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 PDFInfo
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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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Claims (12)
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 一种包括权利要求3所述重组载体的基因工程菌。
- 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。
- 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。
- 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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CN110157715A (zh) * | 2019-05-15 | 2019-08-23 | 陈超 | 一种敲除ZmPAD1基因提升高直链玉米产量的实验方法 |
CN110157715B (zh) * | 2019-05-15 | 2023-03-31 | 陈超 | 一种敲除ZmPAD1基因提升高直链玉米产量的实验方法 |
CN114591882A (zh) * | 2022-03-03 | 2022-06-07 | 东北林业大学 | 一种刺五加细胞瞬时转化的方法 |
CN114703224A (zh) * | 2022-04-09 | 2022-07-05 | 吉林省农业科学院 | 一种利用基因编辑技术创制高类胡萝卜素甜玉米的方法 |
CN114703224B (zh) * | 2022-04-09 | 2023-06-20 | 吉林省农业科学院 | 一种利用基因编辑技术创制高类胡萝卜素甜玉米的方法 |
CN117487847A (zh) * | 2023-10-31 | 2024-02-02 | 中国热带农业科学院橡胶研究所 | 一种获得橡胶树纯合基因编辑植株的方法 |
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