Gene editing vector of sinorhizobium, gene editing method of sinorhizobium and sinorhizobium editing strain

Abstract

The invention relates to a sinorhizobium gene editing vector, a sinorhizobium gene editing method and a sinorhizobium editing strain. The sinorhizobium gene editing vector comprises the following gene elements: a plasmid skeleton, an inducible promoter Plac, an spCas9 gene, gRNA, and an upstream homologous arm and a downstream homologous arm of a target gene. The sinorhizobium gene editing vector contains the spCas9 gene, an inducible promoter Plac is adopted to drive the expression of the spCas9 gene, but the Plac has a certain degree of leakage in sinorhizobium, so that an IPTG inducer does not need to be added to induce the expression of the spCas9 protein, and the expression of the sinorhizobium gene editing vector can be obtained through the leakage of the Plac promoter. The expression of the spCas9 protein is enough to cause the editing of the CRISPR system on the genome of the sinorhizobium, so that the sinorhizobium mutant strain can be obtained.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a gene editing vector of Sinorhizobium and a gene editing method thereof, and an edited strain of Sinorhizobium. Background Art

[0002] Sinorhizobium meliloti belongs to the Gram-negative α-proteobacteria and is an intracellular nitrogen-fixing symbiont of legumes. It contains a series of genes involved in the synthesis of vitamin B12. Efficient production of vitamin B12 can be achieved by genetic manipulation of Sinorhizobium. However, traditional gene editing technology for Sinorhizobium has the disadvantages of low efficiency, redundant operation steps, and inability to obtain mutant strains without screening markers. These disadvantages have created technical barriers to the research and application of Sinorhizobium.

[0003] In 2016, Johannes et al. used Cre / loxP site-specific recombination to edit the Sinorhizobium genome. This method can achieve the deletion and insertion of large fragments of the Sinorhizobium genome. In 2020, Carlos Eduardo Flores-Tinoco et al. mutated the Sinorhizobium genome by transposon mutagenesis, and identified some nitrogen fixation-related genes through this method. In 2021, Longxiang Wang et al. used the CRISPR / Cas9 system and various deaminases to achieve single-base editing of the bacterial genome. Under the guidance of gRNA, the system successfully achieved the conversion of adenine to guanine, cytosine to thymine, and cytosine to guanine without forming double-strand breaks. Although large fragments of the Sinorhizobium genome can be deleted and inserted through Cre / loxP site-specific recombination, this method introduces exogenous DNA fragments into Sinorhizobium, and this method is time-consuming and labor-intensive. Transposon mutagenesis is the use of the transposon system to randomly mutate any gene in the genome. The disadvantage of this technology is that it cannot achieve targeted editing of specific genes, and screening is time-consuming and labor-intensive. The CRISPR / Cas9 system and deaminase-mediated single-base editing can only edit a certain base in the Sinorhizobium genome, and cannot achieve knockout or knockin of the entire gene. Summary of the invention

[0004] Based on this, the present application provides a Sinorhizobium gene editing vector, which is used to achieve single gene knockout of Sinorhizobium with high gene editing efficiency.

[0005] A Sinorhizobium gene editing vector, comprising the following gene elements: a plasmid backbone, an inducible promoter Plac, a spCas9 gene, a gRNA, an upstream homology arm and a downstream homology arm of a target gene.

[0006] The above-mentioned Sinorhizobium gene editing vector contains the spCas9 gene, and the inducible promoter Plac is used to drive the expression of the spCas9 gene. However, Plac has a certain degree of leakage in Sinorhizobium, so there is no need to add IPTG inducer to induce the expression of spCas9 protein. Through the leakage of the Plac promoter, the expression of spCas9 protein is sufficient to cause the CRISPR system to edit the Sinorhizobium genome, and the Sinorhizobium edited strain can be obtained. The above-mentioned Sinorhizobium gene editing vector can efficiently and conveniently achieve single gene knockout in Sinorhizobium based on the CRISPR / spCas9 system.

[0007] In some embodiments, the target gene includes, for example, the tdk gene.

[0008] In some of these embodiments, the gRNA includes at least one of the sequences shown in SEQ ID NO.20, SEQ ID NO.23 and SEQ ID NO.26.

[0009] In some of the embodiments, a repetitive sequence is connected between two adjacent gene elements in the plasmid backbone, the inducible promoter Plac, the spCas9 gene, the gRNA, the upstream homologous arm of the target gene, and the downstream homologous arm of the target gene.

[0010] In some embodiments, the length of the repeated sequence is 30 bp.

[0011] In some embodiments, the plasmid backbone is a pSJ-backbone plasmid backbone, and the nucleotide sequence of the pSJ-backbone plasmid backbone is shown in SEQ ID NO.1;

[0012] The nucleotide sequence of the inducible promoter Plac is shown in SEQ ID NO.4;

[0013] The nucleotide sequence of the spCas9 gene is shown in SEQ ID NO.5.

[0014] A gene editing method for Sinorhizobium rhizobium comprises the following steps:

[0015] constructing the above-mentioned Sinorhizobium gene editing vector;

[0016] Transforming the Sinorhizobium gene editing vector into Escherichia coli competent cells, and culturing to obtain Escherichia coli containing the Sinorhizobium gene editing vector;

[0017] The Sinorhizobium and the Escherichia coli containing the Sinorhizobium gene editing vector are mixed, transferred to an antibiotic-free LB solid plate for conjugation, and then screened using an LB solid plate containing a screening marker to obtain a Sinorhizobium edited strain.

[0018] In some embodiments, the culture medium containing the selection marker is an antibiotic resistance medium without an inducing agent.

[0019] In some of the embodiments, the step of constructing the Sinorhizobium gene editing vector includes: assembling each of the gene elements by Gibson technology to obtain the Sinorhizobium gene editing vector.

[0020] In some embodiments, the Sinorhizobium is a wild-type Sinorhizobium, and the mixed volume ratio of the Sinorhizobium and the Escherichia coli containing the Sinorhizobium gene editing vector is 0.5:1 to 2:1;

[0021] And / or, the Escherichia coli competent cells are WM6026 Escherichia coli.

[0022] In some of the embodiments, after the step of screening using an LB solid plate containing a screening marker, the following step is also included: transferring the screened Sinorhizobium edited strain to the antibiotic-free LB liquid culture medium for culture, and then performing PCR identification to obtain the Sinorhizobium edited strain.

[0023] A Sinorhizobium edited strain prepared by the above gene editing method. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the pSJ-HRs-Plac-spCas9-P23119-gRNA plasmid map;

[0025] Figure 2 This is the result of PCR detection of tdk gene knockout;

[0026] Figure 3 This is a bar graph of the knockout efficiency of three different gRNAs of the tdk gene;

[0027] Figure 4 This is a diagram showing the phenotypic identification of the tdk gene knockout strain. DETAILED DESCRIPTION

[0028] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation of the present invention is described in detail below in conjunction with specific embodiments and drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0029] One embodiment of the present application provides a Sinorhizobium gene editing vector, which can achieve single gene knockout of Sinorhizobium with high gene editing efficiency.

[0030] In some embodiments, the Sinorhizobium gene editing vector comprises the following genetic elements: a plasmid backbone, an inducible promoter Plac, a spCas9 gene, a gRNA, an upstream homology arm and a downstream homology arm of the target gene.

[0031] The CRISPR / spCas9 (Clustered Regularly Interspaced Short PalindromicRepeats-CRISPR associated protein spCas9) gene editing system is a CRISPR type II system. The principle of the CRISPR / spCas9 system: ① Recognition component: The CRISPR sequence is transcribed to form an sgRNA with targeted recognition function; ② Cutting component: The spCas9 protein is a nuclease that forms a complex with the sgRNA. As the sgRNA specifically recognizes and binds to a specific DNA sequence, the spCas9 protein cuts off the targeted DNA chain, causing a gap in the gene and achieving the break of the target gene; then the cell uses a repair system such as homologous recombination or non-homologous end joining to repair the gene and achieve the purpose of gene editing. However, these designs are not enough to make the CRISPR / spCas9 system work efficiently in Sinorhizobium. We further optimized the promoter driving the spCas9 gene, the concentration of promoter inducer, and the length of the homology arm, as detailed below: ① This study tested two inducible promoters, Plac and Pvan. The results showed that when the Plac promoter induced the expression of the spCas9 gene, the gene editing efficiency was relatively high; ② Using a lower concentration of inducer to induce the expression of the spCas9 gene is conducive to the occurrence of gene editing; ③ Due to the limitation of the plasmid size, the longest homology arm should not exceed 2K.

[0032] This system has the following advantages: ① The inducible promoter Plac is used to drive the expression of the spCas9 gene in the CRISPR / spCas9 system, which strictly controls the expression level of the spCas9 protein and is conducive to maximizing the editing efficiency of the CRISPR / spCas9 system; ② By screening homologous arms of different lengths and assembling the homologous arms of the optimal length into the CRISPR / spCas9 system, the gene editing efficiency obtained is relatively high.

[0033] The above-mentioned Sinorhizobium gene editing vector of the present application contains the spCas9 gene, and the inducible promoter Plac is used to drive the expression of the spCas9 gene. However, Plac has a certain degree of leakage in Sinorhizobium, so there is no need to add IPTG inducer to induce the expression of spCas9 protein. Through the leakage of the Plac promoter, the expression of spCas9 protein is sufficient to cause the CRISPR system to edit the Sinorhizobium genome, and the Sinorhizobium edited strain can be obtained. The above-mentioned Sinorhizobium gene editing vector can efficiently and conveniently achieve single gene knockout of Sinorhizobium based on the CRISPR / spCas9 system.

[0034] In some embodiments, the target gene includes a tdk gene. Further, the gRNA includes at least one of the sequences shown in SEQ ID NO.20, SEQ ID NO.23 and SEQ ID NO.26. It should be noted that the target gene is not limited to the tdk gene, and other genes in Sinorhizobium can also be gene edited to design the corresponding gRNA. It should be noted that the tool used for gRNA design is the CHOPCHOP online tool, and its specific website is: http: / / chopchop.cbu.uib.no / .

[0035] In some of the embodiments, a repetitive sequence is connected between two adjacent gene elements in the plasmid backbone, the inducible promoter Plac, the spCas9 gene, the gRNA, the upstream homology arm of the target gene, and the downstream homology arm of the target gene. Further, the length of the repetitive sequence is 30 bp.

[0036] In some of these embodiments, the plasmid backbone is a pSJ-backbone plasmid backbone.

[0037] Specifically, the nucleotide sequence of the pSJ-backbone plasmid backbone is shown in SEQ ID NO. 1. The nucleotide sequence of the inducible promoter Plac is shown in SEQ ID NO. 4. The nucleotide sequence of the spCas9 gene is shown in SEQ ID NO. 5.

[0038] By using the above-mentioned Sinorhizobium gene editing vector of the present application, single gene knockout can be achieved in Sinorhizobium, and streamlined gene editing is facilitated. By using the Sinorhizobium gene editing vector for gene editing, a Sinorhizobium edited strain without a screening marker can be obtained.

[0039] An embodiment of the present application also provides a gene editing method of Sinorhizobium, comprising the following steps S110-S130:

[0040] S110, constructing the above-mentioned Sinorhizobium gene editing vector;

[0041] S120, transforming the Sinorhizobium gene editing vector into Escherichia coli competent cells, and culturing to obtain Escherichia coli containing the Sinorhizobium gene editing vector;

[0042] S130. Mix Sinorhizobium and Escherichia coli containing the Sinorhizobium gene editing vector, transfer to an antibiotic-free LB solid plate for conjugation, and then use an LB solid plate containing a screening marker for screening to obtain the Sinorhizobium edited strain (i.e., Sinorhizobium after gene editing).

[0043] The above-mentioned gene editing method of Sinorhizobium can perform process-based gene editing on Sinorhizobium, and the edited strain of Sinorhizobium can be obtained without adding inducers, which makes the operation simpler and more convenient.

[0044] The specific description of the Sinorhizobium gene editing vector is provided above and will not be repeated here.

[0045] In some of the embodiments, the step of constructing a Sinorhizobium gene editing vector includes: assembling various gene elements by Gibson technology to obtain a Sinorhizobium gene editing vector.

[0046] In some embodiments, the competent E. coli cells are WM6026 E. coli. Specifically, S120 includes: taking 5 microliters of the assembled Sinorhizobium gene editing vector, adding it to 100 microliters of WM6026 competent cells, placing it on ice for 30 minutes, heat shocking it at 42°C for 90 seconds, placing it on ice for 5 minutes, adding 1000 microliters of LB liquid culture medium, then placing it in a 37°C incubator, 200 rpm, for 1 hour, and finally applying it to an LB solid plate containing DAP and gentamicin resistance for culture to obtain E. coli containing the Sinorhizobium gene editing vector.

[0047] In some embodiments, Sinorhizobium is wild-type Sinorhizobium, and the mixed volume ratio of Sinorhizobium and Escherichia coli containing the Sinorhizobium gene editing vector is 0.5: 1 to 2: 1. Further, the mixed volume ratio of Sinorhizobium and Escherichia coli containing the Sinorhizobium gene editing vector is 1:1.

[0048] In some embodiments, the culture medium containing the selection marker is LB solid plates containing antibiotic resistance and no induction agent.

[0049] Specifically, S130 includes: culturing WM6026 Escherichia coli containing plasmid and wild-type Sinorhizobium to the logarithmic phase, respectively, so that the OD600 of the two strains is between 0.4-0.6, mixing them according to a volume ratio of 1:1, centrifuging at 5000rpm for 5 minutes, pouring out the supernatant, using 100 microliters of LB liquid to suspend the bacterial mixed precipitate, transferring it to an antibiotic-free LB solid plate containing DAP, and culturing it at 28°C for 6 hours. Use an inoculation loop to scrape the bacterial lawn, resuspend it in 1 ml of liquid LB culture medium, and culture it at 28°C and 220rpm for 2 hours, and then aspirate 150 microliters to apply it on a gentamicin-resistant LB plate, and culture it at 28°C for 3 days.

[0050] In some embodiments, after the screening step using a culture medium containing a screening marker, the following step is also included: the screened Sinorhizobium edited strain is transferred to a liquid LB culture medium without antibiotics for culture, and then PCR identification is performed to obtain a Sinorhizobium edited strain without a screening marker and an exogenous plasmid. The specific steps include: picking the Sinorhizobium edited strain into an LB liquid culture medium without antibiotics, culturing for 3 days at 28°C and 220rpm, diluting and plating on an LB solid plate without antibiotics on the 4th day, and identifying by plasmid-specific primers to obtain a Sinorhizobium edited strain without a screening marker and an exogenous plasmid.

[0051] The above-mentioned gene editing method of Sinorhizobium can realize process-based gene editing and obtain an edited Sinorhizobium strain without exogenous plasmids.

[0052] In the above gene editing method, the inducible promoter Plac is used to drive the expression of the spCas9 gene. Plac has a certain degree of leakage in Sinorhizobium, so this method can induce the expression of spCas9 protein without adding IPTG inducer. Through the leakage of Plac promoter, the expression of spCas9 protein is sufficient to cause the CRISPR system to edit the Sinorhizobium genome.

[0053] One embodiment of the present application also provides an edited strain of Sinorhizobium meliloti, which is prepared by the above-mentioned gene editing method.

[0054] The following are specific embodiments.

[0055] Unless otherwise specified, the pharmaceutical reagents and instruments used in the examples are all conventionally selected in the art. Experimental methods without specific conditions in the examples are usually carried out under conventional conditions, such as those described in literature or books or methods recommended by kit manufacturers.

[0056] The sequences of genes and primers involved in Example 1 are shown in Table 1. In Table 1, pJ23119 is the promoter of each sgRNA in the example, driving the expression of each sgRNA in the example; sgRNA scaffold is composed of sgRNA together with gRNA sequence. In gene editing, gRNA plays a role in targeting DNA, and sgRNA scaffold sequence plays a role in binding spCas protein; in Table 1, the capital letter part is the corresponding designed homology arm.

[0057] Table 1 Sequences of genes and primers involved in Example 1

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] Example 1 Knockout of wild-type Sinorhizobium tdk gene

[0065] (1) gRNA design: The CHOPCHOP online tool was used for design. The specific website is: http: / / chopchop.cbu.uib.no / .

[0066] Specifically, the targeted gene of this embodiment includes the tdk gene, and the specific sequences of the designed gRNA are shown in SEQ ID NO.20, SEQ ID NO.23 and SEQ ID NO.26 in Table 1.

[0067] (2) Plasmid design:

[0068] See the vector map designed in this example for Figure 1, mainly includes 6 parts, namely pSJ-backbone plasmid skeleton, IPTG inducible promoter Plac, spCas9 gene, gRNA and homology arm. Among them, gRNA and repair template (homology arm) can be replaced according to the target gene. The sequences of each element are shown in Table 1 gRNA: SEQ ID NO.20, SEQ ID NO.23 and SEQID NO.26; repair template: SEQ ID NO.10 and SEQ ID NO.13.

[0069] The main method used to assemble each element into a plasmid is the Gibson assembly method. The specific steps are as follows:

[0070] (a) Design primers to linearize the pSJ-backbone plasmid skeleton and Plac. After linearization, there will be a 30bp repeat sequence between the two elements (this needs to be considered before designing primers. The primers used for linearization are detailed in Table 1). Then assemble according to the operating instructions of the seamless cloning kit of Novozymes. The reaction system is 20 μl, of which the pSJ-backbone plasmid skeleton is 0.1 pmol, the Plac induction system element is 0.2 pmol, 2 μl ExnaseⅡ, 4 μl 5X CEⅡBuffer, and water is added to make it 20 μl. Then place it in a PCR instrument and react at 37℃ for 30 minutes. Then, the assembly product is transformed into DH5α competent cells and screened on LB gentamicin resistance plates to obtain the plasmid pSJ-Plac.

[0071] (b) Design primers to linearize the plasmid pSJ-Plac and spCas9 genes, and also make a 30 bp repeat sequence between the two fragments (the primers used are detailed in Table 1). Assemble according to the method of step (a) above, the molar ratio of the two elements is 1:1, and the other methods are the same as step (a) above to obtain the pSJ-Plac-spCas9 plasmid.

[0072] (c) Design primers to assemble plasmid pSJ-Plac-spCas9, P23119-sgRNA scaffold, upstream homology arms and downstream homology arms of the target gene (the primers used are detailed in Table 1). After linearization of plasmid pSJ-Plac-spCas9, P23119-sgRNA scaffold and upstream and downstream homology arms, there is a 30 bp repeat sequence between each element. The assembly is performed using the method of step (a) above, and the molar ratio of each element is 1:1:1:1, and the total molar number does not exceed 0.2 pmol. The other methods are the same as the method of step (a) above, and the pSJ-Plac-spCas9-P23119-sgRNA scaffold-HRs plasmid can be obtained.

[0073] (d) The gRNA sequence was designed on the primers, and circular PCR was performed on the plasmid pSJ-Plac-spCas9-P23119-sgRNAscaffold-HRs to obtain linear fragments. After that, the linear fragments were transformed into DH5α competent cells, and the gRNA was connected between P23119 and sgRNAscaffold by screening on LB gentamicin resistance plates to obtain the gene editing vector pSJ-Plac-spCas9-P23119-sgRNA-HRs.

[0074] (3) Genome editing of Sinorhizobium rhizobium:

[0075] The gene editing vector was transformed into WM6026 Escherichia coli competent cells to obtain WM6026 Escherichia coli containing the gene editing vector, and then the vector was mixed with wild-type Sinorhizobium (wild-type Sinorhizobium is from the literature: Broad-Host-Range Expression Vectors with Tightly Regulated Promoters and Their UseTo Examine the Influence of TraR and TraM Expression on Ti Plasmid QuorumSensing,Applied and Environmental Microbiology, SR Khan , JGaines , RMRoop , SKFarrand Etc. (editor) were cultured to the logarithmic phase, so that the OD 600 of the two strains was between 0.4-0.6, and mixed according to a volume ratio of 1:1, centrifuged at 5000rpm for 5 minutes, the supernatant was poured off, and 100 microliters of LB liquid medium were used to suspend the bacterial mixed precipitate, which was transferred to an antibiotic-free LB solid plate containing DAP and cultured at 28°C for 6 hours. Use an inoculation loop to scrape the bacterial lawn, elute it in 1 ml of liquid LB liquid medium, culture it at 28°C and 220rpm for 2 hours, and draw 150 microliters to apply it on a gentamicin-resistant LB solid plate, culture it at 28°C for 3 days, and wait for plaques to grow on the plate. The plaques on the LB plate were directly identified by PCR to obtain the edited strain of Sinorhizobium. The principle of PCR primer design is that the upstream and downstream primers are designed in the genomic part outside the homology arm. The specific primers are shown in Table 1.

[0076] (4) Discarding the gene editing vector in the Sinorhizobium editing strain:

[0077] The edited strain of Sinorhizobium was placed in an antibiotic-free LB liquid culture medium, cultured at 28°C, 220rpm for 3 days, and on the 4th day, the bacterial solution was gradiently diluted (100 times, 1000 times, 10000 times) and plated on an antibiotic-free LB solid plate and cultured in a 28°C incubator. Use the specific primers on the gene editing vector (see Table 1 for specific primers) for amplification to determine whether the gene editing vector is lost. If the gene editing vector is lost, no bands are produced after PCR amplification with specific primers; if the gene editing vector exists, a band is produced after PCR amplification with specific primers. By identifying the gene editing vector-specific primers, the edited strain of Sinorhizobium without exogenous plasmids can be obtained.

[0078] (5) Result detection

[0079] PCR combined with gel electrophoresis was used to identify tdk gene knockout PCR. The test results were as follows Figure 2 As shown; the PCR method was used to detect the knockout efficiency of three different gRNAs of the tdk gene, and the results are shown Figure 3 As shown; the phenotypic identification of the tdk gene knockout mutant of Sinorhizobium using the nutritional metabolic deficiency method is shown in Figure 4 shown. Figure 4 In the figure, the first culture dish shows the result of phenotypic identification using PYE solid medium without 5-fluorodeoxyuridine, and the second culture dish shows the result of phenotypic identification using PYE solid medium containing 5-fluorodeoxyuridine.

[0080] from Figure 2 It can be seen that after knocking out the tdk gene, the PCR amplification band in the mutant strain is significantly smaller than the PCR amplification band in the wild-type strain. Figure 3 It can be seen that the knockout efficiency of the tdk gene is affected by the position of the gRNA. The knockout efficiency at different gRNA positions is different, but the overall knockout efficiency is very high. Figure 4 It can be seen that the wild-type Sinorhizobium cannot grow on a PYE solid medium containing 5-fluorodeoxyuridine (5FudR), while the tdk gene knockout mutant strain can grow on a PYE solid medium containing 5-fluorodeoxyuridine. It can be seen that this example achieves the knockout of the tdk gene in Sinorhizobium and identifies the mutant phenotype.

[0081] The Sinorhizobium gene editing vector and editing method of the present application achieve single gene knockout of Sinorhizobium, and construct an efficient and traceless gene editing system for process-based gene editing, which can obtain mutant strains without screening markers.

[0082] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0083] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A Sinorhizobium gene editing vector, characterized in that: The Sinorhizobium gene editing vector includes the following gene elements: a plasmid backbone, an inducible promoter Plac, a spCas9 gene, a gRNA, an upstream homology arm and a downstream homology arm of the target gene.

2. The Sinorhizobium gene editing vector according to claim 1, characterized in that: The target gene includes, for example, the tdk gene; Furthermore, the gRNA includes at least one of the sequences shown as SEQ ID NO.20, SEQ ID NO.23 and SEQ ID NO.

26.

3. The Sinorhizobium gene editing vector according to claim 1, characterized in that: A repetitive sequence is connected between two adjacent gene elements in the plasmid backbone, the inducible promoter Plac, the spCas9 gene fragment, the gRNA, the upstream homology arm of the target gene, and the downstream homology arm of the target gene; Furthermore, the length of the repeated sequence is 30 bp.

4. The Sinorhizobium gene editing vector according to any one of claims 1 to 3, characterized in that The plasmid backbone is a pSJ-backbone plasmid backbone, and the nucleotide sequence of the pSJ-backbone plasmid backbone is shown in SEQ ID NO.1; The nucleotide sequence of the inducible promoter Plac is shown in SEQ ID NO.4; The nucleotide sequence of the spCas9 gene fragment is shown in SEQ ID NO.

5.

5. A gene editing method for Sinorhizobium, characterized in that: The steps include: Constructing the Sinorhizobium gene editing vector according to any one of claims 1 to 4; Transforming the Sinorhizobium gene editing vector into Escherichia coli competent cells, and culturing to obtain Escherichia coli containing the Sinorhizobium gene editing vector; The Sinorhizobium and the Escherichia coli containing the Sinorhizobium gene editing vector are mixed, transferred to an antibiotic-free LB solid plate for conjugation, and then screened using an LB solid plate containing a screening marker to obtain a Sinorhizobium edited strain.

6. The gene editing method according to claim 5, characterized in that The medium containing the selection marker is LB solid plates containing antibiotic resistance and no inducer.

7. The gene editing method according to claim 5, characterized in that The step of constructing the Sinorhizobium gene editing vector includes: assembling each of the gene elements by Gibson technology to obtain the Sinorhizobium gene editing vector.

8. The gene editing method according to claim 5, characterized in that The Sinorhizobium is a wild-type Sinorhizobium, and the mixed volume ratio of the Sinorhizobium and the Escherichia coli containing the Sinorhizobium gene editing vector is 0.5:1 to 2:1; And / or, the Escherichia coli competent cells are WM6026 Escherichia coli.

9. The gene editing method according to any one of claims 5 to 8, characterized in that: After the step of screening using an LB solid plate containing a screening marker, the method further includes the following steps: transferring the screened Sinorhizobium edited strain to the antibiotic-free LB liquid culture medium for culturing, and then performing PCR identification to obtain the Sinorhizobium edited strain.

10. An edited strain of Sinorhizobium rhizobium, characterized in that: Prepared by the gene editing method according to any one of claims 4 to 9.

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