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WO2019014917A1 - 一种基因编辑系统及应用其对植物基因组进行编辑的方法 - Google Patents

一种基因编辑系统及应用其对植物基因组进行编辑的方法 Download PDF

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WO2019014917A1
WO2019014917A1 PCT/CN2017/093837 CN2017093837W WO2019014917A1 WO 2019014917 A1 WO2019014917 A1 WO 2019014917A1 CN 2017093837 W CN2017093837 W CN 2017093837W WO 2019014917 A1 WO2019014917 A1 WO 2019014917A1
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cas9
gene
sgrna
transcription unit
promoter
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PCT/CN2017/093837
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French (fr)
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韩方普
冯超
王锐
柏晗
袁静
张晶
苏汉东
刘亚林
郭宪瑞
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中国科学院遗传与发育生物学研究所
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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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

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  • the present disclosure relates to the field of plant genetic engineering technology, and in particular to a gene editing system and a method for editing the plant genome.
  • Transgenic technology is to obtain certain excellent traits such as insect-resistant and herbicide-resistant by introducing exogenous DNA fragments of interest into the genome of plants by artificial means.
  • the characteristics of transgenic technology make the obtained transgenic plants contain exogenous genetic material, so the safety of genetically modified foods is also a concern of the public.
  • the Genome editing technology developed in recent years offers new opportunities for crop functional genomics research and breeding.
  • the main genome editing techniques include three types: zinc finger nuclease (ZFN), Transcription activator like effector nuclease (TALEN), and clustered regular intervals of short back. Repeated regular interspaced short palindromic repeats/CRISPR associated protein 9, CRISPR/Cas9. All three techniques can generate DNA double strand break (DSB) at specific target sites in the genome, and obtain the insertion or deletion of genomic-specific sites by means of the endogenous DSB repair mechanism. Implement the editing of the genome. Due to the relatively simple and efficient use of the CRISPR/Cas9 system, it has been widely used in plant basic and applied research. The CRISPR/Cas9 system has been successfully applied in major crops such as rice, wheat, corn, and soybeans, demonstrating a strong potential for breeding applications.
  • ZFN zinc finger nuclease
  • TALEN Transcription activator like effector nuclease
  • CRISPR/Cas9 Clustered regular intervals of short back. Repeated
  • the editing efficiency of the CRISPR/Cas9 system is a factor that must be considered in crop basic research and applied research. High editing efficiency not only saves manpower and resource costs, but also makes large-scale gene editing of the whole genome easy to implement, thus promoting the discovery of new genes or genetic loci.
  • the editing efficiency of the CRISPR/Cas9 system is mainly affected by the following factors: A promoter for driving expression of Cas9, a promoter for driving expression of sgRNA, a target site for editing, and the like. Targeted optimization of these factors (such as finding promoters that are more efficiently expressed in callus) will have the potential to improve the efficiency of gene editing.
  • the CRISPR/Cas9 system is used in the practice of crops to drive the expression of the Cas9 gene mainly using two constitutively expressed promoters: the 35S promoter of tobacco mosaic virus and the ubiquitin gene promoter of maize.
  • the average frequency of homozygous or biallelic mutant plants reported in the existing reports is ⁇ 1% (35S), 13% (Ubi) and 30% (Ubi), respectively.
  • the editing efficiency reported in wheat is around 10% and is based on the gene gun transformation system. There are no reports on wheat based on the Agrobacterium transformation system.
  • the present disclosure provides a novel gene editing system comprising a sgRNA (Single Guide RNA) transcription unit, and DMC1p-Cas9 transcription consisting of a sequentially linked maize-derived DMC1 gene promoter and a Cas9 gene.
  • a unit wherein the target site recognized by the sgRNA conforms to a 5'-Nx-NGG-3' or 5'-CCN-Nx-3' sequence rule, and N represents any one of A, T, C, and G, 14 ⁇ X ⁇ 30, and X is an integer, and N X represents X consecutive deoxyribonucleotides.
  • DMC1 gene promoter is represented by DMC1p, and the sequence thereof is preferably as shown in SEQ ID NO.
  • the present disclosure also provides a recombinant vector comprising the aforementioned gene editing system.
  • the present disclosure also provides a method of constructing the aforementioned recombinant vector, comprising the steps of:
  • the sequence of the DMC1 gene promoter is preferably as shown in SEQ ID NO.
  • the promoter of the DMC1 gene is preferably cloned by using the genomic DNA of maize B73 as a template and the primers shown in SEQ ID NO. 2 and SEQ ID NO. 3 as amplification primer pairs.
  • the PCR reaction is carried out to obtain a fragment having the sequence shown in SEQ ID NO. 1, which is the DMC1 gene promoter.
  • the sequence is sequentially ligated to the Cas9 gene, preferably by adopting the following method: replacing the obtained DMC1 gene promoter with the 35S promoter in the 35S-Cas9-SK vector,
  • the pDMC1-Cas9-SK recombinant vector was obtained, and the DMC1p-Cas9 transcription unit was transferred to the binary vector pTF101.1 using two restriction sites of Xma I and EcoR I to obtain a recombinant vector pDMC1-Cas9.
  • Step (2) preferably adopts the following procedure: the DNA sequence of the target site conforming to the 5'-Nx-NGG-3' or 5'-CCN-Nx-3' sequence is ligated into the pU3-sgRNA vector by primer annealing.
  • the transcription unit of sgRNA was subcloned into the recombinant vector pDMC1-Cas9 by Hind III cleavage site to obtain a transcript unit including sgRNA, and DMC1p- consisting of a SGC-derived DMC1 gene promoter and a Cas9 gene.
  • Recombinant vector for the Cas9 transcription unit Recombinant vector for the Cas9 transcription unit.
  • the present disclosure also provides a method for identifying the editing activity of the aforementioned recombinant vector, comprising: transforming the recombinant vector into a plant protoplast, and determining the editing activity of the recombinant vector by identifying the efficiency of genome editing in the protoplast .
  • the present disclosure also provides a genetically engineered bacterium comprising the aforementioned recombinant vector.
  • the present disclosure also provides a method for editing a plant genome using the aforementioned gene editing system, comprising: converting the aforementioned genetically engineered bacteria into a recipient plant tissue to obtain an edited transgenic plant material.
  • the plant is preferably a monocotyledonous plant, more preferably corn or wheat.
  • the recipient plant tissue is preferably an immature immature embryo.
  • the plant genome was edited using the gene editing system of the present disclosure, and a plant having a high proportion of homozygous or biallelic mutants in the T0 generation was realized in maize; for plants with a low mutation ratio obtained by the T0 generation, selfing
  • the progeny T1 generation can also produce new mutant plants; for wheat Agrobacterium-based transformation systems, chimeric plants containing a certain proportion of mutations can be obtained, and plants with homozygous or biallelic mutants are expected to be obtained in the T1 generation.
  • the application of this promoter to the plant genome editing system has not been reported prior to the present disclosure, and the present disclosure is a pioneer.
  • the improved CRISPR/Cas9 system of the present disclosure is expected to improve genome editing efficiency in monocots, promote crop genetic improvement, and basic research.
  • Figure 1 shows the resistant callus of 4 transgenic events obtained in the fourth example of the maize gene 1 (international general number GRMZM2G027059) (3 samples each) using PCR (Polymerase Chain Reaction)-enzyme digestion The method of performing mutation identification results.
  • Figure 2 shows the phenotypic results of the homozygous or biallelic mutants obtained in Example 4.
  • Figure 3 shows the results of PCR-enzyme electrophoresis of the T1 generation plant mutation identification in Example 4.
  • Figure 4 shows the results of deletion mutations in the T0 transgenic wheat plants of Example 4.
  • the inventors of the present disclosure have explored a new promoter for driving the expression of Cas9 gene in maize through a large number of screening experiments and repeated practice verification, and constructed a recombinant vector convenient for its application. Genetically engineered strains. Agrobacterium transformation experiments have shown that the present disclosure can significantly improve the efficiency of gene editing using the CRISPR/Cas9 system in plants, particularly monocot corn, compared to other promoters already reported in the prior art, in transgenic T0 generations. 100% of the resistant callus can be regenerated to obtain homozygous or biallelic mutant plants, and the proportion of homozygous or biallelic mutant plants in all regenerated plants is above 65%. The present disclosure is also expected to obtain homozygous or biallelic mutant plants of multiple gene loci in the wheat T1 generation.
  • Plasmid 35S-Cas9-SK is disclosed in the literature "Feng Z.Y. et al., Efficient genome editing in plants using a CRISPR/Cas system. Cell Res 2013";
  • the plasmid pU3-sgRNA is disclosed in the literature "Feng C. et al., Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System. J Genet. Genomics 2016”;
  • the plasmid pTF101.1 is disclosed in the literature "Paz M. M. et al., Assessment of conditions affecting Agrobacterium mediated Systems transformation using the cotyledonary node explant. Euphytica 2004";
  • Agrobacterium strain EHA105 can be purchased from commercial companies, such as Youbao Bio Company, product number: ST1140;
  • HiII The maize variety HiII is disclosed in the literature "Armstrong CL, Green CE & Phillips RLD and and germplasm germplasm with high type II culture formation response. Maize Genet. Coop. News Lett. 65, 92-93 (1991)"; Access to the Maize Genetics and Genomics Database (MaizeGDB) website;
  • Maize variety B73 is disclosed in the literature "Russell W.A. Registration of B70 and B73parental lines of maize. Crop Sci. 12, 721 (1972)”; publicly available from the Maize Genetics and Genomics Database (MaizeGDB) website;
  • DNA extraction kit (Cat. No. DP305-03), Plasmid Extraction Kit (DP103-03), EsayGeno Rapid Recombination Cloning Kit (Cat. No. VI201-02), etc. were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.;
  • the T vector for PCR product cloning (Cat. No. CT111-01) was purchased from Beijing Quanjin Biotechnology Co., Ltd.;
  • the full length of the maize DMC1 gene (gene ID: GRMZM2G109618) was obtained from the Maize Genetics and Genomics Database (MaizeGDB) website.
  • the primers dmc-F (the sequence is shown in SEQ ID NO. 2) and dmc-R (the sequence is shown in SEQ ID NO. 3) were designed as amplification primers according to the full-length sequence at the 5' non-coding region of the gene.
  • Maize B73 Za mays L.
  • a 3614 bp fragment (sequence shown in SEQ ID NO. 1) was obtained by PCR amplification, which is adjacent to the start codon.
  • the above 3614 bp fragment DNA was used as a template for PCR amplification.
  • the amplified fragment was then integrated into the 35S-Cas9-SK (XhoI digestion) vector to replace the 35S promoter using the EsayGeno Rapid Recombination Cloning Kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) to obtain recombinant pDMC1- Cas9-SK vector.
  • the DMC1p-Cas9 transcription unit was then subcloned into the binary vector pTF101.1 using two cleavage sites of Xma I and EcoR I to obtain the recombinant vector pDMC1-Cas9.
  • the maize genome conforms to 5'-Nx-NGG-3' or 5'-CCN-Nx-3' (N represents any of A, T, C, and G, 14 ⁇ X ⁇ 30, and X is an integer,
  • the site where N X represents X consecutive deoxyribonucleotides) sequence is selected as the target site for editing by the CRISPR/Cas9 system.
  • the DNA sequence of the target site can be ligated into the pU3-sgRNA vector by primer annealing (Bbs I digestion).
  • the transcription unit of the sgRNA was then subcloned into the aforementioned pDMC1-Cas9 vector by a Hind III restriction site.
  • the pDMC1-Cas9 vector was then transferred into Agrobacterium strain EHA105, and the resulting recombinant genetically engineered strain was used for transformation of maize.
  • the main solution is formulated as follows:
  • Enzymatic hydrolysate 1.5% cellulase, 0.4% eductase R10, 0.4 M mannitol, 20 mM potassium chloride, 20 mM fatty acid methyl ester sulfonate, pH 5.7. 10 mM chlorine was added after 10 min in a 55 ° C water bath. Calcium, 0.1% bovine serum albumin and 5 mM ⁇ -mercaptoethanol;
  • Seedlings were germinated under dark conditions of 25 ° C, and the youngest leaves were cut to 0.5 mm wide pieces until the seedlings grew to about 10 cm;
  • the recombinant vector pDMC1-Cas9 obtained in Example 1 was transferred into Escherichia coli strain DH5 ⁇ to prepare a plasmid.
  • the plasmid was extracted using a plasmid extraction kit produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. Take 190 ⁇ l of protoplast solution, add 10 ⁇ l of plasmid (greater than 5 ⁇ g), add 200 ⁇ l of 40% PEG solution, mix gently with a pipette tip, and place at 25 ° C for 18 min;
  • the method of genetic transformation of maize mainly refers to the existing reports (Frame B.R. et al., Agrobacterium-mediated transformation of maize embryos using a standard binary vector system. Plant Physiol. 2002).
  • the transformed acceptor material is HiII.
  • the reagent preparation and main steps are as follows:
  • N6 vitamin storage solution 1000 ⁇ : 2.0 g/L glycine, 1.0 g/L vitamin B1, 0.5 g/L vitamin B6, 0.5 g/L niacin, filter sterilization;
  • MS vitamin storage solution 1000 ⁇ : 2.0 g/L glycine, 0.5 g/L vitamin B1, 0.5 g/L vitamin B6, 0.05 g/L niacin, filter sterilization;
  • Infecting medium 4.0g/L N6 salt, 1ml/L N6 vitamin stock solution, 1.5mg/L 2,4-D, 0.7g/L L-valine, 68.4g/L sucrose, 36.0 g/L glucose, pH 5.2, filter sterilization, adding 100.0 ⁇ M acetosyringone;
  • Co-cultivation medium 4.0 g/L N6 salt, 1.5 mg/L 2,4-D, 0.7 g/L L-valine, 30.0 g/L sucrose, 3.0 g/L plant gel, pH 5 .8; after autoclaving, add 1ml / L N6 vitamin stock solution, 100.0 ⁇ M acetosyringone, 300.0mg / L L-cysteine, 5.0 ⁇ M silver nitrate;
  • Resting medium 4.0 g/L N6 salt, 1.5 mg/L 2,4-D, 0.7 g/L L-valine, 30.0 g/L sucrose, 0.5 g/L fatty acid methyl ester sulfonic acid Salt, 8.0g/L agar, pH 5.8; after autoclaving, add 1ml/L N6 vitamin stock solution, 100.0mg/L cefotaxime, 100.0mg/L vancomycin, 5.0 ⁇ M silver nitrate;
  • Selection medium I 4.0 g/L N6 salt, 1.5 mg/L 2,4-D, 0.7 g/L L-valine, 30.0 g/L sucrose, 0.5 g/L fatty acid methyl sulfonic acid Salt, 8.0g/L agar, pH 5.8; add 1ml/L N6 vitamin stock solution after autoclaving, 100.0mg/L cefotaxime, 100.0mg/L vancomycin, 5.0 ⁇ M silver nitrate, 1.5mg/ L herbicide;
  • Selection medium II 4.0 g/L N6 salt, 1.5 mg/L 2,4-D, 0.7 g/L L-valine, 30.0 g/L sucrose, 0.5 g/L fatty acid methyl ester sulfonic acid Salt, 8.0g/L agar, pH 5.8; add 1ml/L N6 vitamin stock solution after autoclaving, 100.0mg/L cefotaxime, 100.0mg/L vancomycin, 5.0 ⁇ M silver nitrate, 3.0mg/ L herbicide;
  • Regeneration medium I 4.3 g / L MS salt, 1.0 ml / L MS vitamin stock solution, 100.0 mg / L inositol, 60.0 g / L sucrose, 3.0 g / L plant gel, pH 5.8; high temperature After autoclaving, 100.0 mg/L cefotaxime and 3.0 mg/L herbicide were added;
  • Regeneration medium II 4.3 g/L MS salt, 1.0 ml/L MS vitamin stock solution, 100.0 mg/L inositol, 30.0 g/L sucrose, 3.0 g/L plant gel, pH 5.8; high temperature Autoclaved.
  • the recombinant EHA105 genetically engineered strain obtained in Example 1 was inoculated into 10 ml of YEP medium one day before the infection experiment, and cultured in a shaker at 28 ° C, shaking at 200 rpm for 16-20 h.
  • the Agrobacterium was collected by centrifugation and resuspended in the infecting medium (OD 550 value 0.3-0.4). Immature immature embryos (1.5-2.0 mm) were excised from maize ears, and the immature embryos were then placed in 2 ml EP tubes containing Agrobacterium infecting medium, inverted 5-10 times and allowed to stand for 5 min.
  • the infested immature embryos are then placed on sterile filter paper and transferred to the co-culture medium with the scutellum of the young embryos facing up. Then, it was cultured in an incubator at 20 ° C for 3 days in the dark.
  • the selected resistant calli were cultured to a sufficient size, transferred to regeneration medium I, and placed in an incubator for 14 days in the dark at 25 °C.
  • the callus containing the somatic embryos was then transferred to regeneration medium II, and cultured in an incubator at 25 ° C until all the seedlings were regenerated.
  • Example 2 The transformed protoplasts of Example 2 or the transgenic maize callus, the sample of the plant leaves, and the transgenic wheat sample of Example 3 were collected. Genomic DNA was extracted using a DNA extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.);
  • PCR amplification was carried out using the primers of the corresponding target sites, and a part of the obtained PCR products were subjected to restriction enzyme digestion (purchased from NEB) using a corresponding target site, and then subjected to enzymatic digestion, and then subjected to agarose digestion.
  • Gel electrophoresis (Fig. 1); Fig. 1 shows the results of mutation identification of resistant callus (3 samples each) of 4 transgenic events obtained with maize gene 1 (International GQZM2G027059) as target genes; Amplification of 651 bp DNA containing the target site Fragments, if the sequence is not mutated, can be digested into two fragments of 501 bp and 150 bp, and vice versa.
  • the samples corresponding to each lane in Figure 1 are from left to right: 4 transgenic events #1, #3, #4, #6 (3 samples each), control and Marker, as can be seen from Figure 1 Three of the wounded samples contained homozygous or biallelic mutations.
  • the PCR product can be sent directly to the sequencing company for sequencing; for other types of samples containing the mutation, the uncut unbroken mutant sequence band is cloned and cloned. Go to the commercial T-vector and pick the monoclonal delivery company for sequencing;
  • the gene mutation theoretically showed a whitened phenotype.
  • a total of 10 transgenic events were obtained in two batches of transformation experiments, 9 of which were obtained with albino phenotypes and identified as homozygous or biallelic mutants (Fig. 2); Plants with yellowing of the leaves were obtained and identified as biallelic mutants. Therefore, the transgenic event obtained by regenerating a homozygous or biallelic mutant plant was 100%; the proportion of homozygous or biallelic mutant plants in all regenerated plants was greater than 65%.
  • the T1 generation was obtained by selfing or hybridization of the plants with lower mutation ratio in the T0 generation.
  • Figure 3 shows a T0 generation plant and The results of identification of target gene mutations in wild-type hybrid progeny; the left side is the result of T0 generation plant identification, the corresponding samples of lanes are transgenic T0 plants, control and Marker; the right side is the result of T1 generation plant identification, and the corresponding samples of lanes are in turn 8 transgenic plants, controls and Marker for the T1 generation.
  • Table 1 shows the mutant genotype statistics in Example 4.
  • GRMZM2G456570 For maize gene 2 (internationally known as GRMZM2G456570), it is known that this gene mutation will theoretically produce a lethal phenotype, which has been confirmed in other species, transforming a large number of young embryos but only obtaining two In the callus of the resistance event, the transformation efficiency was significantly reduced. This has, to a certain extent, indicated that homozygous or biallelic mutations have occurred in most of the positive transgenic events. In addition, mutation identification of the callus of two positive events revealed that the two samples were chimeric mutants, and the mutation ratio was also 50% or more.
  • the present disclosure provides a novel recombinant vector for plant gene editing.
  • monocots, especially maize have shown that a high proportion of homozygous or biallelic mutant plant material can be obtained in maize in the T0 generation using the recombinant vector based on the Agrobacterium genetic transformation system.
  • the proportion of mutations in some plants in the T1 generation obtained by selfing or cross-breeding was significantly increased.

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Abstract

提供了一种基因编辑系统及应用其对植物基因组进行编辑的方法。该基因编辑系统包括sgRNA转录单元，以及由顺次连接的来源于玉米的DMC1基因启动子和Cas9基因组成的DMC1p-Cas9转录单元，其中，sgRNA识别的靶位点符合5'-Nx-NGG-3'或者5'-CCN-Nx-3'序列规则，N代表A、T、C和G中的任何一种，14≤X≤30，且X为整数，Nx 表示X个连续的脱氧核糖核苷酸。

Description

一种基因编辑系统及应用其对植物基因组进行编辑的方法 技术领域
本公开涉及植物基因工程技术领域，具体涉及一种基因编辑系统及应用其对植物基因组进行编辑的方法。
背景技术
传统的育种技术主要依赖于自然界发生的遗传变异，通过杂交和回交等手段获得拥有优良性状的作物品种。但是自然的变异往往是随机的、有限的。最近几十年发展的转基因技术为作物育种提供了良好的机遇，基因工程育种也取得了巨大的成绩。转基因技术是通过将外源的目的DNA片段用人工的手段将其导入到植物的基因组从而获得某些优良性状如抗虫、抗除草剂等。转基因技术的特点使得获得的转基因植物含有外源的遗传物质，因此转基因食品的安全也为公众所担忧。近些年发展起来的基因组编辑(Genome editing)技术为作物功能基因组研究以及育种提供了新的机遇。
目前主要的基因组编辑技术包括三种，即：锌指核酸酶(Zinc finger nuclease，ZFN)、类转录激活因子效应物核酸酶(Transcription activator like effector nuclease，TALEN)和成簇的规律间隔的短回文重复序列及其相关系统(Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9，CRISPR/Cas9)。这三种技术均能实现在基因组中的特异的靶位点产生DNA双链断裂(Double strand break，DSB)，借助细胞内源的DSB修复机制，获得基因组特异位点的插入或缺失等变异，实现基因组的编辑。由于CRISPR/Cas9系统的利用相对简单和高效，目前已经被广泛用于植物的基础以及应用研究。CRISPR/Cas9系统在主要的农作物如水稻、小麦、玉米、大豆等均已经获得了成功的应用，展现出了强大的育种应用潜力。
CRISPR/Cas9系统的编辑效率对其在作物基础研究以及应用研究中是必须考虑的一个因素。高的编辑效率不但可以节省人力和资源成本，也使得全基因组的大规模基因编辑容易实现，因而促进新的基因或遗传位点的发掘。而CRISPR/Cas9系统的编辑效率主要受到以下一些因素的影响：如 用于驱动Cas9表达的启动子，用于驱动sgRNA表达的启动子，编辑的靶位点等等。对这些因素进行有针对性的优化(如寻找在愈伤组织中更高效表达的启动子)将有提高基因编辑效率的潜力。目前CRISPR/Cas9系统对作物的实践研究中用于驱动Cas9基因表达的主要用到两种组成型表达的启动子：烟草花叶病毒的35S启动子和玉米的ubiquitin基因启动子。在玉米中，基于农杆菌的转化体系，已有的报道中产生纯合或双等位突变体植株的平均频率分别为<1％(35S)、13％(Ubi)和30％(Ubi)。小麦中已报道的编辑效率在10％左右，且基于基因枪转化体系。基于农杆菌转化体系的在小麦中目前还未见有报道。因此，有必要在农作物中发掘和探索新的启动子用于驱动Cas9基因的表达实现更高效的基因组编辑，从而节约时间和人力成本，也使得作物全基因组的大规模编辑更容易实现，促进CRISPR/Cas9系统在作物的基础和应用研究中发挥更强大的作用。
公开内容
(一)要解决的技术问题
本公开的目的是提供一种新的基因编辑系统及应用其对植物基因组进行编辑的方法，以期解决上述现有技术中存在的至少部分技术问题。
(二)技术方案
为实现上述目的，本公开提供一种新的基因编辑系统，其包括sgRNA(Single Guide RNA)转录单元，以及由顺次连接的来源于玉米的DMC1基因启动子和Cas9基因组成的DMC1p-Cas9转录单元，其中，sgRNA识别的靶位点符合5’-Nx-NGG-3’或者5’-CCN-Nx-3’序列规则，N代表A、T、C和G中的任何一种，14≤X≤30，且X为整数，NX表示X个连续的脱氧核糖核苷酸。
其中，所述DMC1基因启动子表示为DMC1p，其序列优选如SEQ ID NO.1所示。
本公开还提供一种包括前述基因编辑系统的重组载体。
本公开还提供前述重组载体的构建方法，其包括如下步骤：
(1)克隆获得DMC1基因启动子并将其与Cas9基因顺次连接，组成DMC1p-Cas9转录单元；
(2)构建识别靶位点的sgRNA转录单元，并将DMC1p-Cas9转录单元和sgRNA转录单元同时导入双元载体，得到重组载体，其中，sgRNA识别的靶位点符合5’-Nx-NGG-3’或者5’-CCN-Nx-3’序列规则，N代表A、T、C和G中的任何一种，14≤X≤30，且X为整数，NX表示X个连续的脱氧核糖核苷酸。
步骤(1)中，所述DMC1基因启动子的序列优选如SEQ ID NO.1所示。
步骤(1)中，克隆获得DMC1基因启动子优选采用如下方法：以玉米B73的基因组DNA为模板，以序列如SEQ ID NO.2和SEQ ID NO.3所示的引物为扩增引物对进行PCR反应，扩增得到序列如SEQ ID NO.1所示的片段，即为DMC1基因启动子。
步骤(1)中，在克隆获得DMC1基因启动子之后，所述将其与Cas9基因顺次连接优选采用如下方法：将得到的DMC1基因启动子替换35S-Cas9-SK载体中的35S启动子，得到pDMC1-Cas9-SK重组载体，再利用Xma I和EcoR I两个酶切位点将DMC1p-Cas9转录单元转移到双元载体pTF101.1，得到重组载体pDMC1-Cas9。
步骤(2)优选采用如下操作：将符合5’-Nx-NGG-3’或者5’-CCN-Nx-3’序列规则的靶位点的DNA序列通过引物退火连入pU3-sgRNA载体中，然后通过Hind III酶切位点将sgRNA的转录单元亚克隆到重组载体pDMC1-Cas9中，得到包括sgRNA转录单元，以及由顺次连接的来源于玉米的DMC1基因启动子和Cas9基因组成的DMC1p-Cas9转录单元的重组载体。
本公开还提供一种对前述重组载体的编辑活性进行鉴定的方法，其包括：将所述重组载体转化植株原生质体，通过鉴定在原生质中发生基因组编辑的效率从而确定所述重组载体的编辑活性。
本公开还提供一种包括前述重组载体的基因工程菌。
本公开还提供一种应用前述基因编辑系统对植物基因组进行编辑的方法，其包括：将前述基因工程菌转化受体植物组织，获得被编辑的转基因植物材料。
其中，所述植物优选单子叶植物，更优选玉米或小麦。
其中，所述受体植物组织优选未成熟的幼胚。
(三)有益效果
应用本公开的基因编辑系统对植物基因组进行编辑，在玉米中实现了在T0代获得高比例的纯合或双等位突变体的植株；对于T0代获得的含突变比例低的植株，自交的后代T1代也能产生新的突变植株；对于小麦基于农杆菌的转化体系可以获得含有一定比例突变的嵌合体植株，预期在T1代可以获得纯合或双等位突变体的植株。对该启动子应用于植物基因组编辑系统，在本公开之前未有报道，本公开属于首创。本公开的改良后CRISPR/Cas9系统将有望提高单子叶植物中的基因组编辑效率，促进作物遗传改良以及基础研究。
附图说明
图1显示实施例4中以玉米基因1(国际通用编号为GRMZM2G027059)为靶基因获得的4个转基因事件的抗性愈伤组织(各取3份样品)利用PCR(Polymerase Chain Reaction)-酶切的方法进行突变鉴定的结果。
图2显示实施例4中获得的纯合或双等位基因的突变体的表型结果。
图3显示实施例4中T1代植株突变鉴定的PCR-酶切后电泳结果。
图4显示实施例4中T0代转基因小麦植株中缺失突变的结果。
具体实施方式
为使本公开的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本公开作进一步的详细说明。
本公开的发明人在研究的过程中，通过大量的筛选实验和反复的实践验证，在玉米中发掘了一个新的启动子用于驱动Cas9基因的表达，同时构建了方便其应用的重组载体和基因工程菌株。农杆菌的转化实验表明，和现有技术中已报道的其他启动子相比，本公开能够显著提高在植物特别是单子叶植物玉米中利用CRISPR/Cas9系统进行基因编辑的效率，在转基因T0代100％的抗性愈伤能再生获得纯合或双等位突变体植株，纯合或双等位突变体植株在所有再生获得的植株中的比例在65％以上。本公开同时有望在小麦T1代获得多基因位点的纯合或双等位突变体植株。
下述各实施例中，所使用的实验材料的来源如下：
质粒35S-Cas9-SK在文献“Feng Z.Y.et al.,Efficient genome editing in plants using a CRISPR/Cas system.Cell Res 2013”中公开过；
质粒pU3-sgRNA在文献“Feng C.et al.,Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9System.J Genet.Genomics 2016”中公开过；
质粒pTF101.1在文献“Paz M.M.et al.,Assessment of conditions affecting Agrobacterium mediated soybean transformation using the cotyledonary node explant.Euphytica 2004”中公开过；
农杆菌菌株EHA105可以从商业公司购买，如优宝生物公司，产品编号：ST1140；
玉米品种HiII在文献“Armstrong C.L.，Green C.E.&Phillips R.L.Development and availability of germplasm with high type II culture formation response.Maize Genet.Coop.News Lett.65，92-93(1991)”中公开过；公众可从玉米遗传学和基因组学数据库(MaizeGDB)网站获取；
玉米品种B73在文献“Russell W.A.Registration of B70and B73parental lines of maize.Crop Sci.12，721(1972)”中公开过；公众可从玉米遗传学和基因组学数据库(MaizeGDB)网站获取；
小麦品种苏麦3号记载于“江苏太湖地区农科所等.小麦赤霉病最优抗源-苏麦3号.江苏农业科学,1988”一文，公众可从中国科学院遗传与发育生物学研究所获得；
DNA提取试剂盒(货号：DP305-03)、质粒提取试剂盒(DP103-03)、EsayGeno快速重组克隆试剂盒(货号：VI201-02)等购自天根生化科技(北京)有限公司；
PCR产物克隆用的T载体(货号：CT111-01)购自北京全式金生物技术有限公司；
限制性内切酶购自纽英伦生物技术(北京)有限公司；
引物序列由赛默飞世尔科技(中国)有限公司合成；
测序由北京睿博兴科生物技术有限公司完成；
实验中用到的其他材料试剂或仪器设备若未作特殊说明，均表示为本领域常规市售可得。
实施例1 玉米中DMC1基因启动子的克隆以及pDMC1-Cas9重组载体的构建
从玉米遗传学和基因组学数据库(MaizeGDB)网站获取玉米DMC1基因的序列全长(基因ID为GRMZM2G109618)。根据全长的序列在该基因的5’端非编码区设计引物dmc-F(序列如SEQ ID NO.2所示)和dmc-R(序列如SEQ ID NO.3所示)作为扩增引物对，以玉米B73(Zea mays L.)基因组DNA为模板，PCR扩增获得一个3614bp长的片段(序列如SEQ ID NO.1所示)，该片段紧邻起始密码子。利用引物dmc-F2(序列如SEQ ID NO.4所示)和dmc-R2(序列如SEQ ID NO.5所示)作为扩增引物对，上述3614bp长的片段DNA为模板进行PCR扩增，然后利用EsayGeno快速重组克隆试剂盒(购自天根生化科技(北京)有限公司)将扩增的片段整合到35S-Cas9-SK(XhoⅠ酶切)载体中替换35S启动子，得到重组的pDMC1-Cas9-SK载体。然后利用Xma Ⅰ和EcoR Ⅰ两个酶切位点将DMC1p-Cas9转录单元亚克隆到双元载体pTF101.1上，得到重组载体pDMC1-Cas9。
玉米基因组中符合5’-Nx-NGG-3’或者5’-CCN-Nx-3’(N代表A、T、C和G中的任何一种，14≤X≤30，且X为整数，NX表示X个连续的脱氧核糖核苷酸)序列规则的位点被选择为CRISPR/Cas9系统编辑的靶位点。靶位点的DNA序列可以通过引物退火连入pU3-sgRNA载体(Bbs Ⅰ酶切)。然后通过Hind Ⅲ酶切位点将sgRNA的转录单元亚克隆到前述的pDMC1-Cas9载体。然后将pDMC1-Cas9载体转入农杆菌菌株EHA105，得到的重组基因工程菌株用于玉米的转化。
实施例2 玉米原生质体转化
玉米原生质体转化的方法参见已有的报道(Feng C.et al.,Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System.J Genet.Genomics 2016)。本实施例中所用玉米材料为B73。试剂配制及主要的步骤如下：
主要溶液配方如下：
(1)酶解液：1.5％纤维素酶，0.4％离析酶R10，0.4M甘露醇，20mM氯化钾，20mM脂肪酸甲酯磺酸盐，pH5.7。55℃水浴10min后加入10mM的氯化钙，0.1％的牛血清蛋白以及5mM的β巯基乙醇；
(2)W5溶液：154mM氯化钠，5mM氯化钾，125mM氯化钙，2mM脂肪酸甲酯磺酸盐，pH5.7；
(3)MMg溶液：4mM脂肪酸甲酯磺酸盐，0.4M甘露醇，15mM氯化镁，pH5.7；
(4)40％PEG溶液：40％PEG4000，100mM氯化钙，0.6M甘露醇。
实验步骤如下：
(1)25℃黑暗条件下萌发幼苗，待幼苗长至10cm左右选取最幼嫩的叶片切成0.5mm宽的碎片；
(2)切好的叶片放入新配制的酶解液中真空抽气0.5h，然后在摇床上40rpm酶解4h，最后在80rpm条件下处理5分钟；
(3)用41μm的尼龙网滤膜过滤酶解后的原生质体到圆底离心管中，100g离心3min，吸取上清，用W5溶液洗涤2次；
(4)洗涤后的原生质体在冰上放置30min，然后离心，吸去上清，加入适量的MMg溶液重悬至细胞数为5×105g/ml；
(5)将实施例1获得的重组载体pDMC1-Cas9转入大肠杆菌菌株DH5α，以备抽提质粒。用天根生化科技(北京)有限公司生产的质粒提取试剂盒抽提质粒。取190μl原生质体溶液，加入10μl质粒(大于5μg)，再加入200μl 40％PEG溶液，用枪头轻轻混匀，25℃放置18min；
(6)加入1.4ml W5溶液，颠倒混匀，100g离心3min，弃掉上清，加入1.5ml W5溶液重悬，然后将原生质体悬液转移到6孔板中，28℃黑暗下培养1.5d。
实施例3 农杆菌介导的玉米遗传转化
玉米遗传转化的方法主要参考已有的报道(Frame B.R.et al.,Agrobacterium-mediated transformation of maize embryos using a standard binary vector system.Plant Physiol.2002)。转化的受体材料为HiII。试剂配制及主要的步骤如下：
主要溶液及培养基配方如下：
(1)N6维生素贮存液(1000×)：2.0g/L甘氨酸，1.0g/L维生素B1，0.5g/L维生素B6，0.5g/L烟酸，过滤除菌；
(2)MS维生素贮存液(1000×)：2.0g/L甘氨酸，0.5g/L维生素B1，0.5g/L维生素B6，0.05g/L烟酸，过滤除菌；
(3)侵染培养基：4.0g/L N6盐，1ml/L N6维生素贮存液，1.5mg/L 2,4-D，0.7g/L L-脯氨酸，68.4g/L蔗糖，36.0g/L葡萄糖，pH5.2，过滤除菌，临用添加100.0μM的乙酰丁香酮；
(4)共培养培养基：4.0g/L N6盐，1.5mg/L 2,4-D，0.7g/L L-脯氨酸，30.0g/L蔗糖，3.0g/L植物凝胶，pH5.8；高温高压灭菌后添加1ml/L N6维生素贮存液，100.0μM乙酰丁香酮，300.0mg/L L-半胱氨酸，5.0μM硝酸银；
(5)静息培养基：4.0g/L N6盐，1.5mg/L 2,4-D，0.7g/L L-脯氨酸，30.0g/L蔗糖，0.5g/L脂肪酸甲酯磺酸盐，8.0g/L琼脂，pH5.8；高温高压灭菌后添加1ml/L N6维生素贮存液，100.0mg/L头孢噻肟，100.0mg/L万古霉素，5.0μM硝酸银；
(6)选择培养基I：4.0g/L N6盐，1.5mg/L 2,4-D，0.7g/L L-脯氨酸，30.0g/L蔗糖，0.5g/L脂肪酸甲酯磺酸盐，8.0g/L琼脂，pH5.8；高温高压灭菌后添加1ml/L N6维生素贮存液，100.0mg/L头孢噻肟，100.0mg/L万古霉素，5.0μM硝酸银，1.5mg/L除草剂；
(7)选择培养基II：4.0g/L N6盐，1.5mg/L 2,4-D，0.7g/L L-脯氨酸，30.0g/L蔗糖，0.5g/L脂肪酸甲酯磺酸盐，8.0g/L琼脂，pH5.8；高温高压灭菌后添加1ml/L N6维生素贮存液，100.0mg/L头孢噻肟，100.0mg/L万古霉素，5.0μM硝酸银，3.0mg/L除草剂；
(8)再生培养基I：4.3g/L MS盐，1.0ml/L MS维生素贮存液，100.0mg/L肌醇，60.0g/L蔗糖，3.0g/L植物凝胶，pH5.8；高温高压灭菌后添加100.0mg/L头孢噻肟，3.0mg/L除草剂；
(9)再生培养基II：4.3g/L MS盐，1.0ml/L MS维生素贮存液，100.0mg/L肌醇，30.0g/L蔗糖，3.0g/L植物凝胶，pH5.8；高温高压灭菌。
主要实验步骤如下：
(1)农杆菌侵染
在侵染实验的前一天接种实施例1得到的重组EHA105基因工程菌株到10ml的YEP培养基中，在摇床中28℃，200rpm振荡培养16-20h。离心收集农杆菌，重悬到侵染培养基中(OD550值为0.3-0.4)。从玉米幼穗中剥出未成熟的幼胚(1.5-2.0mm)，幼胚随后置于含有农杆菌的侵染培养基的2ml EP管中，颠倒5-10次然后静置5min。
(2)共培养
侵染后的幼胚随后置于无菌的滤纸上并紧接着转移到共培养的培养基中，幼胚的盾片朝上。然后放到培养箱中20℃黑暗下培养3天。
(3)静息培养
经过3天的共培养后，转移所有的幼胚到静息培养基，置于培养箱中，28℃黑暗下培养7天。
(4)抗性愈伤的选择培养
经过7天的静息培养，转移所有的幼胚到选择培养基I，置于培养箱中，28℃黑暗下培养14天。然后转移所有的愈伤组织到选择培养基II，置于培养箱中28℃黑暗下培养14天。愈伤组织在选择培养基II中重复继代3-5次，直至陆续选出所有的抗性愈伤组织。
(5)抗性愈伤组织的再生
经过选择培养，筛选到的抗性愈伤组织培养到足够大后转移到再生培养基I，置于培养箱中25℃黑暗下培养14天。然后选取含有体细胞胚的愈伤组织转移到再生培养基II，置于培养箱中25℃光照下培养，直到再生出所有的幼苗。
实施例4 基因编辑效率检测
(1)收集实施例2中的已转化原生质体或实施例3中的转基因玉米愈伤、植株叶片的样品以及转基因小麦样品。用DNA抽提试剂盒(购自天根生化科技(北京)有限公司)提取基因组DNA；
(2)用相应靶位点的引物进行PCR扩增，获得的PCR产物取其中一部分用相应靶位点的限制性内切酶(购自NEB公司)做酶切实验，酶切后进行琼脂糖凝胶电泳(图1)；图1显示以玉米基因1(国际通用编号为GRMZM2G027059)为靶基因获得的4个转基因事件的抗性愈伤组织(各取3份样品)突变鉴定结果；通过PCR扩增得到含靶位点的651bp的DNA 片段，如果序列没有突变则可以被酶切成501bp和150bp的两个片段，反之则不能被切开。图1中各泳道对应的样品从左到右为：4个转基因事件#1、#3、#4、#6(各3份样品)，对照和Marker，从图1中可以看出4个愈伤样品中其中3个都含有纯合或双等位突变。
(3)对于含有纯合或双等位基因的突变的样品，PCR产物可以直接送到测序公司进行测序；对于其他类型的含有突变的样品，回收酶切未切开的突变序列条带，克隆到商业化的T载体，然后挑取单克隆送公司测序；
(4)分析酶切和测序的结果，统计突变类型和比例。
对于玉米基因1(国际通用编号为GRMZM2G027059)作为一个标记基因，该基因突变理论上植株会出现白化的表型。两批次的转化实验共获得10个转基因事件，其中的9个事件中均获得了白化表型的植株，并且经鉴定为纯合或双等位基因突变体(图2)；此外一个事件中获得了叶片黄化的植株，经鉴定也是双等位基因突变体。因此，再生获得含有纯合或双等位基因突变植株的转基因事件为100％；所有的再生植株中纯合或双等位基因突变植株比例大于65％。将T0代中突变比例较低的植株自交或杂交获得T1代，鉴定结果表明在T1代部分植株中突变比例较T0代明显提高(图3)，图3显示的为一棵T0代植株和野生型杂交的后代中靶基因突变的鉴定结果；左侧为T0代植株鉴定的结果，泳道对应样品依次为转基因T0植株、对照和Marker；右侧为T1代植株鉴定的结果，泳道对应样品依次为T1代的8棵转基因植株、对照和Marker。
其中，T0代中突变体基因型统计见表1：
表1实施例4中的突变体基因型统计
Figure PCTCN2017093837-appb-000001
Figure PCTCN2017093837-appb-000002
对于玉米基因2(国际通用编号为GRMZM2G456570)，已知该基因突变理论上会产生致死的表型，在其它物种中已证实这种致死的表型，转化了大量的幼胚但是只获得两个抗性事件的愈伤组织，转化效率显著降低。这从一定程度地表明大部分的阳性转基因事件中产生了纯合或双等位基因的突变。另外，对两个阳性事件的愈伤组织进行突变鉴定发现在这两个样品为嵌合突变体，突变比例分别也在50％以上。
对于小麦，我们选择了苏麦3号材料中的fhb1基因等作为靶基因，到目前已经鉴定的T0代6棵转基因植株中其中4棵可以检测到缺失突变(图4)。
综上所述，本公开提供了一种新的用于植株基因编辑的重组载体。通过在单子叶植物特别是玉米中的实验表明：利用该重组载体基于农杆菌的遗传转化体系，玉米中在T0代可以获得高比例的纯合或双等位突变体植株材料。并且对于T0代含低突变比例的植株，自交或杂交得到的T1代中部分植株中突变比例会显著提高。
以上所述的具体实施例，对本公开的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本公开的具体实施例而已，并不用于限制本公开，凡在本公开的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
Figure PCTCN2017093837-appb-000003
Figure PCTCN2017093837-appb-000004
Figure PCTCN2017093837-appb-000005
Figure PCTCN2017093837-appb-000006
Figure PCTCN2017093837-appb-000007

Claims (12)

  1. 一种基因编辑系统，其包括sgRNA转录单元，以及由顺次连接的来源于玉米的DMC1基因启动子和Cas9基因组成的DMC1p-Cas9转录单元，其中，sgRNA识别的靶位点符合5’-Nx-NGG-3’或者5’-CCN-Nx-3’序列规则，N代表A、T、C和G中的任何一种，14≤X≤30，且X为整数，NX表示X个连续的脱氧核糖核苷酸。
  2. 根据权利要求1所述的基因编辑系统，其中，所述DMC1基因启动子的序列如SEQ ID NO.1所示。
  3. 一种包括如权利要求1或2所述的基因编辑系统的重组载体。
  4. 权利要求3所述重组载体的构建方法，其包括如下步骤：
    (1)克隆获得DMC1基因启动子并将其与Cas9基因顺次连接，组成DMC1p-Cas9转录单元；
    (2)构建识别靶位点的sgRNA转录单元，并将DMC1p-Cas9转录单元和sgRNA转录单元同时导入双元载体，得到重组载体，其中，sgRNA识别的靶位点符合5’-Nx-NGG-3’或者5’-CCN-Nx-3’序列规则，N代表A、T、C和G中的任何一种，14≤X≤30，且X为整数，NX表示X个连续的脱氧核糖核苷酸。
  5. 根据权利要求4所述的构建方法，步骤(1)中，所述DMC1基因启动子的序列如SEQ ID NO.1所示。
  6. 根据权利要求5所述的构建方法，步骤(1)中，克隆获得DMC1基因启动子采用如下方法：以玉米B73的基因组DNA为模板，以序列如SEQ ID NO.2和SEQ ID NO.3所示的引物为扩增引物对模板进行PCR反应，扩增得到序列如SEQ ID NO.1所示的片段，即为DMC1基因启动子。
  7. 根据权利要求4所述的构建方法，步骤(1)中，在克隆获得DMC1基因启动子之后，所述将其与Cas9基因顺次连接采用如下方法：将得到的DMC1基因启动子替换35S-Cas9-SK载体中的35S启动子，得到pDMC1-Cas9-SK重组载体，再利用Xma I和EcoR I两个酶切位点将DMC1p-Cas9转录单元转移到双元载体pTF101.1，得到重组载体pDMC1-Cas9。
  8. 根据权利要求7所述的构建方法，步骤(2)采用如下操作：将符合5’-Nx-NGG-3’或者5’-CCN-Nx-3’序列规则的靶位点的DNA序列通过引物退火连入pU3-sgRNA载体中，然后通过Hind III酶切位点将sgRNA的转录单元亚克隆到重组载体pDMC1-Cas9中，得到包括sgRNA转录单元，以及由顺次连接的来源于玉米的DMC1基因启动子和Cas9基因组成的DMC1p-Cas9转录单元的重组载体。
  9. 一种包括权利要求3所述重组载体的基因工程菌。
  10. 一种应用权利要求1所述的基因编辑系统对植物基因组进行编辑的方法，其包括：将权利要求9所述的基因工程菌转化受体植物组织，获得被编辑的转基因植物材料。
  11. 根据权利要求10所述的方法，其中，所述植物为单子叶植物，更优选玉米或小麦。
  12. 根据权利要求10所述的方法，其中，所述受体植物组织为未成熟的幼胚。
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