Gene editing vector and gene editing method of closterium closterium and closterium closterium editing strain

Abstract

The invention relates to a gene editing vector and a gene editing method of clostridium closterium and a clostridium closterium editing strain. The gene editing vector for the clostridium clostridium comprises the following gene elements: a plasmid skeleton, a Pvan promoter, a spCas9M gene, a green fluorescent protein gene GFP, gRNA, and an upstream homologous arm and a downstream homologous arm of a target gene. A CRISPR / spCas9M-GFP (clustered regularly interspaced short palindromic repeats / spCas9M-Green Fluorescent Protein) gene editing system is adopted by the gene editing vector for the closterium closterium, and an efficient, simple, convenient and traceless genome editing system is constructed in the closterium closterium, so that knockout and insertion of genes can be realized.

Description

Technical Field

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

[0002] Caulobacter crescentus is a non-toxic single-celled organism belonging to the class Alpha-Proteobacteria and is Gram-negative. This bacterium lives in aquatic environments with few nutrients, such as rivers, lakes and oceans. The biggest feature of Caulobacter crescentus is that a single cell can form two different types of cells: when they need to find nutrients, they differentiate into motile cells to explore the environment and find resources; when they need to produce offspring and replicate and divide, they become a non-motile petiole-shaped cell. Caulobacter crescentus can synthesize a super "glue" with a much higher bonding effect than artificial glue, showing great potential in industrial applications, especially in the development and application of biological glue.

[0003] In 1991, Ely bert used the sacB negative screening method based on the two-step homologous recombination method to achieve gene knockout in Caulobacter crescentus. In 2020, Mathilde.Guzzo et al. achieved the expression inhibition of multiple genes in Caulobacter crescentus based on the CRISPR / dCas9 technical solution. The system successfully completed the expression inhibition of ctrA and gcrA under the guidance of gRNA. In 2006, Martin Thanbichler and Lucy Shapiro used single homologous recombination to integrate non-replicating plasmids into specific locations of the Caulobacter crescentus genome, achieving gene knock-in of MipZ (Thanbichler and Shapiro2006). In 2011, Beat Christen et al. used transposon mutagenesis to saturate the genome of Caulobacter crescentus and identified essential genes of Caulobacter crescentus.

[0004] The gene knockout method of Caulobacter crescentus based on sacB negative screening is time-consuming and labor-intensive, and the editing efficiency is very low. CRISPR / dCas9 technology is based on the principle that dCas9 inhibits gene transcription, thereby achieving a reduction in gene expression. However, this technology can only inhibit gene expression and cannot be used for gene knockout and gene knock-in. Single homologous recombination technology is based on the principle of integrase and integration site. All elements on the plasmid (including screening markers, such as antibiotic genes, etc.) are inserted into a specific site of the genome under the action of integrase. The disadvantage of this technology is that it cannot perform gene knockout, nor can it obtain an edited strain without a screening marker. Transposon mutagenesis technology uses 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 can only perform insertion mutation inactivation, and cannot knock out the gene reading frame. Summary of the invention

[0005] Based on this, the present application provides a gene editing vector of Bacillus crescentus, which can achieve efficient editing of Bacillus crescentus genes.

[0006] A gene editing vector for Caulobacter crescentus, comprising the following gene elements: a plasmid backbone, a Pvan promoter, a spCas9M gene, a green fluorescent protein gene GFP, a gRNA, an upstream homology arm and a downstream homology arm of a target gene.

[0007] The above-mentioned Caulobacter crescentus gene editing vector adopts the CRISPR / spCas9M-GFP gene editing system to construct an efficient, simple and scarless genome editing system in Caulobacter crescentus, which can achieve gene knockout and insertion.

[0008] In some embodiments, the target gene includes the spmX gene.

[0009] In some of these embodiments, the sequence of the gRNA includes at least one of the sequences shown in SEQ ID NO.29, SEQ ID NO.32 and SEQ ID NO.35.

[0010] In some of the embodiments, a repetitive sequence is connected between two adjacent gene elements in the plasmid backbone, the Pvan promoter, the spCas9M gene, the green fluorescent protein gene GFP, the gRNA, the upstream homology arm of the target gene, and the downstream homology arm of the target gene;

[0011] Furthermore, the length of the repeated sequence is 30 bp.

[0012] In some embodiments, the plasmid backbone is a pSJ-backbone plasmid backbone.

[0013] A gene editing method for Caulobacter crescentus comprises the following steps:

[0014] Constructing the above-mentioned Caulobacter crescentus gene editing vector;

[0015] The gene editing vector of Caulobacter crescentus is transformed into Caulobacter crescentus competent cells, cultured, and screened to obtain a Caulobacter crescentus edited strain.

[0016] In some of the embodiments, the step of constructing the Caulobacter crescentus gene editing vector includes: assembling each of the gene elements by Gibson technology to obtain the Caulobacter crescentus gene editing vector.

[0017] In some embodiments, the gene editing vector of Caulobacter crescentus is transformed into competent cells of Caulobacter crescentus, and the steps of culturing and screening include:

[0018] Transforming the Caulobacter crescentus gene editing vector into the Caulobacter crescentus competent cells, culturing on a resistant plate, and then observing whether the monoclonal colonies emit green fluorescence under a blue light;

[0019] The monoclonal colony that emits green fluorescence under blue light is verified to obtain the edited strain of Bacillus crescentus.

[0020] In some of the embodiments, after the step of verifying the monoclonal colonies that emit green fluorescence under a blue light, the following step is also included: transferring the verified qualified Bacillus crescentus edited strain to an antibiotic-free PYE liquid culture medium for overnight culture, then diluting it step by step and spreading it on an antibiotic-free PYE solid culture medium for culture, and then performing PCR identification to obtain the Bacillus crescentus edited strain that does not carry a gene editing vector.

[0021] A Caulobacter crescentus edited strain is prepared by the above gene editing method. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the plasmid map of Example 1;

[0023] Figure 2 The figure shows the results of PCR detection and sequencing detection of spmX gene knockout;

[0024] Figure 3 This is a bar graph of the knockout efficiency of three different gRNAs of the spmX gene in Example 1;

[0025] Figure 4 This is a diagram showing the phenotypic identification results of the spmX gene knockout mutant;

[0026] Figure 5This is a statistical analysis of the fluorescence localization of the DivJ-mCherry fusion protein in the wild-type Caulobacter crescentus and the spmX gene knockout mutant. DETAILED DESCRIPTION

[0027] 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.

[0028] One embodiment of the present application provides a gene editing vector of Caulobacter crescentus, which can achieve efficient editing of the Caulobacter crescentus genome.

[0029] Specifically, the Caulobacter crescentus gene editing vector includes the following genetic elements: a plasmid backbone, a Pvan promoter, a spCas9M gene, a green fluorescent protein gene GFP, a gRNA, and upstream homology arms and downstream homology arms of the target gene.

[0030] The CRISPR / spCas9 (Clustered Regularly Interspaced Short PalindromicRepeats-CRISPR associated protein spCas9) gene editing system is a CRISPR type II system. The CRISPR / spCas9M-GFP system, in which the spCas9M gene is named after the spCas9 gene is optimized by C. crescentus-codon. GFP green fluorescent protein is fused to the C-terminus of the spCas9M protein to indicate whether the spCas9M protein is expressed. Principle of the CRISPR / spCas9M-GFP system: ① Recognition component: The CRISPR sequence is transcribed to form sgRNA with targeted recognition function; ② Cutting component: spCas9M protein is a nuclease that forms a complex with sgRNA. As sgRNA specifically recognizes and binds to a specific DNA sequence, the spCas9M protein cuts off the targeted DNA double strand, 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. ③Discrimination component: spCas9M-GFP fusion protein emits green fluorescence under blue light, which can determine whether spCas9M protein is expressed. If the clone emits green fluorescence, it means that spCas9M protein is expressed and gene editing is likely to occur in the cell; if the clone does not emit green fluorescence, it means that spCas9M protein is not expressed, indicating that gene editing has not occurred in the cell. However, these designs are not enough to make the CRISPR / spCas9M-GFP system work efficiently in Caulobacter crescentus. We further optimized the promoter, promoter inducer concentration and homology arm length driving the spCas9M gene, as detailed below: ①This study tested two inducible promoters, Pvan and Pxyl, and found that when the Pvan promoter induced the expression of the spCas9M gene, the gene editing efficiency was relatively high; ②When the expression of the spCas9M gene was induced by a lower concentration of inducer, it was conducive to the occurrence of gene editing; ③Limited by the size of the plasmid, the longest homology arm should not exceed 2K.

[0031] The system has the following advantages: ① In the CRISPR / spCas9M-GFP system, the spCas9 gene was codon-optimized for Caulobacter crescentus, which improved the expression efficiency of the spCas9 gene in Caulobacter crescentus; ② In the CRISPR / spCas9M-GFP system, the inducible promoter Pvan was used to drive the expression of the spCas9M gene, which strictly controlled the expression level of the spCas9M gene, which was beneficial to maximizing the editing efficiency of the CRISPR / spCas9M-GFP system; ③ By screening homologous arms of different lengths, the homologous arms of the optimal length were assembled into the CRISPR / spCas9M-GFP system, and the gene editing efficiency obtained was relatively high; ④ In the CRISPR / spCas9M-GFP system, the spCas9M protein was labeled with GFP green fluorescent protein, which could directly determine whether the spCas9M protein was expressed, and then determine the possibility of gene editing in the colony, which indirectly improved the screening efficiency of gene editing strains.

[0032] The above-mentioned Caulobacter crescentus gene editing vector carries the CRISPR / spCas9M-GFP gene editing system, which constructs an efficient, simple and scarless genome editing tool in Caulobacter crescentus, and can achieve gene knockout and insertion.

[0033] In some embodiments, the target gene includes the spmX gene. It should be noted that the target gene is not limited to the spmX gene, and other genes in Caulobacter crescentus can also be gene edited by designing the corresponding gRNA.

[0034] Furthermore, the sequence of the gRNA includes at least one of the sequences shown in SEQ ID NO.29, SEQ ID NO.32 and SEQ ID NO.35.

[0035] In some of the embodiments, a repetitive sequence is connected between two adjacent gene elements in the plasmid backbone, the Pvan promoter, the spCas9M gene, the green fluorescent protein gene GFP, the gRNA, the upstream homology arm of the target gene, and the downstream homology arm of the target gene;

[0036] Furthermore, the length of the repeated sequence is 30 bp.

[0037] In some embodiments, the plasmid backbone is a pSJ-backbone plasmid backbone.

[0038] In some embodiments, the spCas9M gene is a gene after spCas9 gene Caulobacter crescentus-codon optimization. The specific sequence after codon optimization is shown in Table 1.

[0039] The above-mentioned Caulobacter crescentus gene editing vector is an efficient and scarless gene editing system that can achieve gene knockout and gene knock-in in Caulobacter crescentus.

[0040] One embodiment of the present application also provides a gene editing method for Caulobacter crescentus, characterized in that it includes the following steps S110-S120:

[0041] S110, constructing the above-mentioned Caulobacter crescentus gene editing vector;

[0042] S120, transforming the Caulobacter crescentus gene editing vector into Caulobacter crescentus competent cells, culturing, and screening to obtain a Caulobacter crescentus edited strain (i.e., Caulobacter crescentus after gene editing).

[0043] In some of the embodiments, the step of constructing the Caulobacter crescentus gene editing vector includes: assembling each of the gene elements by Gibson technology to obtain the Caulobacter crescentus gene editing vector.

[0044] In some embodiments, the gene editing vector of Caulobacter crescentus is transformed into competent cells of Caulobacter crescentus, and the steps of culturing and screening include:

[0045] Transforming the Caulobacter crescentus gene editing vector into the Caulobacter crescentus competent cells, culturing them on a resistant plate culture medium, and then observing whether the monoclonal colonies emit green fluorescence under a blue light;

[0046] The monoclonal colony that emits green fluorescence under blue light is verified to obtain the edited strain of Bacillus crescentus.

[0047] Specifically, the steps of electrotransforming Caulobacter crescentus include: taking 500ng of the successfully assembled plasmid and adding it to 100 microliters of Caulobacter crescentus competent cells. After gently mixing, transfer to a 2mm electroporation cup and let it stand on ice for 20 minutes. Select the BIORAD ECO2 program, 2.5kV electrotransformation, quickly add 1ml of PYE liquid culture medium, and rejuvenate at 30℃ for 3h. Centrifuge at 6000rpm for 3min, remove 900 microliters of supernatant, resuspend the bacterial pellet with the remaining 100ul liquid, and apply the resuspended liquid to a PYE resistant plate (Kan).

[0048] The steps of screening the edited strain of Bacillus crescentus include: three days after coating on a kan-resistant plate, observing whether the clone emits green fluorescence under a blue light, and streaking the single clone emitting green fluorescence into small squares on a new PYE-resistant plate (Kan), performing PCR verification the next day, and obtaining the edited strain of Bacillus crescentus.

[0049] In some embodiments, after the step of verifying the monoclonal colonies that emit green fluorescence under a blue light, the following steps are also included: transferring the verified qualified Caulobacter crescentus edited strain to an antibiotic-free liquid PYE medium for overnight culture, then diluting it step by step and spreading it on an antibiotic-free solid medium for culture, and then performing PCR identification to obtain the Caulobacter crescentus edited strain that does not carry a gene editing vector. The purpose of dropping the plasmid of the Caulobacter crescentus edited strain is to obtain a mutant strain with a clean background without any screening markers, which is of great significance for the subsequent research on the Caulobacter crescentus edited strain.

[0050] One embodiment of the present application also provides a Caulobacter crescentus edited strain, which is prepared by the above-mentioned gene editing method.

[0051] The following are specific embodiments.

[0052] Unless otherwise specified, the 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.

[0053] 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; sgRNAscaffold is composed of sgRNA together with gRNA sequence. In gene editing, gRNA plays a role in targeting DNA target, and sgRNAscaffold sequence plays a role in binding spCas9M protein; in Table 1, the capital letter part is the corresponding designed homology arm.

[0054] Table 1 Sequences of genes and primers involved in Example 1

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] Example 1 Gene Editing of Caulobacter crescentus (1) Optimization of spCas9 Gene Codons:

[0064] The tool used for the spCas9 gene codon optimization in this patent is the ExpOptimizer online tool, and its specific website is: https: / / novopro.cn / tools / codon-optimization.html. The specific sequence after codon optimization is shown in Table 1.

[0065] (2) Design of gRNA:

[0066] The design was performed using the CHOPCHOP online tool, the specific URL of which is: http: / / chopchop.cbu.uib.no / .

[0067] Specifically, the targeted gene of this embodiment is the spmX gene, and three gRNAs are designed, whose specific sequences include sequences shown in SEQ ID NO.29, SEQ ID NO.32 and SEQ ID NO.35.

[0068] (3) Plasmid design:

[0069] See the plasmid map designed in this example for Figure 1 , mainly includes 6 parts, namely pSJ-backbone plasmid skeleton, Pvan promoter, spCas9M gene fragment (i.e. after codon optimization), green fluorescent protein gene GFP, gRNA and upstream and downstream homology arms of target gene. Among them, gRNA and repair template (upstream and downstream homology arms of target gene) can be replaced according to different target genes. The sequences of each element are shown in Table 1. 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 Pvan. After linearization, there will be a 30bp repetitive sequence between the two elements (this needs to be considered before designing primers). Then assemble them. The system is 20 μl, of which the pSJ-backbone plasmid skeleton is 0.1 pmol, the Pvan induction system element is 0.2 pmol, 2 μl ExnaseⅡ, 4 μl 5X CEⅡBuffer, and water is added to make up to 10 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 karatomycin resistance plates to obtain the plasmid pSJ-Pvan.

[0071] (b) Design primers to linearize the obtained plasmid pSJ-Pvan and spCas9M gene fragment, and also make a 30bp repetitive sequence between the two fragments. Assemble them according to the method of the above step (a), the molar ratio of the two elements is 1:1, and the other methods are the same as the above step (a), and the pSJ-Pvan-spCas9M plasmid can be obtained.

[0072] (c) After designing primers to linearize the plasmids pSJ-Pvan-spCas9M and GFP, a 30 bp repetitive sequence was also present between the two fragments. The two components were assembled according to the method of step (a) above, with a molar ratio of 1:1. The other methods were the same as step (a) above, and the pSJ-Pvan-spCas9M-GFP plasmid was obtained.

[0073] (d) Design primers to assemble plasmid pSJ-Pvan-spCas9M-GFP, P23119-sgRNAscaffold, upstream homology arms and downstream homology arms of the target gene. After linearization of plasmid pSJ-Pvan-spCas9M-GFP, P23119-sgRNAscaffold 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-P23119-sgRNAscaffold-HRs-Pvan-spCas9M-GFP plasmid can be obtained.

[0074] (e) The gRNA sequence is designed on the primer, and circular PCR is performed on the plasmid pSJ-P23119-sgRNA scaffold-HRs-Pvan-spCas9M-GFP to obtain a linear fragment. After that, the linear fragment is transformed into DH5α competent cells, and the gRNA is connected between P23119 and sgRNA scaffold by screening on LB resistance (Kan) plates to obtain the gene editing vector pSJ-P23119-sgRNA-HRs-Pvan-spCas9M-GFP. The plasmid map is shown in Figure 1 shown.

[0075] (4) Gene editing of Caulobacter crescentus:

[0076] (a) Take 500ng of the successfully assembled plasmid (pSJ-P23119-sgRNA-HRs-Pvan-spCas9M-GFP) and add it to 100μl of Caulobacter crescentus (wild-type Caulobacter crescentus comes from the literature: Phase separation modulates the assembly and dynamics of a polarity-related scaffold-signaling hub, Nature Communications, edited by Wei Tan, Sihua Cheng, Yingying Li, etc.) competent cells. After gently mixing, transfer to a 2mm electroporation cup and let stand on ice for 20min. Select the BIORAD ECO2 program, 2.5kV electroporation, quickly add 1ml of PYE liquid culture medium, and rejuvenate at 30℃ for 3h. Centrifuge at 6000rpm for 3min, remove 900μl of supernatant, resuspend the bacterial pellet in the remaining 100μl of liquid, and spread the resuspended liquid on a PYE resistant plate (Kan).

[0077] (b) After three days, check whether there are monoclonal colonies growing. Under a blue light, streak the monoclonal colonies that emit green fluorescence on a new PYE resistance (kan) plate, and perform PCR verification the next day to obtain the edited strain of Caulobacter crescentus.

[0078] (5) Plasmid loss in editing strains of Caulobacter crescentus:

[0079] The purpose of losing the plasmid of the edited strain of Caulobacter crescentus is to obtain an edited strain with a clean background without any screening markers, which is of great significance for the subsequent research of the edited strain of Caulobacter crescentus. Specifically, the edited strain of Caulobacter crescentus was selected into the PYE liquid culture medium without antibiotics, and cultured overnight at 28°C, 220rpm. The next day, the bacterial solution was diluted 1000 times, and 100 microliters were spread on the PYE solid plate without antibiotics and cultured at 30°C. After 3 days, the clones on the plate were detected using specific primers on the plasmid to determine whether the plasmid was lost. If the plasmid is lost, no bands are produced after PCR amplification with specific primers; if the plasmid exists, bands are produced after PCR amplification with specific primers.

[0080] (6) Result detection

[0081] The spmX gene in Caulobacter crescentus was knocked out by the above steps (1)-(5). PCR detection and sequencing detection were performed on the spmX gene knockout, and the results were as follows Figure 2 As shown, Figure 2 The results of the knockout test of the spmX gene in Agrobacterium using gRNA1 are shown in Figure 2. The knockout efficiency of the spmX gene using three different gRNAs was tested using PCR. Figure 3 As shown; the spmX gene knockout mutant was phenotypically identified, and the results are shown Figure 4-5 Fluorescence microscopy was used to detect the fluorescence localization of DivJ-mCherry fusion protein in wild-type Caulobacter crescentus and spmX gene knockout mutants. The results are shown in Figure 4 shown.

[0082] from Figure 2 It can be seen that the knockout of the scaffolding protein spmX gene was achieved in Caulobacter crescentus. Using the technology of the present invention, the knockout efficiency reached a maximum of 87.5%.

[0083] The gene editing vector and editing method of Caulobacter crescentus of the present application realize single gene and double gene knockout and gene knock-in in Caulobacter crescentus, construct an efficient and traceless gene editing system, realize process-based gene editing, and can obtain mutant strains without screening markers. The system greatly improves the efficiency of gene editing, fills the technical vacuum in this field, and plays an important role.

[0084] 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.

[0085] 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 gene editing vector of Caulobacter crescentus, characterized in that: The gene editing vector of Caulobacter crescentus includes the following gene elements: a plasmid backbone, a Pvan promoter, a spCas9M gene, a green fluorescent protein gene GFP, a gRNA, and an upstream homology arm and a downstream homology arm of the target gene.

2. The gene editing vector of Caulobacter crescentus according to claim 1, characterized in that The target gene includes the spmX gene.

3. The gene editing vector of Caulobacter crescentus according to claim 2, characterized in that The sequence of the gRNA includes at least one of the sequences shown in SEQ ID NO.29, SEQ ID NO.32 and SEQ ID NO.

35.

4. The gene editing vector of Caulobacter crescentus according to any one of claims 1 to 3, characterized in that A repetitive sequence is connected between two adjacent gene elements in the plasmid backbone, the Pvan promoter, the spCas9M gene, the green fluorescent protein gene GFP, 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.

5. The gene editing vector of Caulobacter crescentus according to any one of claims 1 to 3, characterized in that The plasmid backbone is the pSJ-backbone plasmid backbone.

6. A gene editing method for Caulobacter crescentus, characterized in that: The steps include: Constructing the Caulobacter crescentus gene editing vector as described in any one of claims 1 to 5; The gene editing vector of Caulobacter crescentus is transformed into Caulobacter crescentus competent cells, cultured, and screened to obtain a Caulobacter crescentus edited strain.

7. The gene editing method according to claim 6, characterized in that: The step of constructing the Caulobacter crescentus gene editing vector comprises: assembling each of the gene elements by Gibson technology to obtain the Caulobacter crescentus gene editing vector.

8. The gene editing method according to claim 6, characterized in that: The steps of transforming the Caulobacter crescentus gene editing vector into the Caulobacter crescentus competent cells, culturing and screening include: Transforming the Caulobacter crescentus gene editing vector into the Caulobacter crescentus competent cells, culturing them on a resistant plate culture medium, and then observing whether the monoclonal colonies emit green fluorescence under a blue light; The monoclonal colony that emits green fluorescence under blue light is verified to obtain the edited strain of Bacillus crescentus.

9. The gene editing method according to any one of claims 5 to 8, characterized in that: After the step of verifying the monoclonal colonies that emit green fluorescence under a blue light, the following steps are also included: transferring the verified qualified Bacillus crescentus edited strain to an antibiotic-free PYE liquid culture medium for overnight culture, then gradually diluting and spreading it on an antibiotic-free PYE solid culture medium for culture, and then performing PCR identification to obtain the Bacillus crescentus edited strain that does not carry a gene editing vector.

10. An edited strain of Caulobacter crescentus, characterized in that Prepared by the gene editing method according to any one of claims 6 to 9.

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