Gene editing vector of caulobacter crescentus and gene editing method therefor, and caulobacter crescentus editing strain

Abstract

The present invention relates to a gene editing vector of Caulobacter crescentus and a gene editing method therefor, and a Caulobacter crescentus editing strain. The gene editing vector of Caulobacter crescentus comprises the following gene elements: a plasmid backbone, a Pvan promoter, an spCas9M gene, a green fluorescent protein (GFP) gene, a gRNA, and an upstream homologous arm and downstream homologous arm of a target gene. The gene editing vector of Caulobacter crescentus uses a CRISPR / spCas9M-GFP gene editing system to construct an efficient, simple and scarless genome editing system in Caulobacter crescentus, which can realize gene knockout and insertion.

Description

Gene editing vector of caulobacter crescentus, gene editing method thereof and caulobacter crescentus edited strain TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a gene editing vector of caulobacter crescentus, a gene editing method thereof and a caulobacter crescentus edited strain. BACKGROUND

[0002] Caulobacter crescentus is a non-toxic single-cell organism, belonging to the class of alpha-proteobacteria and gram-negative. This bacterium lives in water environments with little nutrients, such as rivers, lakes and oceans. The most notable feature of caulobacter crescentus is that a single cell can form two different types of cells: when they need to find nutrients, they will differentiate into motile cells to explore the environment and find resources; when they need to produce offspring and undergo binary fission, they will become a kind of non-motile leaf stalk cell. Caulobacter crescentus can synthesize a super "glue" with a much higher adhesion effect than artificial glue, which has great potential in industrial applications, especially in the development and application of biological glue.

[0003] In 1991, Elybert used the method of two-step homologous recombination to realize the gene knockout of caulobacter crescentus based on the negative screening of sacB. In 2020, Mathilde Guzzo et al. realized the expression inhibition of multiple genes of caulobacter crescentus based on the technical scheme of CRISPR / dCas9. This 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 a non-replicative plasmid into a specific position of the genome of caulobacter crescentus, realizing the gene knock-in of MipZ (Thanbichler and Shapiro 2006). In 2011, Beat Christen et al. used transposon mutation to realize the saturation mutation of the genome of caulobacter crescentus, realizing the identification of essential genes of caulobacter crescentus.

[0004] The operation process of the sacB negative screening-based C. crescentus gene knockout method is time-consuming and laborious, and the editing efficiency is very low. The CRISPR / dCas9 technology is based on the principle of dCas9 inhibiting gene transcription, thereby realizing the reduction of gene expression. However, this technology can only realize the inhibition of gene expression and cannot be used for gene knockout and gene knock-in. The single homologous recombination technology is based on the principle of integrase and integration site, and all elements (including screening markers such as antibiotic genes) on the plasmid are inserted into the specific site of the genome under the action of integrase. The disadvantage of this technology is that it cannot perform gene knockout and cannot obtain an editing strain without screening markers. The transposon mutation technology randomly mutates any gene in the genome by using a transposon system. The disadvantage of this technology is that it cannot realize directional editing of specific genes, and can only make insertion mutation inactivation and cannot perform gene reading frame knockout.

[0005] SUMMARY

[0006] Therefore, the present application provides a C. crescentus gene editing vector, which can realize efficient editing of C. crescentus genes.

[0007] A C. crescentus gene editing vector, the C. crescentus gene editing vector comprising the following gene elements: a plasmid backbone, a Pvan promoter, a spCas9M gene, a green fluorescent protein gene GFP, a gRNA, an upstream homologous arm of a target gene, and a downstream homologous arm of the target gene.

[0008] The above-mentioned C. crescentus gene editing vector adopts a CRISPR / spCas9M-GFP gene editing system, constructs an efficient, simple and traceless genome editing system in C. crescentus, and can realize gene knockout and insertion.

[0009] In some embodiments, the target gene comprises a spmX gene.

[0010] In some embodiments, the sequence of the gRNA comprises at least one of the sequences shown in SEQ ID NO. 29, SEQ ID NO. 32 and SEQ ID NO. 35.

[0011] In some embodiments, among the plasmid backbone, the Pvan promoter, the spCas9M gene, the green fluorescent protein gene GFP, the gRNA, the upstream homologous arm of the target gene and the downstream homologous arm of the target gene, a repeat sequence is connected between two adjacent gene elements.

[0012] Further, the length of the repeat sequence is 30 bp.

[0013] In some embodiments, the plasmid backbone is a pSJ-backbone plasmid backbone.

[0014] A gene editing method of Caulobacter crescentus, comprising the following steps:

[0015] constructing the above-mentioned gene editing vector of Caulobacter crescentus;

[0016] transforming the gene editing vector of Caulobacter crescentus into a competent cell of Caulobacter crescentus, culturing, screening, and obtaining an edited strain of Caulobacter crescentus.

[0017] In some embodiments, the step of constructing the gene editing vector of Caulobacter crescentus comprises: assembling each of the gene elements by Gibson technology to obtain the gene editing vector of Caulobacter crescentus.

[0018] In some embodiments, the step of transforming the gene editing vector of Caulobacter crescentus into the competent cell of Caulobacter crescentus, culturing, and screening comprises:

[0019] transforming the gene editing vector of Caulobacter crescentus into the competent cell of Caulobacter crescentus, culturing on a resistant plate, and then observing whether a single colony emits green fluorescence under a blue light lamp;

[0020] verifying the single colony emitting green fluorescence under the blue light lamp to obtain the edited strain of Caulobacter crescentus.

[0021] In some embodiments, after the step of verifying the single colony emitting green fluorescence under the blue light lamp, the method further comprises the following steps: transferring the verified edited strain of Caulobacter crescentus to an antibiotic-free PYE liquid medium for overnight culture, then gradient dilution and coating on an antibiotic-free PYE solid medium for culture, and then performing PCR identification to obtain the edited strain of Caulobacter crescentus without carrying the gene editing vector.

[0022] An edited strain of Caulobacter crescentus prepared by the above-mentioned gene editing method. BRIEF DESCRIPTION OF DRAWINGS

[0023] FIG. 1 is a plasmid map of Example 1;

[0024] FIG. 2 is a result map of PCR detection and sequencing detection of spmX gene knockout;

[0025] FIG. 3 is a column chart of knockout efficiency of three different gRNAs of spmX gene in Example 1;

[0026] FIG. 4 is a result map of phenotype identification of spmX gene knockout mutant;

[0027] FIG. 5 is a result map of statistical analysis of fluorescence localization of DivJ-mCherry fusion protein in wild-type Caulobacter crescentus and spmX gene knockout mutant. 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 Bacillus crescentis, which enables efficient editing of the Bacillus crescentis genome.

[0030] Specifically, the Crested Bacillus gene editing vector includes the following gene elements: plasmid backbone, Pvan promoter, spCas9M gene, green fluorescent protein gene GFP, gRNA, upstream homologous arm and downstream homologous arm of the target gene.

[0031] CRISPR / spCas9(Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein spCas9) gene editing system is a CRISPR type II system. CRISPR / spCas9M-GFP system, in which spCas9M gene is a crescent-shaped rod bacterium-codon optimized spCas9 gene. The GFP green fluorescent protein is fused to the C-terminal of the spCas9M protein, which is used to indicate whether the spCas9M protein is expressed. The principle of CRISPR / spCas9M-GFP system: ① recognition component: CRISPR sequence is transcribed to form sgRNA with targeted recognition function; ② cutting component: spCas9M protein is a endonuclease, which forms a complex with sgRNA. With the specific recognition and binding of sgRNA and specific DNA sequence, spCas9M protein cuts the targeted DNA double strand, making the gene gapped, and achieving the target gene disruption; then the cell repairs the gene through repair system such as homologous recombination or non-homologous end joining, etc., to achieve the purpose of gene editing. ③ discrimination 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, which indicates that gene editing does not occur in the cell. However, these designs are not enough to make CRISPR / spCas9M-GFP system work efficiently in crescent-shaped rod bacterium. We further optimized the promoter driving spCas9M gene, the concentration of promoter inducer and the length of homologous arm, which are described as follows: ① In this study, two inducible promoters, Pvan and Pxyl, were tested, and the results showed that the Pvan promoter induced spCas9M gene expression with relatively high gene editing efficiency; ② Using a lower concentration of inducer to induce spCas9M gene expression is conducive to the occurrence of gene editing; ③ Limited by the size of the plasmid, the length of the homologous arm should not exceed 2K.

[0032] The system has the following advantages: ① In the CRISPR / spCas9M-GFP system, the spCas9 gene is codon-optimized for C. lunata, thereby improving the expression efficiency of the spCas9 gene in C. lunata; ② In the CRISPR / spCas9M-GFP system, the expression of the spCas9M gene is driven by the inducible promoter Pvan, thereby strictly controlling the expression amount of the spCas9M gene and facilitating the maximization of the editing efficiency of the CRISPR / spCas9M-GFP system; ③ By screening homologous arms of different lengths, the homologous arm of the optimal length is assembled into the CRISPR / spCas9M-GFP system, and the obtained gene editing efficiency is relatively high; ④ In the CRISPR / spCas9M-GFP system, the spCas9M protein is labeled with the GFP green fluorescent protein, and it can be directly judged whether the spCas9M protein is expressed, and then the possibility of gene editing of the colony is judged, thereby indirectly improving the screening efficiency of the gene editing strain.

[0033] The above-mentioned C. lunata gene editing vector carries the CRISPR / spCas9M-GFP gene editing system, and a high-efficiency, simple and scarless genome editing tool is constructed in C. lunata, which can realize gene knockout and insertion.

[0034] 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 C. lunata can also be subjected to gene editing, and a corresponding gRNA can be designed.

[0035] Further, 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.

[0036] In some embodiments, a repeat 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 homologous arm of the target gene and the downstream homologous arm of the target gene.

[0037] Further, the length of the repeat sequence is 30 bp.

[0038] In some embodiments, the plasmid backbone is a pSJ-backbone plasmid backbone.

[0039] In some embodiments, the spCas9M gene is a C. lunata-codon-optimized gene of the spCas9 gene. The specific sequence of the codon-optimized gene is shown in Table 1.

[0040] The above-mentioned Caulobacter crescentus gene editing vector is a high-efficiency, non-scarring gene editing system, which can realize gene knockout and gene knock-in in Caulobacter crescentus.

[0041] The embodiment of the present application also provides a gene editing method of Caulobacter crescentus, which is characterized by comprising the following steps S110-S120:

[0042] S110, constructing the above-mentioned Caulobacter crescentus gene editing vector;

[0043] S120, transforming the Caulobacter crescentus gene editing vector into a Caulobacter crescentus competent cell, culturing, screening, and obtaining a Caulobacter crescentus editing strain (i.e. the Caulobacter crescentus after gene editing).

[0044] In some embodiments, 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.

[0045] In some embodiments, the step of transforming the Caulobacter crescentus gene editing vector into the Caulobacter crescentus competent cell, culturing, and screening comprises:

[0046] The Caulobacter crescentus gene editing vector is transformed into the Caulobacter crescentus competent cell, cultured on a resistant plate medium, and then observed under a blue light lamp to observe whether a single clone colony emits green fluorescence;

[0047] The single clone colony emitting green fluorescence under the blue light lamp is verified to obtain the Caulobacter crescentus editing strain.

[0048] Specifically, the step of electrotransforming the Caulobacter crescentus comprises: taking 500 ng of the successfully assembled plasmid, adding 100 microliters of Caulobacter crescentus competent cells, mixing gently, transferring to a 2mm electroshock cup, and standing on ice for 20 min. The BIORAD ECO2 program is selected for 2.5kV electrotransformation, 1ml of PYE liquid medium is quickly added, and the bacteria are resuscitated at 30°C for 3h. Centrifugation is performed at 6000rpm for 3min, 900 microliters of supernatant is removed, the remaining 100ul of liquid is used to resuspend the bacterial precipitate, and the resuspension is coated on a PYE resistant plate (Kan).

[0049] The step of screening the Caulobacter crescentus editing strain comprises: after coating on the kan resistant plate for three days, observing whether the clone emits green fluorescence under the blue light lamp, and dividing the single clone emitting green fluorescence into small squares on a new PYE resistant plate (Kan), and performing PCR verification the next day to obtain the Caulobacter crescentus editing strain.

[0050] In some embodiments, the step of verifying the monoclonal colony emitting green fluorescence under blue light further comprises the following steps after the step of verifying the monoclonal colony emitting green fluorescence under blue light: transferring the verified new moon-shaped handle bacillus editing strain to an antibiotic-free liquid PYE medium for overnight culture, then gradient dilution and coating on an antibiotic-free PYE solid medium for culture, and then PCR identification to obtain the new moon-shaped handle bacillus editing strain without carrying a gene editing vector. The purpose of plasmid loss of the new moon-shaped handle bacillus editing strain is to obtain a mutant strain with a clean background without any selection marker, which is of great significance for the subsequent study of the new moon-shaped handle bacillus editing strain.

[0051] The embodiment of the present application also provides a new moon-shaped handle bacillus editing strain prepared by the above gene editing method.

[0052] The following are specific examples.

[0053] In the examples, reagents and instruments are used as conventional choices in the art unless otherwise specified. The experimental methods not specified in the examples are usually carried out according to conventional conditions, for example, the conditions described in the literature, books or the methods recommended by the kit manufacturers.

[0054] 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 examples, which drives the expression of each sgRNA in the examples; sgRNA scaffold is the sgRNA together with gRNA sequence, which plays a role in targeting DNA target in gene editing, and sgRNA scaffold sequence plays a role in binding spCas9M protein; in Table 1, the capital letter part is the corresponding designed homologous arm.

[0055] Table 1 Sequences of genes and primers involved in Example 1

[0056] Example 1 Gene editing of new moon-shaped handle bacillus

[0057] (1) spCas9 gene codon optimization:

[0058] The tool used for spCas9 gene codon optimization in the present patent is 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.

[0059] (2) Design of gRNA:

[0060] The design was performed using CHOPCHOP online tool, the specific website of which is: http: / / chopchop.cbu.uib.no / .

[0061] Specifically, the target gene of the present embodiment is spmX gene, and three gRNAs are designed, and the specific sequences thereof include the sequences shown in SEQ ID NO. 29, SEQ ID NO. 32 and SEQ ID NO. 35.

[0062] (3) Design of plasmid:

[0063] The plasmid map designed in the present embodiment is shown in FIG. 1, which mainly comprises 6 parts, which are pSJ-backbone plasmid backbone, Pvan promoter, spCas9M gene fragment (i.e. after codon optimization), green fluorescent protein gene GFP, gRNA and target gene upstream and downstream homologous arms. Among them, gRNA and repair template (target gene upstream and downstream homologous arms) can be replaced according to different target genes. The sequences of each element are shown in Table 1. The main method for assembling each element into a plasmid is Gibson assembly method, and the specific operation steps are as follows:

[0064] (a) Design primers to linearize pSJ-backbone plasmid backbone and Pvan, which will have a 30bp repeat sequence between the two elements after linearization (this needs to be considered before designing primers), then assemble, the system is 20 microliters, of which pSJ-backbone plasmid backbone is 0.1 pmol, Pvan induction system element is 0.2 pmol, 2 microliters of Exnase II, 4 microliters of 5X CE II Buffer, and water is added to 10 microliters, then put into PCR instrument, 37°C reaction for 30 min, then transform the assembly product into DH5α competent cells, and screen on LB kanamycin resistant plate to obtain plasmid pSJ-Pvan.

[0065] (b) Design primers to linearize the obtained plasmid pSJ-Pvan and spCas9M gene fragment, which will also have a 30bp repeat sequence between the two fragments, and 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 pSJ-Pvan-spCas9M plasmid.

[0066] (c) Design primers to linearize plasmid pSJ-Pvan-spCas9M and GFP, and then assemble them according to the method of step (a) above, with a 30 bp repeat sequence between the two fragments. The molar ratio of the two components is 1:1. Other methods are the same as those described in step (a) above. The pSJ-Pvan-spCas9M-GFP plasmid can be obtained.

[0067] (d) Design primers to assemble plasmid pSJ-Pvan-spCas9M-GFP, P23119-sgRNA scaffold, and the upstream and downstream homologous arms of the target gene. Linearize the plasmid pSJ-Pvan-spCas9M-GFP, P23119-sgRNA scaffold, and the upstream and downstream homologous arms, and then assemble them using the method of step (a) above, with a 30 bp repeat sequence between each component. The molar ratio of each component is 1:1:1:1, and the total molar number is not more than 0.2 pmol. Other methods are the same as those described in step (a) above. The pSJ-P23119-sgRNA scaffold-HRs-Pvan-spCas9M-GFP plasmid can be obtained.

[0068] (e) Design gRNA sequences on primers, and perform loop PCR on plasmid pSJ-P23119-sgRNA scaffold-HRs-Pvan-spCas9M-GFP to obtain a linear fragment. Then, linearize the fragment and transform DH5α competent cells, and screen on LB resistance (Kan) plates to achieve the connection of gRNA between P23119 and sgRNA scaffold, and obtain the gene editing vector pSJ-P23119-sgRNA-HRs-Pvan-spCas9M-GFP. The plasmid map is shown in Figure 1.

[0069] (4) Caudovirales gene editing:

[0070] (a) Take 500 ng of assembled plasmid (pSJ-P23119-sgRNA-HRs-Pvan-spCas9M-GFP) and add it to 100 microliters of Caulobacter crescentus (wild type Caulobacter crescentus is from the literature: Phase separation modulates the assembly and dynamics of a polarity-related scaffold-signaling hub, Nature Communications, Wei Tan, Sihua Cheng, Yingying Li et al. Eds.) competent cells. Mix gently and transfer to a 2 mm electroporation cuvette and place on ice for 20 min. Select the BIORAD ECO2 program, 2.5 kV electroporation, quickly add 1 ml of PYE liquid medium, and recover at 30°C for 3 h. Centrifuge at 6000 rpm for 3 min, remove 900 microliters of supernatant, resuspend the bacterial pellet in the remaining 100 microliters of liquid, and spread the resuspension on a PYE resistant plate (Kan).

[0071] (b) After three days, check if single colonies have grown. Under blue light, single colonies that emit green fluorescence are cut into small squares on a new PYE resistant (kan) plate, and PCR verification is performed the next day to obtain the Caulobacter crescentus editing strain.

[0072] (5) Caulobacter crescentus editing strain plasmid loss:

[0073] The purpose of the Caulobacter crescentus editing strain plasmid loss is to obtain an editing strain with a clean background without any selection markers, which is of great significance for the subsequent study of the Caulobacter crescentus editing strain. Specifically, the Caulobacter crescentus editing strain is picked into PYE liquid medium without antibiotics and incubated overnight at 28°C and 220 rpm. The next day, the bacterial solution is diluted 1000-fold, and 100 microliters are spread on a PYE solid plate without antibiotics and incubated at 30°C. After 3 days, the colonies on the plate are detected using specific primers on the plasmid to determine whether the plasmid has been lost. If the plasmid is lost, no band is produced after PCR amplification of the specific primers; if the plasmid exists, a band is produced after PCR amplification of the specific primers.

[0074] (6) Results detection

[0075] The spmX gene in C. crescentus was knocked out by the above steps (1)-(5). The spmX gene knockout was detected by PCR and sequencing, and the results are shown in Figure 2, which is the detection result of spmX gene knockout in Agrobacterium using gRNA1. The knockout efficiency of three different gRNAs of spmX gene was detected by PCR method, and the results are shown in Figure 3; the spmX gene knockout mutant was phenotypically identified, and the results are shown in Figures 4-5. The fluorescence localization of DivJ-mCherry fusion protein in wild type C. crescentus and spmX gene knockout mutant was detected by fluorescence microscopy, and the results are shown in Figure 4.

[0076] From Figure 2, it can be seen that the scaffold protein spmX gene is knocked out in C. crescentus, and the knockout efficiency is up to 87.5% using the present technology.

[0077] The C. crescentus gene editing vector and editing method of the present application realize single gene, double gene knockout and gene knock-in in C. crescentus, construct a high-efficiency, scar-free gene editing system, realize process-based gene editing, and obtain a mutant strain without a screening marker. The system greatly improves the efficiency of gene editing, fills the technical vacuum in this field, and has an important role.

[0078] The technical features of the above-described embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, but as long as the combinations of the technical features do not exist contradictory, they should be considered as the scope of the present disclosure.

[0079] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as limiting the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of the present patent should be subject to the appended claims.

Claims

1. A Caulobacter crescentus gene editing vector, characterized in that, The Caulobacter crescentus gene editing vector comprises the following genetic elements: a plasmid backbone, a Pvan promoter, a spCas9M gene, a green fluorescent protein gene GFP, a gRNA, an upstream homologous arm of a target gene, and a downstream homologous arm of the target gene.

2. The Caulobacter crescentus gene editing vector of claim 1, wherein, The target gene comprises a spmX gene.

3. The Caulobacter crescentus gene editing vector of claim 2, wherein, The sequence of the gRNA comprises at least one of the sequences shown in SEQ ID NO. 29, SEQ ID NO. 32, and SEQ ID NO.

35.

4. The Caulobacter crescentus gene editing vector according to any one of claims 1 to 3, characterized in that, Among the plasmid backbone, the Pvan promoter, the spCas9M gene, the green fluorescent protein gene GFP, the gRNA, the upstream homologous arm of the target gene, and the downstream homologous arm of the target gene, a repeat sequence is connected between two adjacent genetic elements. Further, the repeat sequence has a length of 30 bp.

5. The Caulobacter crescentus gene editing vector of any one of claims 1-3, wherein, The plasmid backbone is a pSJ-backbone plasmid backbone.

6. A method of gene editing of Caulobacter crescentus, comprising, The method comprises the following steps: constructing the Caulobacter crescentus gene editing vector according to any one of claims 1-5; transforming the Caulobacter crescentus gene editing vector into a Caulobacter crescentus competent cell, culturing, screening, and obtaining a Caulobacter crescentus editing strain.

7. The method of gene editing of claim 6, wherein, The step of constructing the Caulobacter crescentus gene editing vector comprises assembling each of the genetic elements by Gibson technology to obtain the Caulobacter crescentus gene editing vector.

8. The method of gene editing of claim 6, wherein, The steps of transforming the Caulobacter crescentus gene editing vector into the Caulobacter crescentus competent cell, culturing, and screening comprise: transforming the Caulobacter crescentus gene editing vector into the Caulobacter crescentus competent cell, culturing on a resistant plate medium, and then observing whether a single colony emits green fluorescence under a blue light lamp; verifying the single colony emitting green fluorescence under the blue light lamp to obtain the Caulobacter crescentus editing strain.

9. The method of genetic editing according to any one of claims 5-8, wherein, After the step of verifying the single colony emitting green fluorescence under the blue light lamp, the method further comprises the following steps: transferring the verified Caulobacter crescentus editing strain to an antibiotic-free PYE liquid medium for overnight culture, then gradually diluting and coating on an antibiotic-free PYE solid culture medium for culture, and then performing PCR identification to obtain the Caulobacter crescentus editing strain not carrying the gene editing vector.

10. A Caulobacter crescentus editing strain, characterized in that, The method is prepared by any one of claims 6-9. The method is prepared by any one of claims 6-9.

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