Gene editing vector of sinorhizobium meliloti, gene editing method thereof and sinorhizobium meliloti editing strain
By using the CRISPR/spCas9 system and the inducible promoter Plac-driven gene editing vector for Rhizobium sinense, the problems of low efficiency and complex operation in traditional techniques have been solved, and efficient single gene knockout and marker-free mutant strains of Rhizobium sinense have been achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-04
- Publication Date
- 2026-05-15
AI Technical Summary
Traditional gene editing technology for Rhizobium sinense is inefficient, involves cumbersome procedures, and cannot produce mutant strains without selection markers, thus limiting its research and application.
A gene editing vector for Rhizobium sinense using the CRISPR/spCas9 system was developed. The expression of the spCas9 gene was driven by the inducible promoter Plac, and efficient single-gene knockout of Rhizobium sinense was achieved by optimizing the homologous arm length, avoiding the use of IPTG inducer.
This study achieved efficient and convenient single-gene knockout of Rhizobium sinense, simplified the operation process, obtained edited strains without selection markers, and improved gene editing efficiency.
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Figure CN120099053B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to gene editing vectors of Rhizobium sinense, gene editing methods thereof, and edited strains of Rhizobium sinense. Background Technology
[0002] *Sinorhizobium meliloti*, a Gram-negative alpha-proteobacterium, is an intracellular nitrogen-fixing symbiont in legumes, containing a series of genes involved in vitamin B12 synthesis. Genetic manipulation of *Sinorhizobium meliloti* can achieve efficient vitamin B12 production. However, traditional gene-editing techniques for *Sinorhizobium meliloti* suffer from low efficiency, redundant procedures, and the inability to obtain marker-free mutants. These drawbacks pose technical obstacles to the research and application of *Sinorhizobium meliloti*.
[0003] In 2016, Johannes Researchers have used Cre / loxP site-specific recombination to edit the genome of *Rhizobium sinense*. This method can achieve deletion and insertion of large segments of the *Rhizobium sinense* genome. In 2020, Carlos Eduardo Flores-Tinoco et al. used transposon mutation to mutate the *Rhizobium sinense* genome, identifying some nitrogen-fixing-related genes. In 2021, Longxiang Wang et al. used the CRISPR / Cas9 system and various deaminases to achieve single-base editing of the bacterial genome. This system, guided by gRNA, successfully achieved the conversion of adenine to guanine, cytosine to thymine, and cytosine to guanine without forming double-strand breaks. Although Cre / loxP site-specific recombination can achieve deletion and insertion of large segments of the *Rhizobium sinense* genome, this method introduces exogenous DNA fragments into *Rhizobium sinense*, and is relatively time-consuming and labor-intensive. Transposon mutation involves randomly mutating any gene in the genome using the transposon system. The drawback of this technique is that it cannot achieve targeted editing of specific genes, and the screening process is time-consuming and labor-intensive. The CRISPR / Cas9 system and deaminase-mediated single-base editing can only edit a single base in the *Rhizobium sinense* genome, and cannot achieve knockout or knock-in of the entire gene. Summary of the Invention
[0004] Based on this, this application provides a gene editing vector for Rhizobium sinense, which enables single-gene knockout of Rhizobium sinense with high gene editing efficiency.
[0005] A gene editing vector for Rhizobium sinense, comprising the following gene elements: plasmid backbone, inducible promoter Plac, spCas9 gene, gRNA, upstream homologous arm and downstream homologous arm of target gene.
[0006] The aforementioned *Rhizobium sinense* gene-editing vector contains the spCas9 gene, and its expression is driven by the inducible promoter Plac. However, Plac exhibits a certain degree of leakage in *Rhizobium sinense*, therefore, it is unnecessary to add an IPTG inducer to induce spCas9 protein expression. By exploiting the leakage of the Plac promoter, sufficient expression of spCas9 protein is achieved to trigger CRISPR system editing of the *Rhizobium sinense* genome, thus obtaining an edited *Rhizobium sinense* strain. This *Rhizobium sinense* gene-editing vector, based on the CRISPR / spCas9 system, can efficiently and conveniently achieve single-gene knockout in *Rhizobium sinense*.
[0007] In some embodiments, the target gene includes, for example, the tdk gene.
[0008] In some 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 embodiments, repetitive sequences are connected between 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 repeating 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 Rhizobium sinense includes the following steps:
[0015] Construct the gene editing vector of the above-mentioned Rhizobium sinense;
[0016] The gene-editing vector of *Rhizobium sinense* was transformed into competent *E. coli* cells and cultured to obtain *E. coli* containing the gene-editing vector of *Rhizobium sinense*.
[0017] Rhizobium sinense and Escherichia coli containing the Rhizobium sinense gene editing vector were mixed, transferred to antibiotic-free LB agar plates for conjugation, and then screened using LB agar plates containing selection markers to obtain the Rhizobium sinense edited strain.
[0018] In some embodiments, the culture medium containing the screening marker is an antibiotic-resistant culture medium without an inducer.
[0019] In some embodiments, the step of constructing the *Rhizobium sinense* gene editing vector includes: assembling the individual gene elements using Gibson technology to obtain the *Rhizobium sinense* gene editing vector.
[0020] In some embodiments, the Rhizobium sinense is wild-type Rhizobium sinense, and the mixed volume ratio of the Rhizobium sinense and Escherichia coli containing the gene editing vector of the Rhizobium sinense is 0.5:1 to 2:1.
[0021] And / or, the competent Escherichia coli cells are WM6026 Escherichia coli.
[0022] In some embodiments, after the step of screening using LB solid plates containing screening markers, the method further includes the following step: transferring the screened Rhizobium sinense edited strain to the antibiotic-free LB liquid medium for culture, and then performing PCR identification to obtain the Rhizobium sinense edited strain.
[0023] An edited strain of *Rhizobium sinense* was prepared using the gene editing method described above. Attached Figure Description
[0024] Figure 1 Map of pSJ-HRs-Plac-spCas9-P23119-gRNA plasmid;
[0025] Figure 2 The results of PCR detection for tdk gene knockout;
[0026] Figure 3 A bar chart showing the knockout efficiency of three different gRNAs of the tdk gene;
[0027] Figure 4 Phenotypic identification diagram of tdk gene knockout strain. Detailed Implementation
[0028] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples and accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.
[0029] One embodiment of this application provides a gene editing vector for Rhizobium sinense, which enables single-gene knockout of Rhizobium sinense with high gene editing efficiency.
[0030] In some embodiments, the Rhizobium sinense gene editing vector includes the following gene elements: plasmid backbone, inducible promoter Plac, spCas9 gene, gRNA, upstream homologous arm and downstream homologous arm of target gene.
[0031] The CRISPR / spCas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein spCas9) gene editing system is a CRISPR type II system. The principle of the CRISPR / spCas9 system is as follows: ① Recognition component: The CRISPR sequence is transcribed to form sgRNA with target recognition function; ② Cutting component: The spCas9 protein is a nuclease that binds to the sgRNA. As the sgRNA specifically recognizes and binds to the specific DNA sequence, the spCas9 protein cuts the target DNA strand, creating a gap in the gene and achieving gene editing. Subsequently, intracellular repair systems, such as homologous recombination or non-homologous end joining, repair the gene, achieving the purpose of gene editing. However, these design features are not sufficient to make the CRISPR / spCas9 system highly efficient in *Rhizobium sinense*. We further optimized the promoter driving the spCas9 gene, the promoter inducer concentration, and the homologous arm length, as detailed below: ① This study tested two inducible promoters, Plac and Pvan, and found that the Plac promoter induced spCas9 gene expression with relatively high gene editing efficiency; ② Using a lower concentration of inducer to induce spCas9 gene expression is beneficial for gene editing; ③ Due to the limitation of plasmid size, the longest homologous arm should not exceed 2K.
[0032] The system has the following advantages: ① The CRISPR / spCas9 system uses the inducible promoter Plac to drive the expression of the spCas9 gene, which strictly controls the expression level of spCas9 protein and is conducive to maximizing the editing efficiency of the CRISPR / spCas9 system; ② By screening homologous arms of different lengths, the optimal length homologous arm is assembled into the CRISPR / spCas9 system, resulting in relatively high gene editing efficiency.
[0033] The gene-editing vector for *Rhizobium sinense* described in this application contains the spCas9 gene and uses the inducible promoter Plac to drive its expression. However, Plac exhibits a certain degree of leakage in *Rhizobium sinense*. Therefore, it is not necessary to add an IPTG inducer to induce spCas9 protein expression. The leakage of the Plac promoter results in sufficient spCas9 protein expression to trigger CRISPR system editing of the *Rhizobium sinense* genome, thus obtaining an edited *Rhizobium sinense* strain. The aforementioned gene-editing vector for *Rhizobium sinense*, based on the CRISPR / spCas9 system, can efficiently and conveniently achieve single-gene knockout in *Rhizobium sinense*.
[0034] In some embodiments, the target gene includes, for example, the tdk gene. Further, the gRNA includes at least one sequence from 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; other genes in *Rhizobium sinense* can also be edited, and corresponding gRNAs can be designed. It should be noted that the tool used for gRNA design is the CHOPCHOP online tool, the specific URL of which is: http: / / chopchop.cbu.uib.no / .
[0035] In some embodiments, repetitive sequences are connected between 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. Further, the length of the repetitive sequence is 30 bp.
[0036] In some 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] Using the gene editing vector of *Rhizobium sinense* described in this application, single gene knockout can be achieved in *Rhizobium sinense*, and a streamlined gene editing process can be implemented. Gene editing using this gene editing vector can yield *Rhizobium sinense* edited strains without selection markers.
[0039] One embodiment of this application also provides a gene editing method for Rhizobium sinense, comprising the following steps S110-S130:
[0040] S110. Construct the above-mentioned gene editing vector of Rhizobium sinense;
[0041] S120. Transform the gene-editing vector of Rhizobium sinense into competent Escherichia coli cells, culture them, and obtain Escherichia coli containing the gene-editing vector of Rhizobium sinense.
[0042] S130. Mix *Rhizobium sinense* and *Escherichia coli* containing the *Rhizobium sinense* gene-editing vector, transfer to antibiotic-free LB agar plates for conjugation, and then screen using LB agar plates containing selection markers to obtain the *Rhizobium sinense* edited strain (i.e., gene-edited *Rhizobium sinense*).
[0043] The gene editing method described above for Rhizobium sinense enables streamlined gene editing of Rhizobium sinense, and the edited strains can be obtained without the addition of inducing agents, making the operation simpler and more convenient.
[0044] The detailed description of the gene editing vector of Rhizobium sinense is provided above and will not be repeated here.
[0045] In some embodiments, the steps of constructing the *Rhizobium sinense* gene-editing vector include: assembling various gene elements using Gibson technology to obtain the *Rhizobium sinense* gene-editing vector.
[0046] In some embodiments, the competent Escherichia coli cells are WM6026 Escherichia coli. Specifically, S120 includes: taking 5 μL of the assembled Rhizobium sinense gene-editing vector, adding it to 100 μL 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 μL of LB liquid medium, then incubating it at 37°C, 200 rpm for 1 hour, and finally plating it onto LB solid plates containing DAP and gentamicin resistance for culture to obtain Escherichia coli containing the Rhizobium sinense gene-editing vector.
[0047] In some embodiments, *Rhizobium sinense* is wild-type *Rhizobium sinense*, and the volume ratio of *Rhizobium sinense* to *Escherichia coli* containing the *Rhizobium sinense* gene-editing vector is 0.5:1 to 2:1. Further, the volume ratio of *Rhizobium sinense* to *Escherichia coli* containing the *Rhizobium sinense* gene-editing vector is 1:1.
[0048] In some embodiments, the culture medium containing the selection marker is an antibiotic-resistant and inducible LB solid plate.
[0049] Specifically, S130 includes: culturing plasmid-containing *Escherichia coli* WM6026 and wild-type *Rhizobium sinense* separately to the logarithmic development phase, achieving an OD600 of 0.4-0.6 for both strains; mixing them at a 1:1 volume ratio; centrifuging at 5000 rpm for 5 minutes; discarding the supernatant; suspending the bacterial mixture in 100 μL of LB broth; transferring it to an antibiotic-free LB agar plate containing DAP; and incubating at 28°C for 6 hours. Scraping the bacterial growth with an inoculation loop, resuspending it in 1 mL of liquid LB broth, and incubating at 28°C and 220 rpm for 2 hours; then spreading 150 μL of the culture onto a gentamicin-resistant LB agar plate and incubating at 28°C for 3 days.
[0050] In some embodiments, after the screening step using a culture medium containing selection markers, the following steps are also included: transferring the screened *Rhizobium sinense* edited strains to antibiotic-free liquid LB medium for culture, and then performing PCR identification to obtain *Rhizobium sinense* edited strains without selection markers and exogenous plasmids. Specific steps include: picking the *Rhizobium sinense* edited strains into antibiotic-free LB liquid medium, culturing at 28°C and 220 rpm for 3 days, diluting and plating on antibiotic-free LB solid plates on the 4th day, and identifying them using plasmid-specific primers to obtain *Rhizobium sinense* edited strains without selection markers and exogenous plasmids.
[0051] The gene editing method for *Rhizobium sinense* described above enables streamlined gene editing and can produce *Rhizobium sinense* strains without exogenous plasmids.
[0052] Furthermore, in the aforementioned gene editing method, the inducible promoter Plac is used to drive the expression of the spCas9 gene. Plac exhibits a certain degree of leakage in *Rhizobium sinense*, therefore, this method can induce spCas9 protein expression without the addition of IPTG inducer. Through the leakage of the Plac promoter, the expression of spCas9 protein is sufficient to trigger CRISPR system editing of the *Rhizobium sinense* genome.
[0053] One embodiment of this application also provides an edited strain of *Rhizobium sinense*, prepared by the above-described gene editing method.
[0054] The following are specific examples.
[0055] Unless otherwise specified, the reagents and instruments used in the examples are conventionally selected in the art. Experimental methods not specifying particular conditions in the examples are typically performed under standard conditions, such as those described in literature, books, or recommended by the reagent kit manufacturer.
[0056] The sequences of the 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 the role of targeting DNA, and sgRNAscaffold sequence plays the role of binding spCas protein; in Table 1, the uppercase letters are the corresponding designed homologous arms.
[0057] Table 1. Sequences of the genes and primers involved in Example 1.
[0058]
[0059]
[0060]
[0061]
[0062]
[0063]
[0064] Example 1: Knockout of the tdk gene in wild-type Rhizobium sinense
[0065] (1) gRNA design: The gRNA was designed using the online tool CHOPCHOP, the specific URL of which is: http: / / chopchop.cbu.uib.no / .
[0066] Specifically, the target gene in 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] The carrier pattern designed in this embodiment is shown in [reference]. Figure 1The plasmid mainly consists of six parts: the pSJ-backbone plasmid backbone, the IPTG-inducible promoter Plac, the spCas9 gene, gRNA, and homologous arms. The gRNA and repair template (homologous arms) can be substituted depending on the target gene. The sequences of each element are shown in Table 1: gRNA: SEQ ID NO.20, SEQ ID NO.23, and SEQ ID NO.26; repair template: SEQ ID NO.10 and SEQ ID NO.13.
[0069] The main method used to assemble the various components into a plasmid is the Gibson assembly method, and the specific operation steps are as follows:
[0070] (a) Primers were designed to linearize the pSJ-backbone plasmid backbone and Plac. Linearization resulted in a 30 bp repetitive sequence between the two elements (this needs to be considered before primer design; see Table 1 for details of the linearization primers). Assembly was then performed according to the instructions of the Novizan Seamless Cloning Kit. The reaction volume was 20 μL, containing 0.1 pmol of the pSJ-backbone plasmid backbone, 0.2 pmol of the Plac induction system element, 2 μL of Exnase II, 4 μL of 5X CE II Buffer, and water was added to bring the volume to 20 μL. The mixture was then placed in a PCR instrument and incubated at 37°C for 30 min. The assembled product was then transduced into DH5α competent cells, and the plasmid pSJ-Plac was obtained by screening on LB gentamicin-resistant plates.
[0071] (b) Design primers to linearize the plasmid pSJ-Plac and spCas9 gene, and also make a 30bp repetitive sequence between the two fragments (see Table 1 for primers used). Assemble the two elements according to the method in step (a) above, with a molar ratio of 1:1. Other methods are the same as in step (a) above, and the pSJ-Plac-spCas9 plasmid can be obtained.
[0072] (c) Design primers to assemble plasmids pSJ-Plac-spCas9, P23119-sgRNA scaffold, and the upstream and downstream homologous arms of the target gene (see Table 1 for primers used). After linearization of plasmids pSJ-Plac-spCas9, P23119-sgRNA scaffold, and the upstream and downstream homologous arms, there are 30 bp repetitive sequences between each element. Assemble them using the method in step (a) above, with the molar ratio of each element being 1:1:1:1, and the total molar number not exceeding 0.2 pmol. Other methods are the same as in step (a) above, to obtain the pSJ-Plac-spCas9-P23119-sgRNA scaffold-HRs plasmid.
[0073] (d) The gRNA sequence was designed on primers, and loop PCR was performed on the plasmid pSJ-Plac-spCas9-P23119-sgRNAscaffold-HRs to obtain a linear fragment. Subsequently, the linear fragment was transformed into DH5α competent cells, and selection was performed on LB gentamicin-resistant plates to ligate the gRNA between P23119 and sgRNAscaffold, thus obtaining the gene editing vector pSJ-Plac-spCas9-P23119-sgRNA-HRs.
[0074] (3) Genome editing of Rhizobium sinense:
[0075] The gene-editing vector was transformed into competent cells of *E. coli* WM6026 to obtain *E. coli* containing the gene-editing vector. This *E. coli* was then compared with wild-type *Rhizobium sinense* (wild-type *Rhizobium sinense* is derived from the literature: Broad-Host-Range Expression Vectors with Tightly Regulated Promoters and Their Use; To Examine the Influence of TraR and TraM Expression on Ti Plasmid Quorum Sensing, Applied and Environmental Microbiology). SRKhan , JGaines , RMRoop , SKFarrand Both strains (including the edited strain) were cultured separately to the logarithmic growth phase, ensuring their OD600 was between 0.4 and 0.6. They were then mixed at a 1:1 volume ratio, centrifuged at 5000 rpm for 5 minutes, and the supernatant was discarded. The bacterial mixture was resuspended in 100 μL of LB liquid medium and transferred to antibiotic-free LB agar plates containing DAP. The plates were incubated at 28°C for 6 hours. Bacterial growth was scraped off using an inoculation loop and eluted into 1 mL of liquid LB medium. The plates were incubated at 28°C and 220 rpm for 2 hours. 150 μL of the eluted bacterial growth was then spread onto gentamicin-resistant LB agar plates and incubated at 28°C for 3 days until plaques appeared. The plaques on the LB plates were directly identified by PCR to obtain the edited *Rhizobium sinense* strain. The principle of PCR primer design was that both upstream and downstream primers were designed in the genomic region outside the homologous arms. Specific primers are shown in Table 1.
[0076] (4) Discard the gene editing vectors in the edited strains of *Rhizobium sinense*:
[0077] The edited strain of *Rhizobium sinense* was picked and cultured in antibiotic-free LB broth at 28°C and 220 rpm for 3 days. On the fourth day, the bacterial suspension was serially diluted (100-fold, 1000-fold, 10000-fold) and plated onto antibiotic-free LB agar plates, and incubated at 28°C. Amplification was performed using specific primers for the gene-editing vector (see Table 1 for details) to determine if the gene-editing vector was lost. If the gene-editing vector was lost, no band was produced after PCR amplification with the specific primers; if the gene-editing vector was present, a band was produced after PCR amplification with the specific primers. The *Rhizobium sinense* strain without exogenous plasmids was obtained through identification using gene-editing vector-specific primers.
[0078] (5) Result detection
[0079] PCR combined with gel electrophoresis was used to identify tdk gene knockout PCR. The results are as follows: Figure 2 As shown; the knockout efficiency of three different gRNAs of the tdk gene was detected by PCR, and the results are as follows. Figure 3 As shown; the phenotypic identification of the tdk gene knockout mutant of *Rhizobium sinense* using the nutritional metabolic defect method is as follows: Figure 4 As shown. Figure 4 In the first culture dish, the results of phenotypic identification were obtained using PYE solid medium without 5-fluorodeoxyuridine, and the results of phenotypic identification were obtained using PYE solid medium containing 5-fluorodeoxyuridine.
[0080] from Figure 2 It can be seen that after the tdk gene was knocked out, the PCR amplification band in the mutant strain was significantly smaller than that in the wild-type strain. From... Figure 3 It can be seen that the knockout efficiency of the TDK gene is affected by the location of the gRNA; the knockout efficiency varies at different gRNA locations, but the overall knockout efficiency is very high. From... Figure 4 It was found that wild-type *Rhizobium sinense* could not grow on PYE solid medium containing 5-fluorodeoxyuridine (5FudR), while the tdk gene knockout mutant strain could grow on PYE solid medium containing 5-fluorodeoxyuridine. Therefore, this embodiment demonstrated the successful knockout of the tdk gene in *Rhizobium sinense* and identified the mutant phenotype.
[0081] The gene editing vector and editing method of *Rhizobium sinense* disclosed in this application achieve single gene knockout in *Rhizobium sinense*, and construct an efficient and traceless gene editing system for streamlined gene editing, which can obtain mutant strains without selection markers.
[0082] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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 embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.
Claims
1. A gene-editing vector for *Rhizobium sinense*, characterized in that, The *Rhizobium sinense* gene editing vector includes the following gene elements: a plasmid backbone, an inducible promoter Plac, spCas9 Gene, gRNA, upstream homologous arm and downstream homologous arm of the target gene, wherein the target gene is composed of tdk Genome composition; The gRNA consists of at least one of the sequences shown in SEQ ID NO.23 and SEQ ID NO.26; The nucleotide sequence of the inducible promoter Plac is shown in SEQ ID NO.4; 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 spCas9 The nucleotide sequence of the gene fragment is shown in SEQ ID NO.5; The plasmid backbone is based on the pSJ-backbone, and the inducible promoter Plac drives the downstream... spCas9 The gene is transcribed, and the gRNA is transcribed into a downstream sgRNA by the constitutive promoter pJ23119. tdk The gene is located between the upstream and downstream homologous arms, and the inducible promoter Plac expresses the gene upon induction. spCas9 Gene.
2. The gene editing vector of *Rhizobium sinense* according to claim 1, characterized in that, The plasmid backbone, the inducible promoter Plac, and the spCas9 In the gene fragment, the gRNA, the upstream homologous arm of the target gene, and the downstream homologous arm of the target gene, there are repetitive sequences connected between adjacent gene elements; The length of the repeating sequence is 30 bp.
3. A gene editing method for *Rhizobium sinense*, characterized in that, Includes the following steps: Construct the gene editing vector of *Rhizobium sinense* as described in any one of claims 1-2; The gene-editing vector of *Rhizobium sinense* was transformed into competent *E. coli* cells and cultured to obtain *E. coli* containing the gene-editing vector of *Rhizobium sinense*. Rhizobium sinense and Escherichia coli containing the Rhizobium sinense gene editing vector were mixed, transferred to antibiotic-free LB agar plates for conjugation, and then screened using LB agar plates containing selection markers to obtain the Rhizobium sinense edited strain.
4. The gene editing method according to claim 3, characterized in that, The culture medium containing the selection marker is an LB solid plate containing antibiotic resistance and no inducer.
5. The gene editing method according to claim 3, characterized in that, The steps for constructing the *Rhizobium sinense* gene editing vector include: assembling each of the gene elements using Gibson technology to obtain the *Rhizobium sinense* gene editing vector.
6. The gene editing method according to claim 3, characterized in that, The *Rhizobium sinense* is wild-type *Rhizobium sinense*, and the mixed volume ratio of the *Rhizobium sinense* and *Escherichia coli* containing the gene editing vector of the *Rhizobium sinense* is 0.5:1~2:
1. And / or, the competent Escherichia coli cells are WM6026 Escherichia coli.
7. The gene editing method according to any one of claims 3-6, characterized in that, After the step of screening using LB solid plates containing screening markers, the following step is also included: transferring the screened Rhizobium sinense edited strain to the antibiotic-free LB liquid medium for culture, and then performing PCR identification to obtain the Rhizobium sinense edited strain.