO-A1基因。Miao等(2013)对水稻叶绿素合成基因CAO1和分蘖夹角控制基因LAZY进行编辑，结果表明，有83.3％的T0代转基因植株中CAO1基因相应位点发生了突变，而有高达91.6％的转基因植株中LAZY基因相应位点发生了突变，其中LAZY基因的突变纯合体的比例达到50％，均表现出分蘖夹角变大的表型。 Zhang等(2014)对2个水稻亚种(粳稻日本晴和籼稻Kasalath)11个靶基因中CRISPR/Cas9诱导产生突变的效率、特点、遗传性及特异性等进行分析，T0代转基因植株突变效率高达66.7％，而且超过一半的靶基因位点在T0代获得纯合突变体，突变体后代的遗传传递符合经典的孟德尔定律。Ma等(2015)利用CRISPR/Cas9系统共编辑水稻中46个靶位点，突变率平均为85.4％，大部分为均一的双等位突变(54.9％)和纯合突变(24.7％)，并可遗传到后代。以上研究说明，利用CRISPR/Cas9系统可实现对作物特定基因的高效定点突变。
利用CRISRP/Cas9系统对植物进行定点氨基酸替换和外源基因定点整合的研究，目前只有几例报道。Miao等(2013)通过将GUUS基因与CRISPR/Cas9系统同时转入到水稻中，可检测到GUS活性，说明同源重组发生。Schiml等(2014)将CRISPR/Cas9以及两端含有与spacer相同序列的模板DNA构建到同一个T-DNA中转化拟南芥，通过同源重组DNA修复途径，在AtADH1基因的靶标位点精确插入卡那霉素的抗性基因nptII。Li等(2015)将外源片段和CRISPR/Cas9同时导入大豆中，并在愈伤组织中检测到成功定点修饰的ALS1基因，但尚未获得植株。但将抗潮霉素基因表达盒与CRISPR/Cas9系统同时导入到大豆中，通过PCR及Southern鉴定确定获得同源重组株系，整合基因的遗传符合孟德尔遗传规律。Svitashev等(2015)通过同源重组方法将liguleless-1基因定点重组到玉米基因组中。此外，将CRISPR/Cas9系统和外源修饰片段(双链和单链DNA)同时通过基因枪或农杆菌方法导入玉米中，成功将ALS2的第165位的脯氨酸修饰为丝氨酸(P165S)的植株并获得磺脲类除草剂抗性。
通过基因组编辑介导的同源重组，定点修饰提高重要农作物的除草剂抗性，是目前基因组编辑研究领域的热点之一。传统获得抗除草剂作物方法主要如下：(1)通过外源基因导入(epsps，bar，pmi等)提高农作物的除草剂抗性，已经有多项报道，但因为是属于转基因作物，其在重要粮食作物中的推广应用受到限制，目前尚未有抗除草剂的转基因小麦和水稻商业化生产的报道；此外，抗除草剂基因漂移可能形成超级杂草等转基因生物安全问题而备受关注；(2)植物氨基酸合成中的关键酶一直是新型除草剂研发中重要的靶标酶，通过EMS将作物内源基因突变，提高重要农 作物的除草剂抗性，已经在小麦、大麦和水稻等有相关报道，但因EMS突变的随机性，需要大面积广泛筛选具有除草剂抗性和其它农艺性状未改变的突变体，因此，此技术很难得到广泛应用。
靶标乙酰乳酸合成酶(ALS)抑制剂类除草剂双草醚是目前开发最活跃的领域之一。乙酰乳酸合成酶(Acetolactate synthase,ALS)存在于植物中，它能催化丙酮酸为乙酰乳酸，具有高度专一性和极高的催化效率，从而合成植物体内3种必需的支链氨基酸(缬氨酸、亮氨酸和异亮氨酸)。磺脲类、咪唑啉酮类、嘧啶羧酸类除草剂可抑制ALS活性，破坏植物体内缬氨酸、亮氨酸和异亮氨酸的合成，从而导致植物死亡。Swanson等(1989)利用小孢子化学诱变的方法获得2个抗咪唑啉酮类除草剂的油菜突变体PMl和PM2，PMl和PM2都由ALS基因点突变而成，其中PMl是基因BnALS1的第653位丝氨酸发生突变，PM2是基因BnALS2的第574位色氨酸发生突变(以拟南芥的ALS氨基酸位置计算)。2014年3月，Cibus Global公司利用单核苷酸基因修复技术(Gene Repair OligoNucleotide technology)，成功获得了ALS基因编辑后抗磺脲类除草剂油菜，目前已经在加拿大获得商业化生产(http://www.cibus.com/technology.php)。磺脲类、咪唑啉酮类、嘧啶羧酸类除草剂广泛应用于生产中，具备生物活性高、杀草谱广的优点，并且对人和动物具有安全性(Endo et al.,2007)。
发明公开
本发明提供了一种利用CRISPR-Cas9系统定点修饰ALS基因获得抗除草剂水稻的系统及其应用。
本发明所提供的用于植物基因组定点修饰的系统，含有用于植物基因组定点修饰的载体和供体DNA甲；所述用于植物基因组定点修饰的载体含有Cas9蛋白表达盒、gRNA表达盒和供体DNA乙；所述gRNA表达盒编码两种gRNA，所述两种gRNA分别靶向于目的植物的靶DNA的两个靶位点；所述靶DNA中待定点修饰片段位于所述两个靶位点之间；所述两个靶位点中位于上游的靶位点为上游靶位点，位于下游的靶位点为下游靶位点；
所述供体DNA乙含有所述上游靶位点、所述下游靶位点和位于所述上游靶位点和所述下游靶位点之间的定点修饰片段；所述定点修饰片段为要替换所述待定点修饰片段的DNA片段；
所述供体DNA甲与所述供体DNA乙的核苷酸序列相同。
所述Cas9蛋白表达盒、所述gRNA表达盒和所述供体DNA乙可以存在于同一个质粒上，也可以以任一组合存在于两个质粒上，也可以分别存在于一个质粒上。
上述系统中，所述定点修饰可为氨基酸替换，外源基因定点整合或外源片段定点整合。
上述系统中，所述供体DNA乙还含有用于和所述靶DNA进行同源重组的上游同源臂和下游同源臂，所述上游同源臂位于所述上游靶位点与所述定点修饰片段之间，所述下游同源臂位于所述定点修饰片段与所述下游靶位点之间。
上述系统中，所述植物或所述目的植物可为单子叶植物或双子叶植物。所述单子叶植物可为禾本科植物。进一步的，所述禾本科植物具体可为水稻，例如日本晴水稻。
上述系统中，所述靶DNA可为编码乙酰乳酸合成酶的基因；所述乙酰乳酸合成酶为a1或a2：
a1、氨基酸序列如序列表的序列2所示的蛋白质；
a2、将序列2中进行一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有乙酰乳酸合成酶活性的由a1衍生的蛋白质。
所述靶DNA的核苷酸序列具体如序列表的序列3所示。
所述上游靶位点具体如序列表的序列1的自5’末端第7590-7609位核苷酸所示。所述下游靶位点具体如序列表的序列1的自5’末端第8032-8051位核苷酸所示。所述定点修饰片段具体如序列表的序列1的自5’末端第7716-7979位核苷酸所示。
所述gRNA表达盒包括gRNA表达盒1和gRNA表达盒2。所述gRNA表达盒1编码gRNA1(如gRNAW548L)，所述gRNA表达盒2编码gRNA2(如gRNAS627I)。所述gRNA1靶向于所述上游靶位点，所述gRNA2靶向于所述下游靶位点。
所述Cas9蛋白表达盒包括启动Cas9基因转录的启动子(如Ubiquitin启动子)、Cas9基因和终止Cas9基因转录的终止子(如NOS终止子)。所述gRNA表达盒1包括启动gRNA1编码基因转录的启动子(如水稻U3启 动子)、所述gRNA1编码基因和终止所述gRNA1编码基因转录的终止子(如Poly-A终止子)。所述gRNA表达盒2包括启动gRNA2编码基因转录的启动子(如水稻U3启动子)、所述gRNA2编码基因和终止所述gRNA2编码基因转录的终止子(如Poly-T终止子)。
所述gRNA表达盒1(如gRNAW548L表达盒)具体如序列表的序列1的自5’末端第261-747位核苷酸所示。所述gRNA表达盒2(如gRNAS627I表达盒)具体如序列表的序列1的自5’末端第8328-8814位核苷酸所示。
上述系统中，所述用于植物基因组定点修饰的载体可为重组载体pCXUN-cas9-gRNA548-gRNA627-arm donor，如序列表的序列1所示。序列表的序列1中，第900-7570位核苷酸为所述Cas9蛋白表达盒(第5580-7570位核苷酸为启动所述Cas9蛋白基因转录的Ubiquitin启动子,第1446-5576位核苷酸为所述Cas9蛋白基因，第900-1152位核苷酸为所述终止Cas9蛋白基因转录的NOS终止子)，第261-747位核苷酸为所述gRNA表达盒1(第367-747位核苷酸为启动所述gRNA1编码基因转录的水稻U3启动子，第271-366位核苷酸为所述gRNA1编码基因，第261-270位核苷酸为终止所述gRNA1编码基因转录的Poly-A终止子)，第8328-8814位核苷酸为所述gRNA表达盒2(第8328-8708位核苷酸为启动所述gRNA2编码基因转录的水稻U3启动子，第8709-8804位核苷酸为所述gRNA2编码基因，第8805-8814位核苷酸为终止所述gRNA2编码基因转录的Poly-T终止子)，第7590-8051位核苷酸为所述供体DNA乙(第7590-7609位核苷酸为所述上游靶位点，第7616-7715位核苷酸为所述上游同源臂，第7716-7979位核苷酸为所述定点修饰片段，第7980-8025位核苷酸为所述下游同源臂，第8032-8051位核苷酸为所述下游靶位点)。
上述系统中，所述供体DNA甲可如序列表的序列1的自5’末端第7590-8051位核苷酸所示。
上述系统还可包括进行PCR扩增所需的其它试剂、进行凝胶电泳所需的试剂、PCR仪、电泳仪、凝胶成像系统和照相机。
为解决上述技术问题，本发明还提供了一种用于植物基因组定点修饰的方法。
本发明所提供的一种用于植物基因组定点修饰的方法，包括如下步 骤：向目的植物中导入所述用于植物基因组定点修饰的载体和所述供体DNA甲，得到植物基因组被定点修饰的植物。
上述方法中，所述用于植物基因组定点修饰的载体和所述供体DNA甲的摩尔比可为1：(0-40)，具体可为1:20。
上述方法中，所述定点修饰可为氨基酸替换，外源基因定点整合或外源片段定点整合。
上文中，定点修饰具体可为将乙酰乳酸合成酶(ALS)的第548位色氨酸(W)突变为亮氨酸(L)和第627位丝氨酸(S)突变为异亮氨酸(I)。
本发明还提供了上述用于植物基因组定点修饰的系统的应用，为下述1)-5)中任一种：
1)在植物基因组定点修饰中的应用；
2)在培育基因组被定点修饰的植物中的应用；
3)在培育抗除草剂植物中的应用；
4)在植物育种中的应用；
5)在转基因植物研究中，利用定点修饰后的ALS基因作为筛选标记基因的应用。
上述应用中，所述定点修饰可为氨基酸替换，外源基因定点整合或外源片段定点整合。
上述应用中，所述定点修饰后的ALS基因是将SEQ ID No.2所示的乙酰乳酸合成酶中的第548位色氨酸(W)(野生型中该位置的色氨酸的密码子为TGG)突变为亮氨酸(L)(该位置亮氨酸的密码子为TTG)，第627位丝氨酸(S)(野生型中该位置的丝氨酸的密码子为AGT)突变为异亮氨酸(I)(该位置异亮氨酸的密码子为ATT)，其它氨基酸残基不变得到的乙酰乳酸合成酶定点修饰蛋白的编码基因。
上述应用中，所述除草剂可为嘧啶羧酸类、磺脲类或咪唑啉酮类除草剂，具体可为嘧啶羧酸类除草剂，进一步的，所述嘧啶羧酸类除草剂具体可为双草醚(BS)。
所述植物可为单子叶植物或双子叶植物。所述目的植物可为单子叶植物或双子叶植物。所述单子叶植物可为禾本科植物，所述禾本科植物具体可为水稻，例如日本晴水稻。
实验证明，采用本发明的体外构建含有Cas9蛋白的表达盒、两个靶点的gRNA表达盒、及外源片段arm donor(供体DNA)两端加两个gRNA可识别的靶点序列的重组载体pCXUN-cas9-gRNA548-gRNA627-arm donor与外源片段arm donor通过基因枪方法共转化水稻愈伤组织，能够成功将乙酰乳酸合成酶(ALS)中的第548位色氨酸(W)突变为亮氨酸(L)和第627位丝氨酸(S)突变为异亮氨酸(I)，实现了水稻ALS基因的定点修饰，获得了同源重组的纯合系植株，无脱靶效应，并且同源重组的植株具有抗除草剂双草醚的特性。
附图说明
图1为部分T0代定点修饰水稻的PCR产物酶切鉴定结果。其中，M为DL2000 DNA分子量Marker。
图2为Cas9-arm donor组植株的ALS的第548位氨基酸位点和第627位氨基酸位点附近的核苷酸序列的同义突变结果。
图3为Cas9-arm donor组植株的同源重组类型的测序结果鉴定；其中，WT ALS为野生型水稻植株的ALS的基因；Donor为供体DNA基因型。
图4为检测Cas9-arm donor组植株中是否存在重组载体pCXUN-cas9-gRNA548-gRNA627-arm donor上相关序列的鉴定；其中，A为检测Cas9-arm donor组植株所用引物位置标示图；B为PCR检测Cas9-arm donor组植株中是否含有Cas9蛋白基因的琼脂糖凝胶电泳图；C为PCR检测Cas9-arm donor组植株中是否含有gRNA表达盒的琼脂糖凝胶电泳图；D为PCR检测Cas9-arm donor组植株中随机整合的重组载体pCXUN-cas9-gRNA548-gRNA627-arm donor中的外源片段arm donor是否完整的琼脂糖凝胶电泳图；vector为重组载体pCXUN-cas9-gRNA548-gRNA627-arm donor，WT为野生型组水稻植株的PCR产物酶切鉴定结果。
图5为喷洒双草醚(Bispyribac-sodium)36天后植株的生长情况；其中，1、2和3均为双位点同源重组成功的植株，4、5和6均为野生型水稻植株。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施 例仅为了阐明本发明，而不是为了限制本发明的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，如无特殊说明，均设置三次重复实验，结果取平均值。
下述实施例以日本晴水稻作为将要进行基因组定点修饰的目的植物，以日本晴水稻的乙酰乳酸合成酶基因作为靶DNA(如序列表的序列3所示)，构建将日本晴水稻的乙酰乳酸合成酶的第548位色氨酸(W)(野生型中该位置的色氨酸的密码子为TGG)突变为亮氨酸(L)(该位置亮氨酸的密码子为TTG)，第627位丝氨酸(S)(野生型中该位置的丝氨酸的密码子为AGT)突变为异亮氨酸(I)(该位置异亮氨酸的密码子为ATT)的定点修饰水稻。
日本晴水稻中的乙酰乳酸合成酶基因中还含有EcoR V酶切识别序列，供体DNA(arm donor)中酶切识别序列在不改变氨基酸的情况下被定点突变掉。
日本晴水稻种子为中国农业科学院作物科学研究所国家农作物种质资源保存中心的产品。日本晴水稻又称野生型水稻，用WT表示。
R1固体培养基(pH5.8)：4.3g/L MS&Vitamins盐+30g/L蔗糖+0.5g/L MES+300mg/L酪蛋白氨基酸+2.8g/L L-脯氨酸+2mg/L 2，4-D+4g/L植物凝胶，余量为水。
R4固体培养基(pH5.8)：4.3g/L MS&Vitamins盐+30g/L蔗糖+0.5g/L MES+2g/L酪蛋白氨基酸+30g/L山梨醇+2mg/L激动素+1mg/LNAA+4g/L植物凝胶，余量为水。
R5固体培养基(pH5.8)：2.15g/L MS&Vitamins盐+15g/L蔗糖+0.5g/L MES+2g/L植物凝胶，余量为水。
下述实施例中所用到的引物对序列及其用途如表1所示。
表1 所用引物对的序列及用途
Figure PCTCN2016077337-appb-000001
Figure PCTCN2016077337-appb-000002
实施例1、用于乙酰乳酸合成酶(ALS)基因定点突变的载体的构建
人工合成序列表的序列1自5’末端第7590-8051位所示的双链DNA分子，将其命名为arm donor(供体DNA)。
人工合成重组载体pCXUN-cas9-gRNA548-gRNA627-arm donor(环形质粒)。重组载体pCXUN-cas9-gRNA548-gRNA627-arm donor如序列表的序列1所示。序列表的序列1中，第900-7570位核苷酸组成Cas9蛋白表达盒(第5580-7570位核苷酸组成Ubiquitin启动子，第1446-5576位核苷酸组成Cas9基因，第900-1152位核苷酸组成NOS终止子)，第261-747位核苷酸组成gRNA表达盒1(第367-747位核苷酸组成OsU3启动子，第271-366位核苷酸组成gRNA1编码基因，第261-270位核苷酸组成Poly-A终止子)，第8328-8814位核苷酸组成gRNA表达盒2(第8328-8708位核苷酸组成OsU3启动子，第8709-8804位核苷酸组成gRNA2编码基因，第8805-8814位核苷酸组成Poly-T终止子)，第7590-8051位核苷酸组成arm donor(第7590-7609位核苷酸组成上游靶位点，第7616-7715位核 苷酸组成上游同源臂，第7716-7979位核苷酸组成定点修饰片段，第7980-8025位核苷酸组成下游同源臂，第8032-8051位核苷酸组成下游靶位点)。Cas9蛋白表达盒表达Cas9蛋白。gRNA表达盒1，命名为gRNAW548L表达盒，表达gRNAW548L。gRNA表达盒2，命名为gRNAS627I表达盒，表达gRNAS627I。
实施例2、ALS的第548位和627位双位点氨基酸定点修饰水稻的获得及验证
一、定点修饰水稻的获得
1、选取饱满的日本晴水稻种子，剥去种皮，灭菌洗涤后，均匀的点入R1固体培养基中，28℃持续光照2-3周诱导愈伤组织的形成。
2、完成步骤1后，将诱导得到的愈伤组织用含有0.3M甘露醇和0.3M山梨醇的R1固体培养基高渗处理4-6h，得到高渗处理后的愈伤组织。
3、将重组载体pCXUN-cas9-gRNA548-gRNA627-arm donor和arm-donor按摩尔比1：20混合后采用基因枪轰击步骤2得到的高渗处理的愈伤组织(采用0.6μm的金粉，轰击压力为900psi进行轰击)，得到转化后的愈伤组织。
4、完成步骤3后，将转化后的愈伤组织在含0.3M甘露醇和0.3M山梨醇的R1固体培养基上培养16h，然后转移至含50mg/L潮霉素的R1固体培养基上并28℃光照培养2周，然后转移至含0.4μM Bispyribac-sodium的R1固体培养基上并28℃光照培养2周。
5、完成步骤4后，选取生长良好呈嫩黄色的阳性愈伤组织，用无菌镊子移至含0.4μM Bispyribac-sodium的R4固体培养基中，28℃光照培养至分化出来的幼苗长至2-5mm。
6、完成步骤5后，将幼苗转入R5固体培养基中，28℃光照培养2-3周，然后移栽到土中并置于温室中培养(温度28-30℃，16h光照/8h黑暗)，共获得116株T0代定点修饰水稻，记为Cas9-arm donor组。
7、将步骤2得到的高渗处理的愈伤组织在含0.3M甘露醇和0.3M山梨醇的R1固体培养基上培养16h，然后转移至R1固体培养基上并28℃光照培养2周，然后转移至R1固体培养基上并28℃光照培养2周。
8、完成步骤7后，选取生长良好呈嫩黄色的愈伤组织，用无菌镊子移至R4固体培养基中，28℃光照培养至分化出来的幼苗长至2-5mm。
9、完成步骤8后，将幼苗转入R5固体培养基中，28℃光照培养2-3周，然后移栽到土中并置于温室中培养(温度28-30℃，16h光照/8h黑暗)，获得T0代非定点修饰水稻，记为野生型组。
二、同源重组植株的鉴定
1、同源重组植株的PCR酶切鉴定
随机取52株步骤一获得的Cas9-arm donor组水稻和10株步骤一获得的野生型组水稻进行如下鉴定：
取水稻叶片，使用植物基因组DNA提取试剂盒(天根生化科技(北京)有限公司)提取基因组DNA。以基因组DNA为模板，采用753F和753R组成的引物对对乙酰乳酸合成酶(ALS)基因进行PCR扩增，然后用限制性内切酶EcoR V进行酶切鉴定。PCR反应体系(25μL)：10×PCR Buffer 2.5μL、dNTP 2μL、753F 0.5μL、753R 0.5μL、基因组DNA 1μL、rTaq 0.2μL、ddH2O 18.3μL。PCR反应条件：94℃预变性4min；94℃变性40s、58℃退火40s、72℃延伸1min，共35个循环；最后72℃延伸10min。
由于arm-Donor与水稻自身的ALS基因区段具有很高的同源性，为了排除arm-Donor对同源重组植株的鉴定的干扰，将753F和753R分别设在水稻基因组中与arm-Donor对应的区段的上游和下游。因水稻自身ALS基因中含有限制性内切酶EcoR V的酶切识别序列(gatatc)。arm-Donor中，引入了定点突变，因此arm-Donor为模板的PCR扩增产物不能被限制性内切酶EcoR V切开。以待测植物的基因组DNA为模板，如果PCR扩增产物不能被限制性内切酶EcoR V切开(PCR扩增产物进行限制性内切酶EcoR V酶切后，由于没有被切开，仍为753bp)，待测植株即为重组成功的植株。以待测植物的基因组DNA为模板，如果PCR扩增产物能被限制性内切酶EcoR V切开(为488bp和265bp)，待测植株即为没有重组成功的植株。
部分酶切产物的琼脂糖凝胶电泳图见图1。52株Cas9-arm donor组水稻中，48株不能被限制性内切酶EcoR V切开，4株能被限制性内切酶EcoR V切开。野生型组水稻均可以被限制性内切酶EcoR V切开。
2、测序
取步骤1中随机取的52株Cas9-arm donor组水稻，提取基因组DNA，采用引物对753F和753R组成的引物对进行PCR扩增，然后将PCR扩增产物进行测序。
48株不能被限制性内切酶EcoR V切开均为同源重组成功植株(其中包括B98-1、B98-3、B98-4、B98-5、B99-5、B99-6、B99-7、B99-13、B99-23)，并为纯合株系。Cas9-arm donor组同源重组成功植株的ALS与野生型相比，第548位色氨酸(W)(野生型中该位置的色氨酸的密码子为TGG)均突变为亮氨酸(L)(该位置亮氨酸的密码子为TTG)，第627位丝氨酸(S)(野生型中该位置的丝氨酸的密码子为AGT)均突变为异亮氨酸(I)(该位置异亮氨酸的密码子为ATT)，其它氨基酸均没有变化，但是这两个位点附近的核苷酸发生同义突变。
测序结果表明，4株能被限制性内切酶EcoR V切开的Cas9-arm donor组水稻植株(B99-9、B99-10、B99-11和B99-12)，为一条链在ALS的第548位氨基酸的密码子处发生同源重组、在ALS的第627位氨基酸的密码子处没有发生同源重组，另外一条链在ALS的第548位氨基酸的密码子处和第627位氨基酸的密码子处均发生非同源重组。
Cas9-arm donor组植株的重组结果统计见表2。
表2 重组结果的统计
Figure PCTCN2016077337-appb-000003
注：Ho代表纯合株系，Com-He代表复合杂合株系，“+”代表检测结果为阳性。HR548、HR627-1、HR627-2、HR627-3的测序结果和相应核苷 酸序列见图2。
部分Cas9-arm donor组植株第548位氨基酸位点附近的核苷酸序列和第627位氨基酸位点附近的核苷酸序列见图3(B99-12为杂合型，B99-12-39、B99-12-17分别为两条染色体中相关区间的测序结果)。
3、同源重组植株是否存在重组载体pCXUN-cas9-gRNA548-gRNA627-arm donor上相关序列的鉴定
根据重组载体pCXUN-cas9-gRNA548-gRNA627-arm donor序列，分别设计如下引物对：检测Cas9蛋白基因的引物对(Cas9-F/Cas9-R)，在含有Cas9蛋白基因的植株中扩增得到的片段长度为738bp；检测gRNA表达盒的引物对(U3F/U3R)，在含有gRNA表达盒的植株中扩增得到的片段长度为614bp；检测植株中随机整合的重组载体pCXUN-cas9-gRNA548-gRNA627-arm donor中的外源片段arm donor是否完整的引物对(365F/365R)，在植株中的arm donor全部被编辑时扩增得到的片段长度均为365bp，在植株中的arm donor未被编辑扩增得到的片段长度为841bp，在植株中的arm donor部分被编辑时扩中同时含有365bp和841bp的扩增片段。
取步骤1中随机取的52株Cas9-arm donor组水稻，提取基因组DNA，利用检测Cas9蛋白基因的引物对进行PCR检测。结果表明,除了植株B99-9、B99-10、B99-11和B99-12,其它植株均含有Cas9蛋白基因，PCR扩增产物中含有738bp长度的扩增片段。含有Cas9蛋白基因的植株已经全部同源重组成功并且为纯合株系，所以gRNA无法识别定点修饰后的ALS基因，不存在嵌合体的情况发生。植株B99-9、B99-10、B99-11和B99-12等4颗植株虽然仍有ALS的第627位点没有被编辑，但因没有Cas9蛋白基因的完整序列在内，同样无法再被重新编辑(图4中B)。
取步骤1中随机取的52株Cas9-arm donor组水稻，提取基因组DNA，利用检测gRNA表达盒的引物对进行PCR检测。检测结果表明，所有植株均含有gRNA表达盒(图4中C)，PCR扩增产物中均含有614bp长度的扩增片段。
取步骤1中随机取的52株Cas9-arm donor组水稻，提取基因组DNA，利用检测植株中随机整合的重组载体pCXUN-cas9-gRNA548-gRNA627-arm donor中的外源片段arm donor是否完整的引物对进行PCR检测。检测结 果表明，52株植株中有51株植株全部为大片段缺失类型，即所有外源片段arm donor均被设计的重组载体pCXUN-cas9-gRNA548-gRNA627-arm donor编辑，gRNA548和gRNA627的存在将外源片段arm donor切下并用于定点修饰水稻自身ALS基因，因此为大片段缺失类型。其中,植株B99-9为唯一一株仍然存在841bp长片段PCR扩增产物的植株(图4中D)，测序结果表明，该植株在ALS的第548和627位点的氨基酸的密码子处均发生了非同源重组。
4、重组载体pCXUN-cas9-gRNA548-gRNA627-arm donor的脱靶分析
根据网上预测软件(http://crispr.dbcls.jp/)，分别对gRNA548和gRNA627可能存在的脱靶位点进行预测，并根据可能存在脱靶位点的侧翼序列设计引物对：针对gRNAW548L表达盒的引物对为OFF1F/OFF1R和OFF2F/OFF2R，针对gRNAS627I表达盒的引物对为OFF3F/OFF3R、OFF4F/OFF4R和OFF5F/OFF5R。
取步骤1中随机取的52株Cas9-arm donor组水稻，提取基因组DNA，分别采用上述各个引物对进行PCR检测，引物对OFF1F/OFF1R、OFF2F/OFF2R、OFF3F/OFF3R、OFF4F/OFF4R和OFF5F/OFF5R对脱靶植株的扩增片段长度分别为492bp、606bp、597bp、388bp和382bp。
利用引物对OFF1F/R、OFF2F/R、OFF3F/R、OFF4F/R和OFF5F/R对30颗植株进行PCR扩增，并将PCR扩增产物克隆并测序。检测结果表明，所设计的重组载体pCXUN-cas9-gRNA548-gRNA627-arm donor中gRNA表达和表达得到的gRNA并不存在脱靶情况(表3)。
表3、靶点脱靶分析
Figure PCTCN2016077337-appb-000004
4、同源重组成功的植株对除草剂双草醚(BS)的抗性鉴定
对野生型水稻和步骤一获得的ALS的第548位和627位双位点氨基酸的密码子处同源重组成功的Cas9-arm donor组T0代定点修饰水稻喷洒浓度为100μM的双草醚(Bispyribac-sodium)30-50天后，观察植株的生长状态。
结果如图5所示，喷洒浓度为100μM的双草醚(Bispyribac-sodium)36天后野生型水稻植株枯萎，Cas9-arm donor组T0代定点修饰同源重组成功的植株生长正常。
工业应用
本发明建立了一种利用CRISPR/Cas9系统定点修饰乙酰乳酸合成酶(ALS)基因获得抗除草剂水稻的技术体系，同时为在水稻和其它作物中利用此CRISPR/Cas9系统进行基因的定点修饰、替换和外源基因定点整合提供了依据，为改良其它重要农作物的农艺性状提供基础。

Claims (17)

  1. 用于植物基因组定点修饰的系统，含有用于植物基因组定点修饰的载体和供体DNA甲；
    所述用于植物基因组定点修饰的载体含有Cas9蛋白表达盒、gRNA表达盒和供体DNA乙；
    所述gRNA表达盒编码两种gRNA，所述两种gRNA分别靶向于目的植物的靶DNA的两个靶位点；所述靶DNA中待定点修饰片段位于所述两个靶位点之间；所述两个靶位点中位于上游的靶位点为上游靶位点，位于下游的靶位点为下游靶位点；
    所述供体DNA乙含有所述上游靶位点、所述下游靶位点和位于所述上游靶位点和所述下游靶位点之间的定点修饰片段；所述定点修饰片段为要替换所述待定点修饰片段的DNA片段；
    所述供体DNA甲与所述供体DNA乙的核苷酸序列相同。
  2. 根据权利要求1所述的系统，其特征在于：所述目的植物为单子叶植物或双子叶植物。
  3. 根据权利要求2所述的系统，其特征在于：所述单子叶植物为禾本科植物。
  4. 根据权利要求1-3中任一所述的系统，其特征在于：所述靶DNA为编码乙酰乳酸合成酶的基因。
  5. 如权利要求4所述的系统，其特征在于：所述乙酰乳酸合成酶为如下a1或a2：
    a1、氨基酸序列如序列表的序列2所示的蛋白质；
    a2、将序列2中进行一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有乙酰乳酸合成酶活性的由a1衍生的蛋白质。
  6. 根据权利要求5所述的系统，其特征在于：所述靶DNA如序列表的序列3所示。
  7. 如权利要求6所述的系统，其特征在于：所述上游靶位点如序列表的序列1的自5’末端第7590-7609位核苷酸所示；所述下游靶位点如序列表的序列1的自5’末端第8032-8051位核苷酸所示；所述定点修饰 片段如序列表的序列1的自5’末端第7716-7979位核苷酸所示。
  8. 根据权利要求4所述的系统，其特征在于：所述gRNA表达盒包括gRNA表达盒1和gRNA表达盒2，所述gRNA表达盒1编码gRNA1，所述gRNA表达盒2编码gRNA2，所述gRNA1靶向于所述上游靶位点，所述gRNA2靶向于所述下游靶位点。
  9. 根据权利要求8所述的系统，其特征在于：所述gRNA表达盒1如序列表的序列1的自5’末端第261-747位核苷酸所示；所述gRNA表达盒2如序列表的序列1的自5’末端第8328-8814位核苷酸所示。
  10. 根据权利要求9所述的系统，其特征在于：
    所述用于植物基因组定点修饰的载体如序列表的序列1所示；
    所述供体DNA甲如序列表的序列1的自5’末端第7590-8051位核苷酸所示。
  11. 一种用于植物基因组定点修饰的方法，包括如下步骤：向目的植物中导入权利要求1至10中任一所述的用于植物基因组定点修饰的载体和权利要求1至10中任一所述的供体DNA甲，得到植物基因组被定点修饰的植物。
  12. 根据权利要求11所述的方法，其特征在于：所述用于植物基因组定点修饰的载体和所述供体DNA的摩尔比为1：(0-40)。
  13. 权利要求1-10中任一所述的系统在植物基因组定点修饰中的应用。
  14. 权利要求1-10中任一所述的系统在培育基因组被定点修饰的植物中的应用。
  15. 权利要求1-10中任一所述的系统在培育抗除草剂植物中的应用。
  16. 权利要求1-10中任一所述的系统在植物育种中的应用。
  17. 权利要求1-10中任一所述的系统在转基因植物研究中，利用定点修饰后的ALS基因作为筛选标记基因的应用。
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