CN104450774A - Construction of soybean CRISPR/Cas9 system and application of soybean CRISPR/Cas9 system in soybean gene modification - Google Patents

Abstract

The invention relates to construction of a soybean CRISPR/Cas9 system. The construction disclosed by the invention is mainly characterized in that a core sequence is designed according to a target gene, and the core sequence is constructed into an expression carrier with cas9. The soybean CRISPR/Cas9 system utilizes the core sequence to identify the target gene, the cas9 shears the target gene, and various mutations can be generated in the process of repairing a broken chain by an organism. The invention also provides application of the soybean CRISPR/Cas9 system in soybean gene modification. With the adoption of the construction and the application provided by the invention, the constructed soybean CRISPR/Cas9 system is simple, the soybean gene can be rapidly and efficiently modified, and the problems of the traditional ZFNs system is too complex, wastes time and is low in efficiency are overcome.

Description

Construction of a soybean CRISPR/Cas9 system and its application in soybean genetic modification

technical field

The invention belongs to the field of plant molecular biology, and relates to a site-directed mutation system, in particular to the construction of a soybean CRISPR/Cas9 system and its application in soybean gene modification.

Background technique

Site-specific modification of genes is one of the most important methods in the field of biological research. With the development of science, more and more silencing techniques are developing rapidly. From classical MES random mutagenesis, T-DNA or transposon insertional inactivation to zinc finger structures (ZFs) and transcription activator-like effector nucleases (TALENs) site-directed mutagenesis, these techniques have greatly facilitated the study of gene function. process. However, zinc finger ribonuclease (ZFN) and transcription activator-like effector nuclease (TALENs) technologies need to design specific endonucleases for each target gene, and the construction process is cumbersome, which greatly limits their application range. Compared with other silencing systems, CRISPR site-directed mutagenesis technology has its incomparable advantages, and has gradually been widely used in the research of site-directed gene modification.

The CRISPR/Cas system was first discovered in Escherichia coli, and it is the acquired immune system of bacteria against foreign DNA such as phages. This system mainly relies on the ribonucleoprotein complex formed by crRNA and Cas protein to recognize the PAM on the target sequence to specifically cut the invading phage or plasmid. There are three main types of CRISPR systems, among which the type II system only needs a Cas9 protein, crRNA and tracrRNA to perform its function. Studies have shown that integrating crRNA and tracrRNA into sgRNA does not affect the function of the CRISPR/Cas9 system. In August 2013, three studies on the successful application of the CRISPR/Cas9 system to plant genetic modification were simultaneously published in the journal Nature Biotechnology for the first time. Since then, the CRISPR/Cas9 system has been widely used in the research of Arabidopsis thaliana, tobacco, rice, sorghum, corn and wheat, but the CRISPR/Cas9 system is still blank in the research of soybean crops.

Soybean [Glycine max(L.)Merr.] is one of the important crops and the main source of vegetable protein and edible oil, with high nutritional and economic value. In order to better study gene function, it is often necessary to carry out gene mutation at a specific site, but the existing ZFNs system is too complicated, time-consuming and inefficient, which greatly limits the application of this method. Therefore, it is particularly important to develop a soybean-specific CRISPR/Cas system and apply it in the field of soybean genetic modification.

Contents of the invention

The first object of the present invention is to provide a construction of a soybean CRISPR/Cas9 system, which solves the problems that there is no CRISPR/Cas system in soybean at present, and the ZFNs system is too complicated, time-consuming and inefficient.

The second object of the present invention is to provide the application of the above soybean CRISPR/Cas9 system in soybean genetic modification.

The present invention is realized through the following technical solutions:

One, the construction of a soybean CRISPR/Cas9 system, the method comprises the following steps:

(1) Cas9 is constructed on the pCambia3301 vector;

(2) Artificially synthesize sgRNAs with a soybean-specific U6-10 promoter, with two BsaI restriction sites between the soybean-specific U6-10 promoter and sgRNAs;

(3) Designing a core sequence according to the soybean target gene, and synthesizing primers with a cohesive end complementary sequence formed after digestion with BsaI at both ends according to the core sequence;

(4) The fragment synthesized in step (2) is connected into the pUC57-Kan plasmid, and after the plasmid is extracted, it is completely digested with BsaI endonuclease;

(5) linking the core sequence with cohesive ends formed by annealing the primers synthesized in step (3) to the fragment after digestion in step (4);

(6) Digest the plasmid after ligation in step (5) to obtain the target fragment and construct it on the pCambia3301 vector with cas9 to obtain pCas9-GmU6-sgRNA.

2. Application of the above soybean CRISPR/Cas9 system in soybean genetic modification.

The positive effect of adopting the above technical solution: the soybean CRISPR/Cas9 system constructed by the present invention is simple, and the soybean genetic modification can be completed quickly and efficiently, and overcomes the problems that the existing ZFNs system is too complicated, time-consuming and inefficient; and through Experiments have shown that the combination of soybean CRISPR/Cas9 system with soybean cotyledon rooting method or soybean protoplast method has a higher mutation rate, which is more conducive to the study of soybean genes, especially the functions of genes related to soybean roots or root nodules.

Description of drawings

Fig. 1 is a schematic diagram of the CRISPR/Cas9 carrier of the present invention;

Figure 2 is a gel electrophoresis image of the editing of Glyma06g14180 by the CRISPR/Cas9 system detected by the soybean protoplast method;

In the figure, M: marker; 1: 48 hours after the CRISPR/Cas9 vector was transferred into soybean protoplasts, the genomic DNA was extracted, and Pst I was digested completely, then PCR amplified Glyma06g14180 electrophoresis; 2: Genomic DNA of leaves, Pst I was completely digested After enzyme digestion, PCR amplification of Glyma06g14180 and electrophoresis; 3: Leaf genomic DNA, PCR amplification of Glyma06g14180 and electrophoresis;

Figure 3 is a gel electrophoresis image of the editing of Glyma08g02290 by the CRISPR/Cas9 system detected by the soybean protoplast method;

In the figure, M: marker; 1: 48 hours after the CRISPR/Cas9 vector was transferred into soybean protoplasts, the genomic DNA was extracted, and after BamH I complete digestion, PCR amplified Glyma08g02290 electrophoresis; 2: Leaf genomic DNA, BamH I complete After enzyme digestion, PCR amplification of Glyma08g02290 and electrophoresis; 3: Leaf genomic DNA, PCR amplification of Glyma08g02290 and electrophoresis;

Figure 4 is a gel electrophoresis image of the editing of Glyma12g37050 by the CRISPR/Cas9 system detected by the soybean protoplast method;

In the figure, M: marker; 1: 48 hours after the CRISPR/Cas9 vector was transferred into soybean protoplasts, the genomic DNA was extracted, and EcoR I was digested completely, then PCR amplified Glyma12g37050 electrophoresis; 2: Genomic DNA of leaves, EcoR I completely After enzyme digestion, PCR amplification of Glyma12g37050 and electrophoresis; 3: Leaf genomic DNA, PCR amplification of Glyma12g37050 and electrophoresis;

Figure 5 is a gel electrophoresis image of Glyma06g14180 edited by the CRISPR/Cas9 system detected by the soybean cotyledon node-induced hairy root method;

In the figure, M: marker; wt1: leaf genomic DNA, Glyma06g14180 was amplified by PCR, and electrophoresed with PstI; wt2: leaf genomic DNA, Glyma06g14180 fragment was amplified by PCR; 1-21: induced hairy roots, extracted Genomic DNA, after PCR amplification of Glyma06g14180, was digested with Pst I and electrophoresed; 4 and 7 were positive hairy roots.

Figure 6 is a gel electrophoresis image of Glyma08g02290 edited by the CRISPR/Cas9 system detected by the soybean cotyledonary node hairy root method;

In the figure, M: marker; wt1: leaf genomic DNA, Glyma08g02290 fragment amplified by PCR; wt2: leaf genomic DNA, PCR amplified Glyma08g02290, electrophoresed with BamH I; 1-21: induced hairy root, extracted Genomic DNA of Glyma08g02290 was amplified by PCR, and digested with BamHI and electrophoresed; 11, 12, 13, 18 and 19 were positive hairy roots.

Figure 7 is a gel electrophoresis image of Glyma12g37050 edited by the CRISPR/Cas9 system detected by the soybean cotyledonary node hairy root method;

In the figure, M: marker; wt1: leaf genomic DNA, PCR amplified Glyma12g37050 fragment; wt2: leaf genomic DNA, PCR amplified Glyma12g37050, electrophoresed with EcoR I; 1-21: induced hairy roots, extracted Genomic DNA of Glyma12g37050 was amplified by PCR, and digested with EcoRI and electrophoresed; 1, 5, 8, 14, and 19 were positive hairy roots.

Figure 8 is a schematic diagram of the construction of the CRISPR/Cas9 system with the Arabidopsis-specific U6-26 promoter.

Detailed ways

Sources of biological material of the present invention:

1. Carrier pH-Ubi-cas9-7: Targeted mutagenesis in rice using CRISPR-Cas system, JinMiao, Dongshu Guo, Jinzhe Zhang, Qingpei Huang, Genji Qin, Xin Zhang, Jianmin Wan, Hongya Gu, Li-Jia Qu, Cell Research (2013) 23:1233-1236, the carrier was presented to the applicant of this patent by Professor Qu Lijia;

2. Vector pCambia3301: purchased from Beijing Genia Biotechnology Co., Ltd.;

3. All primers were designed by ourselves and synthesized by Sanbo Yuanzhi Biotechnology Co., Ltd.

Below in conjunction with embodiment and comparative example technical scheme of the present invention will be further described, but should not be interpreted as limiting the present invention:

Example 1

This example is used to illustrate the construction process of soybean CRISPR/Cas9 system.

(1) Pick a single colony containing the pCambia3301 plasmid, inoculate it in 50ml LB liquid medium, add kanapenicillin to the medium to a final concentration of 100ng/ml, and culture overnight in a shaker at 37°C and 200 rpm.

(2) Collect colonies, extract pCambia3301 plasmid by alkaline lysis, measure the plasmid concentration by spectrophotometer, and adjust the concentration to 200ng/ul.

(3) Get the 5ug pCambia3301 plasmid in step (2), and use NcoI and PmlI enzymes (Fermentas) to digest overnight.

(4) The digested product was electrophoresed on a 0.8% agarose gel, and the large DNA fragment after double digestion of the pCambia3301 plasmid was recovered with a gel purification recovery kit (Tiangen). The concentration of the recovered pCambia3301 large fragment DNA was measured by a spectrophotometer, and the concentration was adjusted to 50ng/ul.

(5) Using the pH-Ubi-Cas9-7 plasmid provided by Professor Qu Lijia as a template, the Cas9 gene was amplified with high-fidelity DNA phusion enzyme (NEB) and Cas9-specific primers (Cas9-F: CATGccatggCCCCAAAGAAGAAGCGC and Cas9-RTCAATCGCCGCCGAGTTGTGA).

(6) The amplified Cas9 DNA was purified with a PCR product purification kit (Tiagen), and its concentration was adjusted to 100 ng/ul by a spectrophotometer.

(7) Cas9DNA was digested overnight with NcoI enzyme (Fermenata), and the digested product was purified with a PCR product purification kit (Tiagen), and its concentration was adjusted to 100 ng/ul by a spectrophotometer.

(8) After mixing 100ng Cas9DNA derived from (7) with 100ng pCambia3301 large fragment DNA derived from step (4), add 1 unit of T4 DNA ligase for 16°C ligation overnight to form pCambia3301-Cas9 plasmid.

(9) Take 2 ul of the ligation product of step (8), mix it with 50 ul of DH10β competent cells, place it on ice for 30 minutes, add 800 ul of LB liquid medium for 90 seconds at 42°C, and incubate at 37°C and 200 rpm for 50 minutes. Centrifuge at 3000 rpm, remove the supernatant, resuspend the precipitated DH10β cells, spread evenly in the medium containing 100ng/ml kanapenicillin, and culture overnight at 37°C.

(10) Pick a single colony, use pCambia3301 primers (GATCCCTAAGCTTGGCAAGTTCC) and Cas9 gene primers (AGTTGGGCGATCAGATTCTC) to carry out colony PCR to verify whether Cas9 is connected to pCambia3301, pick positive clones in 50ml, kanapenicillin to LB with a final concentration of 100ng/ml In liquid culture medium, cultivate overnight at 37°C in a shaker at 200 rpm.

(11) Collect colonies, extract the pCambia3301-Cas9 plasmid by alkaline lysis, measure the concentration of the plasmid by a spectrophotometer, and adjust the concentration to 200ng/ul for later use.

(12) sgRNAs with soybean-specific U6-10 promoter were artificially synthesized in Genscript Company, and the fragment was located between the EcoRI and HindIII restriction sites of pUC57-Kan plasmid to form pUC57-GmU6-sgRNA plasmid. There are two BsaI enzyme cutting sites between the soybean-specific U6-10 promoter and sgRNAs; the two BsaI enzyme cutting sites work together to remove all the enzyme cutting site sequences after enzyme digestion;

(13) The pUC57-GmU6-sgRNA plasmid was extracted by alkaline lysis, and the plasmid concentration was adjusted to 100 ng/ul with a spectrophotometer for later use.

(14) Take 1 ug of the plasmid prepared in step (13), digest it with BsaI enzyme at 37°C overnight, purify it with a PCR product purification kit (Tiagen), and adjust the plasmid concentration to 100 ng/ul with a spectrophotometer for later use.

(15) Design primers complementary to the core sequence according to the soybean target gene, and the two ends of the core sequence are synthesized with sequences complementary to the cohesive ends formed after digestion with BsaI. Take 1ug of complementary primers in 10ul TE, 90°C for 5 minutes, and then slowly lower the temperature to make the complementary primers pair to form double-stranded DNA with BsaI enzyme sticky ends.

(16) Take 1 ug of the double-stranded DNA refolded in step (15) and 100 ng of the pUC57-GmU6-sgRNA plasmid in step (14), and ligate overnight at 16° C. under the action of T4 DNA ligase.

(17) Take 2 ul of the ligation product of step (16), mix it with 50 ul of DH10β competent cells, place it on ice for 30 minutes, add 800 ul of LB liquid medium for 90 seconds at 42°C, and incubate at 37°C and 200 rpm for 50 minutes. Centrifuge at 3000 rpm, remove the supernatant, resuspend the precipitated DH10β cells, spread evenly in the medium containing 100ng/ml kanapenicillin, and culture overnight at 37°C.

(18) Pick a single colony, use M13F (cgccagggttttcccagtcacgac) and M13R (agcggataacaatttcacacagga) primers to perform colony PCR amplification to verify the insert. Pick positive clones for sequencing (Sanbo Polygala Biotechnology Co., Ltd.) to verify the integrity of the sequence.

(19) Pick the clone with the correct sequence in step (18), inoculate it in 50 ml of LB liquid medium with a final concentration of kanapenicillin of 100 ng/ml, and culture overnight at 37° C. in a shaker at 200 rpm. After the plasmid was extracted by alkaline lysis, the concentration of the plasmid was adjusted to 100 ng/ul with a spectrophotometer.

(20) Take 100 ng of pCambia3301-Cas9 plasmid DNA from step (11) and 100 ng of pUC57-GmU6-sgRNA plasmid DNA from step (18), and double digest with EcoRI and HindIII enzymes (Fermentas) at 37°C overnight. The digested products were purified using a PCR product purification kit (Tiagen).

(21) Take an equal volume of pCambia3301-Cas9 and pUC57-GmU6-sgRNA obtained from the digestion and purification of step (20), add T4 DNA ligase, and ligate overnight at 16°C.

(22) Take 2ul of the ligation product of step (21), mix it with 50ul of DH10β competent cells, place it on ice for 30 minutes, add 800ul LB liquid medium for 90 seconds at 42°C, and incubate at 37°C and 200 rpm for 50 minutes. Centrifuge at 3000 rpm, remove the supernatant, resuspend the precipitated DH10β cells, spread evenly in the medium containing 100ng/ml kanapenicillin, and culture overnight at 37°C.

(23) Pick a single colony, use pCambia3301 multiple cloning site-specific primers Pdmsf: (GGCTCGTATGTTGTGTGGAA) and pDMSR: (GCCTCTTCGCTATTACGCC) to perform colony PCR amplification to verify the insert. The obtained positive clone is the pCas9-GmU6-sgRNA plasmid, which is the soybean CRISPR/Cas9 system. In this system, the core sequence designed according to the target gene is used to identify the target gene, cas9 is used to recognize the NGG sequence in the sequence, and cuts at 3-5 bp upstream of NGG, causing DNA strand breaks, while the organism is repairing DNA. During the process, a variety of mutations will be generated to edit and modify the gene.

(24) Pick the positive clone pCas9-GmU6-sgRNA in step (23), inoculate it in 50 ml of LB liquid medium with a final concentration of kanapenicillin of 100 ng/ml, and culture overnight at 37° C. in a shaker at 200 rpm. After extracting the plasmid by alkaline lysis, use a spectrophotometer to adjust the plasmid concentration to 1 ug/ul for later use.

Example 2

This example is used to illustrate the functional verification of soybean CRISPR/Cas9 system for soybean gene modification.

Three soybean genes Glyma06g14180, Glyma08g02290 and Glyma12g37050 were selected as research objects, and the primer synthesis sequences are shown in Table 1:

Table 1: Design of primers for soybean target genes

Since the CRISPR/Cas9 system is constructed based on the core sequence designed based on the soybean target gene, the core sequence in the CRISPR/Cas9 system can recognize the soybean target gene, thereby providing a location for the modification and editing function of cas9. In Table 1, the italics are the enzyme cleavage site, the bold is the PAM sequence, that is, NGG, cas9 cuts from 3-5bp upstream of NGG, so the cleavage happens exactly at the enzyme cleavage site, which provides convenience for later verification experiments Conditions, that is, by using enzyme digestion test, it can be seen whether a mutation has occurred.

According to the CRISPR/Cas9 system suitable for these three genes constructed by these three genes, the activity of the CRISPR/Cas9 system was detected by soybean protoplast and cotyledonary node hairy root method respectively.

1. Detection of activity of CRISPR/Cas9 system by soybean protoplasts

(1) Take fresh soybean leaves, cut them into pieces and put them into lysate (0.5% cellulose R10, 0.5% macerozyme R10, 0.1% pectolase Y23, 0.6M mannitol, 10mM MES pH5.7, 20mM KCl, 10mM CaCl2 and 0.1% BSA) .

(2) After mixing, vacuum filter for 30 minutes.

(3) Cleavage for about 6 hours under the condition of 30 rpm.

(4) Filter with a 100 μm nylon membrane, centrifuge at 100 g for 3 minutes, and collect the supernatant.

(5) Add an equal volume of W5 solution (2mM MES pH 5.7, 154mM NaCI, 125mM CaCl ₂ , 5mM KCl), mix slowly, and ice-bath for 30 minutes.

(6) Centrifuge at 100 g for 3 minutes, resuspend in MMG solution (4 mM MES pH 5.7, 0.4 mM mannitol, 100 mM CaCl ₂ ), and wait for transformation.

(7) Add 10 μl of the pCas9-GmU6-sgRNA plasmid DNA in step (24) of Case 1 to 100 μl of the protoplast solution resuspended in MMG, mix well, then add 110 μl of 40% PEG, and mix slowly. Let stand at room temperature for 30 minutes.

(8) After pipetting 800 μl of W5 solution for rinsing, add 1 ml of WI solution (4mM MES pH5.7, 0.5mMmannitol, 20mM CaCl ₂ ), and incubate in the dark for 48 hours at room temperature.

(9) Protoplast cells were collected, and the protoplast genomic DNA was extracted by CTAB method.

(10) Complete digestion of the extracted genomic DNA with different endonucleases (Glyma06g14180, Glyma08g02290, and Glyma12g37050 corresponding endonucleases are Pst I, BamHI, and EcoRI, respectively), and the gene is amplified and electrophoresed with gene-specific primers Detection (Figure 2-4). The results show that when the restriction site of the gene is mutated, the restriction site disappears, and when the corresponding endonuclease is used for digestion, the mutated DNA is complete DNA, and gene-specific primers can be used to amplify the gene-specific DNA. However, after the control DNA was digested, the DNA of the gene was broken due to the existence of the restriction site, and the gene-specific band could not be amplified with the gene-specific primers.

(11) Cloning the PCR product of step (10) into the pEasy-T vector (Transgene), picking positive clones for sequencing to verify the mutation site. See SEQ ID No.1-SEQ ID No.16 for the three gene editing situations of the soybean protoplast method.

2. Detecting the activity of CRISPR/Cas9 system by the method of inducing hairy roots from soybean cotyledon nodes

(1) The Williams 82 soybean seeds were treated with chlorine gas disinfection, and the sterilized seeds were sown in a sterilized humid box, and cultured for 5 days at 25°C with 16 hours of light/8 hours of darkness.

(2) Transform the constructed pCas9-GmU6-sgRNA and pCas9-AtU6-sgRNA plasmids into Agrobacterium rhizogenes K599 by liquid nitrogen quick-freezing method, in the presence of 100ng/ml kanapenicillin, 50ng/ml streptomycin and 50ng/ml spectinomycin-resistant solid MS medium cultured at 28°C for 3 days.

(3) The colonies in step (2) were picked up with the needle of a syringe, and Agrobacterium K599 was directly inserted into the hypocotyl of the cotyledonary nodes of the soybean seedlings growing healthily in step (1). Soybean seedlings infected with K599 were placed in a sealed high-humidity culture box at 25°C, 16 hours light/8 hours dark culture for 2-3 weeks.

(4) When the hairy root grows to 5-10 cm from the hypocotyl of the cotyledonary node, a single hairy root is cut, and the hairy root genomic DNA is extracted by CTAB method.

(5) Genomic DNA is amplified with gene-specific primers, and the amplified PCR products are digested with corresponding endonucleases (the endonucleases corresponding to Glyma06g14180, Glyma08g02290, and Glyma12g37050 are PstI, BamHI, and EcoRI, respectively) electrophoresis Detection (Figure 5-7). Using soybean leaf genomic DNA as a control, the results have three situations:

Two bands are displayed: the 3-5 nucleotide sequence upstream of the PAM of the target gene has not changed or other non-predicted sequences have changed;

Only one band is shown: double mutation. Because soybean is a diploid plant, when the sister chromosomes of the genome are mutated, due to the change of the enzyme cleavage site, the endonuclease cannot perform enzymatic cleavage, so a band is displayed;

Three bands are shown: single mutation. Single mutation refers to a mutation on one chromosome, while the other paired chromosome has no mutation. Therefore, during enzyme digestion, one chromosomal DNA can be digested and two bands are displayed, while the other chromosomal DNA cannot be digested and one band is displayed. There are three strips in total.

(6) The mutated DNA band was recovered with a gel recovery kit (Tiagen), cloned into the pEasy-T vector (Transgene), and positive clones were picked for sequencing to verify the mutated site. It can be seen from the figure that each target gene shows multiple mutations, that is, the bands are different. For statistics on the editing of the target gene by the CRISPR/Cas9 system, see the gene fragments in the sequence listing for details. The soybean gene processed by the CRISPR/Cas9 system has indeed undergone various mutations such as deletion, addition, and substitution. See SEQ ID No.17-SEQ ID No.28 for the three gene editing methods of soybean cotyledonary node hairy root method.

It can be shown that the soybean protoplast method, the soybean cotyledon node hair root method, and the CRISPR/Cas9 system can cause multiple mutations to the target gene, and the mutation rate is high.

comparative example

Using the method of Example 1, a pCRISPR/Cas9 system with an Arabidopsis-specific U6-26 promoter was also constructed, as shown in FIG. 8 . The constructed pCas9-AtU6-sgRNA is also mutated to the soybean genome obtained by the two methods of soybean protoplast method and soybean cotyledonary node hairy root method, and the processing method is the same as above. turn out,

The activity of the pCas9-GmU6-sgRNA system is much higher than that of the pCas9-AtU6-sgRNA system, and the target gene mutation rate is as high as 20.2%. See Table 2 for details, indicating that the system constructed in this patent application is suitable for soybean gene editing modification.

Table 2 Comparison of the results of mutation treatment of three target genes with two systems

The soybean CRISPR/Cas9 system constructed by the present invention is simple, and the soybean genetic modification can be completed quickly and efficiently, which overcomes the problems that the existing ZFNs system is too complicated, time-consuming and inefficient.

Claims (2)

1. a structure for soybean CRISPR/Cas9 system, is characterized in that: the method comprises the following steps:

(1) cas9 is building up on pCambia3301 carrier;

(2) synthetic is with the sgRNAs of the special U6-10 promotor of soybean, with two BsaI restriction enzyme sites between the special U6-10 promotor of soybean and sgRNAs;

(3) according to soybean goal gene design core sequence, according to core sequence synthesis two ends with the primer of sticky end complementary sequence cutting rear formation with BsaI enzyme;

(4) fragment that step (2) is synthesized is connected into pUC57-Kan plasmid, after extracting plasmid, with BsaI restriction endonuclease complete degestion;

(5) core sequence with sticky end be combined into after primer annealing step (3) synthesized, the fragment after cutting with step (4) enzyme is connected;

(6) enzyme cut step (5) connect after plasmid, obtain object fragment and be building up on the pCambia3301 carrier with cas9, obtain pCas9-GmU6-sgRNA.

2. the application of soybean CRISPR/Cas9 system according to claim 1 in soybean gene is modified.