Gene editing vector of c. lunata, gene editing method thereof and c. lunata editing strain

Abstract

The present application relates to a gene editing vector of Caulobacter crescentus, a gene editing method thereof and a Caulobacter crescentus editing strain. The gene editing vector of the Caulobacter crescentus comprises 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 and a downstream homologous arm of a target gene. The gene editing vector of the Caulobacter crescentus adopts a CRISPR / spCas9M-GFP gene editing system, constructs a high-efficiency, simple and scarless genome editing system in the Caulobacter crescentus, and can realize gene knockout and insertion.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to gene editing vectors of *Stipa crescentis*, gene editing methods thereof, and editable strains of *Stipa crescentis*. Background Technology

[0002] *Caulobacter crescentus* is a non-toxic, single-celled organism belonging to the class Alpha-Proteobacteria and is Gram-negative. This bacterium lives in nutrient-poor aquatic environments such as rivers, lakes, and oceans. The most distinctive feature of *Caulobacter crescentus* is that a single cell can differentiate into two different cell types: when they need to find nutrients, they differentiate into motile cells to explore their environment and find resources; when they need to reproduce and reproduce, they become non-motile, petiole-like cells. *Caulobacter crescentus* can synthesize a super glue with adhesive properties far exceeding those of artificial glues, demonstrating enormous potential in industrial applications, especially in the development and application of bio-adhesives.

[0003] In 1991, Ely bert used a two-step homologous recombination method and sacB negative screening to achieve gene knockout in *Stereomycium crescentis*. In 2020, Mathilde Guzzo et al. used CRISPR / dCas9 technology to suppress the expression of multiple genes in *Stereomycium crescentis*, successfully suppressing ctrA and gcrA expression under gRNA guidance. In 2006, Martin Thanbichler and Lucy Shapiro used single homologous recombination to integrate a non-replicating plasmid into a specific location in the *Stereomycium crescentis* genome, achieving MipZ gene knock-in (Thanbichler and Shapiro 2006). In 2011, Beat Christen et al. used transposon mutations to perform saturation mutations on the *Stereomycium crescentis* genome, enabling the identification of essential genes in *Stereomycium crescentis*.

[0004] The SacB negative screening-based gene knockout method for *Styrax lunulata* is time-consuming, labor-intensive, and has very low editing efficiency. CRISPR / dCas9 technology, based on the principle of dCas9 repressing gene transcription, reduces gene expression. However, this technology can only suppress gene expression and cannot be used for gene knockout or knock-in. Single homologous recombination technology, based on integrase and integration sites, inserts all elements on the plasmid (including selection markers, such as antibiotic genes) into a specific site in the genome under the action of integrase. The disadvantage of this technology is that it cannot perform gene knockout or obtain edited strains without selection markers. Transposon mutation 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 it can only perform insertion mutation inactivation, not gene reading frame knockout. Summary of the Invention

[0005] Based on this, this application provides a gene editing vector for Bacillus crescentis, which enables efficient editing of Bacillus crescentis genes.

[0006] A gene editing vector for *Stylosus crescentis*, comprising the following gene elements: a plasmid backbone, a Pvan promoter, a spCas9M gene, a green fluorescent protein gene (GFP), gRNA, an upstream homologous arm and a downstream homologous arm of a target gene.

[0007] The aforementioned *Stipa lunata* gene editing vector utilizes the CRISPR / spCas9M-GFP gene editing system to construct an efficient, simple, and scarless genome editing system in *Stipa lunata*, enabling gene knockout and insertion.

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

[0009] In some 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 embodiments, repetitive sequences are connected between 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.

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

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

[0013] A gene editing method for Bacillus crescentis includes the following steps:

[0014] Construct the above-mentioned gene editing vector for *Stipa crescentis*;

[0015] The gene-editing vector of *Stibulobacterium crescentis* was transformed into competent *Stibulobacterium crescentis* cells, cultured, and screened to obtain the edited strain of *Stibulobacterium crescentis*.

[0016] In some embodiments, the step of constructing the *Sterculia crescentis* gene editing vector includes: assembling the individual gene elements using Gibson technology to obtain the *Sterculia crescentis* gene editing vector.

[0017] In some embodiments, the steps of transforming the *Stipa lunata* gene-editing vector into *Stipa lunata* competent cells, culturing, and screening include:

[0018] The gene-editing vector of *Stylosus crescentis* was transformed into competent *Stylosus crescentis* cells, cultured on resistant plates, and then observed under blue light to see if single colonies emitted green fluorescence.

[0019] The edited strain of *Stylosus crescentis* was obtained by verifying the monoclonal colonies that fluoresced green under blue light.

[0020] In some embodiments, after verifying the single-clonal colonies that fluoresce green under blue light, the following steps are also included: transferring the verified Crestedia crescentis-edited strain to antibiotic-free PYE liquid medium for overnight culture, then serially diluting and plating it onto antibiotic-free PYE solid medium for culture, and then performing PCR identification to obtain the Crestedia crescentis-edited strain without the gene editing vector.

[0021] An edited strain of *Stylosus crescentis* was prepared using the gene editing method described above. Attached Figure Description

[0022] Figure 1 The plasmid map of Example 1;

[0023] Figure 2 Figures showing the PCR and sequencing results for spmX gene knockout;

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

[0025] Figure 4 Phenotypic identification results of spmX gene knockout mutants;

[0026] Figure 5The figure shows the results of the fluorescence localization statistical analysis of the DivJ-mCherry fusion protein in wild-type Bacillus crescentis and spmX gene knockout mutant. Detailed Implementation

[0027] 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.

[0028] One embodiment of this application provides a gene editing vector for Bacillus crescentis, which enables efficient editing of the Bacillus crescentis genome.

[0029] 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.

[0030] The CRISPR / spCas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein spCas9) gene editing system is a CRISPR type II system. The CRISPR / spCas9M-GFP system, where spCas9M is a codon-optimized name for the spCas9 gene (e.g., *Stipa lunata*). A GFP (green fluorescent protein) is fused to the C-terminus of the spCas9M protein to indicate its expression. The principle of the CRISPR / spCas9M-GFP system is as follows: ① Recognition component: The CRISPR sequence is transcribed to form sgRNA with target recognition function; ② Cutting component: The spCas9M protein is an endonuclease that binds to the sgRNA. As the sgRNA specifically recognizes and binds to the specific DNA sequence, the spCas9M protein cuts the targeted DNA double 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. ③ Discriminant Component: The spCas9M-GFP fusion protein fluoresces green under blue light, indicating whether the spCas9M protein is expressed. If the clone fluoresces green, it means that the spCas9M protein is expressed, and gene editing is likely to occur in the cell; if the clone does not fluoresce green, it means that the spCas9M protein is not expressed, and gene editing has not occurred in the cell. However, these designs are not sufficient to make the CRISPR / spCas9M-GFP system work efficiently in *Stipa crenata*. We further optimized the promoter driving the spCas9M gene, the promoter inducer concentration, and the homologous arm length, as detailed below: ① This study tested two inducible promoters, Pvan and Pxyl, and found 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 gene editing; ③ Due to the limitation of plasmid size, the longest homologous arm should not exceed 2K.

[0031] This system has the following advantages: ① In the CRISPR / spCas9M-GFP system, the spCas9 gene codon was optimized in *Stipa crescentis*, improving the expression efficiency of the spCas9 gene in *Stipa crescentis*; ② The CRISPR / spCas9M-GFP system uses the inducible promoter Pvan to drive the expression of the spCas9M gene, strictly controlling the expression level of the spCas9M gene, which is conducive to maximizing the editing efficiency of the CRISPR / spCas9M-GFP system; ③ By screening homologous arms of different lengths, the optimal length homologous arms are assembled into the CRISPR / spCas9M-GFP system, resulting in relatively high gene editing efficiency; ④ In the CRISPR / spCas9M-GFP system, the spCas9M protein is labeled with GFP (green fluorescent protein), which can directly determine whether the spCas9M protein is expressed, thereby determining the possibility of gene editing in the colony, indirectly improving the screening efficiency of gene-editing strains.

[0032] The aforementioned *Stipa lunata* gene editing vector carries the CRISPR / spCas9M-GFP gene editing system, enabling the construction of an efficient, simple, and scarless genome editing tool in *Stipa lunata*, capable of 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; other genes in *Sterculia crescentis* can also be edited by designing corresponding gRNAs.

[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 embodiments, repetitive sequences are connected between 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.

[0036] Furthermore, the length of the repeating 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 the spCas9 gene from *Stipa crescentis* with optimized codons. The specific sequence of the optimized codons is shown in Table 1.

[0039] The aforementioned *Stipa crescentis* gene editing vector is a highly efficient and scarless gene editing system that can achieve gene knockout and gene knock-in in *Stipa crescentis*.

[0040] An embodiment of this application also provides a gene editing method for Bacillus crescentis, characterized by comprising the following steps S110-S120:

[0041] S110. Construct the above-mentioned *Stylosus* gene editing vector;

[0042] S120. The gene-editing vector of *Stylosus crescentis* is transformed into competent *Stylosus crescentis* cells, cultured, and screened to obtain the *Stylosus crescentis* edited strain (i.e., gene-edited *Stylosus crescentis*).

[0043] In some embodiments, the step of constructing the *Sterculia crescentis* gene editing vector includes: assembling the individual gene elements using Gibson technology to obtain the *Sterculia crescentis* gene editing vector.

[0044] In some embodiments, the steps of transforming the *Stipa crescentis* gene-editing vector into *Stipa crescentis* competent cells, culturing, and screening include:

[0045] The gene-editing vector of *Stylos lunulae* was transformed into competent *Stylos lunulae* cells, cultured on resistant agar plates, and then observed under blue light to see if single colonies emitted green fluorescence.

[0046] The edited strain of *Stylosus crescentis* was obtained by verifying the monoclonal colonies that fluoresced green under blue light.

[0047] Specifically, the steps for electroporation of *Stipa crescentis* include: adding 500 ng of the successfully assembled plasmid to 100 μL of competent *Stipa crescentis* cells. After gentle mixing, transfer to a 2 mm electroporation cuvette and incubate on ice for 20 min. Select the BIORAD ECO2 program, electroporate at 2.5 kV, quickly add 1 ml of PYE liquid medium, and incubate at 30°C for 3 h. Centrifuge at 6000 rpm for 3 min, discard 900 μL of supernatant, resuspend the bacterial pellet in the remaining 100 μL of liquid, and plate the resuspended solution onto a PYE antibody plate (Kan).

[0048] The steps for screening edited strains of *Sterculia crescentis* include: plating the sample onto a Kan resistant plate for three days, observing under a blue light whether the clones emit green fluorescence, and marking small squares on a new PYE resistant plate (Kan) for verification by PCR the next day to obtain edited strains of *Sterculia crescentis*.

[0049] In some embodiments, after the step of verifying the monoclonal colonies that fluoresce green under blue light, the following steps are also included: transferring the verified *S. crescentis* edited strain to antibiotic-free liquid PYE medium for overnight culture, then serially diluting and plating it onto antibiotic-free PYE solid medium for further culture, followed by PCR identification to obtain the *S. crescentis* edited strain without the gene-editing vector. The purpose of discarding the plasmid in the *S. crescentis* edited strain is to obtain a mutant strain with a clean background and no selection markers, which is of great significance for subsequent research on *S. crescentis* edited strains.

[0050] One embodiment of this application also provides a *Bacillus lunulatus* edited strain, prepared by the above-described gene editing method.

[0051] The following are specific examples.

[0052] 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.

[0053] 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; sgRNAscaffold is formed together with the gRNA sequence to form sgRNA. In gene editing, gRNA plays the role of targeting DNA targets, and sgRNAscaffold sequence plays the role of binding spCas9M protein; in Table 1, the uppercase letters are the corresponding designed homologous arms.

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

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] Example 1 Gene editing of Bacillus crescentis (1) codon optimization of spCas9 gene:

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

[0065] (2) gRNA design:

[0066] Use the CHOPCHOP online tool for design; its URL is: http: / / chopchop.cbu.uib.no / .

[0067] Specifically, the target gene in this embodiment is the spmX gene, and three gRNAs were designed, with specific sequences including those shown in SEQ ID NO.29, SEQ ID NO.32 and SEQ ID NO.35.

[0068] (3) Plasmid design:

[0069] The plasmid map designed in this embodiment is shown in [reference]. Figure 1 The plasmid mainly consists of six parts: the pSJ-backbone backbone, the Pvan promoter, the spCas9M gene fragment (i.e., after codon optimization), the green fluorescent protein gene GFP, gRNA, and upstream and downstream homologous arms of the target gene. The gRNA and repair template (upstream and downstream homologous arms of the target gene) can be replaced depending on the target gene. The sequences of each element are shown in Table 1. The Gibson assembly method is mainly used to assemble the elements into a single plasmid. The specific steps are as follows:

[0070] (a) To design primers, first linearize the pSJ-backbone plasmid backbone 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 the components in a 20 μL system, which contains 0.1 pmol of pSJ-backbone plasmid backbone, 0.2 pmol of Pvan induction system elements, 2 μL of Exnase II, 4 μL of 5X CE II Buffer, and water to make up to 10 μL. Then place the system in a PCR instrument and react at 37℃ for 30 min. After that, transform the assembled product into DH5α competent cells and screen them on LB kanamycin resistance plates to obtain the plasmid pSJ-Pvan.

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

[0072] (c) After designing primers to linearize plasmids pSJ-Pvan-spCas9M and GFP, a 30bp repeat sequence is created between the two fragments. The two elements are then assembled 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-Pvan-spCas9M-GFP plasmid can be obtained.

[0073] (d) Primers were designed to assemble plasmids pSJ-Pvan-spCas9M-GFP, P23119-sgRNAscaffold, and the upstream and downstream homologous arms of the target gene. After linearization of plasmids pSJ-Pvan-spCas9M-GFP, P23119-sgRNAscaffold, and the upstream and downstream homologous arms, there were 30 bp repetitive sequences between each element. The assembly was performed using the method described 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 were the same as in step (a) above, to obtain the plasmid pSJ-P23119-sgRNAscaffold-HRs-Pvan-spCas9M-GFP.

[0074] (e) The gRNA sequence was designed on primers, and loop PCR was performed on the plasmid pSJ-P23119-sgRNA scaffold-HRs-Pvan-spCas9M-GFP to obtain a linear fragment. Subsequently, the linear fragment was transformed into DH5α competent cells, and selection was performed on LB resistant (Kan) plates to achieve gRNA ligation between P23119 and sgRNA scaffold, obtaining the gene editing vector pSJ-P23119-sgRNA-HRs-Pvan-spCas9M-GFP, whose plasmid map is shown below. Figure 1 As shown.

[0075] (4) Gene editing in Bacillus crescentis:

[0076] (a) Take 500 ng of the successfully assembled plasmid (pSJ-P23119-sgRNA-HRs-Pvan-spCas9M-GFP) and add it to 100 μL of *S. crescentis* (wild-type *S. crescentis* is derived 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, et al.) competent cells. Gently mix and transfer to a 2 mm electroporation cuvette, incubate on ice for 20 min. Select the BIORAD ECO2 program, electroporate at 2.5 kV, quickly add 1 ml of PYE liquid medium, and incubate at 30 °C for 3 h. Centrifuge at 6000 rpm for 3 min, discard 900 μL of supernatant, resuspend the bacterial pellet in the remaining 100 μL of liquid, and spread the resuspended solution on a PYE-resistant plate (Kan).

[0077] (b) Three days later, check for the growth of monoclonal colonies. Under blue light, mark small squares of green fluorescent monoclonal colonies on new PYE-resistant (KAN) plates, and perform PCR verification the next day to obtain the edited strain of *Stipa crescentis*.

[0078] (5) Plasmid loss in *Stylosporium crescentis* edited strains:

[0079] The purpose of losing the plasmid in *Stipa crescentis* edited strains is to obtain edited strains with a clean background and no selection markers, which is of great significance for subsequent research on *Stipa crescentis* edited strains. Specifically, the *Stipa crescentis* edited strains were picked and cultured overnight in antibiotic-free PYE liquid medium at 28°C and 220 rpm. The next day, the bacterial culture was diluted 1000-fold, and 100 μL was plated onto antibiotic-free PYE solid plates and cultured at 30°C. After 3 days, the clones on the plate were detected using specific primers for the plasmid to determine if the plasmid was lost. If the plasmid was lost, no band was produced after PCR amplification with the specific primers; if the plasmid was present, a band was produced after PCR amplification with the specific primers.

[0080] (6) Result detection

[0081] The spmX gene in *Sterculia crescentis* was knocked out using steps (1)-(5) above. PCR and sequencing were performed to detect the spmX gene knockout, and the results are as follows: Figure 2 As shown, Figure 2 The results of spmX gene knockout in Agrobacterium using gRNA1 are shown in the figure. The knockout efficiency of three different gRNAs for the spmX gene was detected using PCR, and the results are as follows. Figure 3 As shown; phenotypic identification of the spmX gene knockout mutant was performed, and the results are as follows. Figure 4-5 As shown in the figure. Fluorescence microscopy was used to detect the fluorescence localization of the DivJ-mCherry fusion protein in wild-type *Stipa crescentis* and the spmX gene knockout mutant. The results are as follows. Figure 4 As shown.

[0082] from Figure 2 It is known that the spmX gene of the scaffolding protein was knocked out in Bacillus crescentis. Using the technology of this invention, the knockout efficiency reached up to 87.5%.

[0083] The gene editing vector and editing method of *Stipa crescentis* presented in this application realize single-gene and double-gene knockout and gene knock-in in *Stipa crescentis*, constructing an efficient and traceless gene editing system, realizing streamlined gene editing, and obtaining mutant strains without selection markers. This system greatly improves the efficiency of gene editing, fills the technological vacuum in this field, and plays an important role.

[0084] 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.

[0085] 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 Bacillus crescentis, characterized in that, The *Stylosus lunulatus* 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; The plasmid backbone is the pSJ-backbone plasmid backbone. The target gene is the spmX gene. The sequence of the gRNA is selected from at least one of the sequences shown in SEQ ID NO.29, SEQ ID NO.32 and SEQ ID NO.35; The base sequence of the upstream homologous arm of the target gene is shown in SEQ ID NO.17, and the base sequence of the downstream homologous arm of the target gene is shown in SEQ ID NO.

20. The gRNA is linked to the plasmid backbone in the form of sgRNA, and the sgRNA includes an sgRNA scaffold sequence and the gRNA; the Bacillus crescentis gene editing vector also includes the pJ23119 promoter, which is located upstream of the sgRNA and is the promoter of the sgRNA; The upstream and downstream homologous arms of the target gene, the Pvan promoter, the spCas9M gene, and the green fluorescent protein gene GFP are all linked to the plasmid backbone. In the plasmid backbone, the sgRNA, the upstream and downstream homologous arms of the target gene, the Pvan promoter, the spCas9M gene, and the green fluorescent protein gene GFP are sequentially linked from upstream to downstream via repetitive sequences.

2. The *Stipa crescentis* gene editing vector according to claim 1, characterized in that, The upstream repeat sequence of the pJ23119 promoter is linked to the plasmid backbone, and the downstream repeat sequence of the green fluorescent protein gene GFP is linked to the plasmid backbone.

3. The *Stipa crescentis* gene editing vector according to any one of claims 1-2, characterized in that, The length of the repeating sequence is 30 bp.

4. A gene editing method for Bacillus crescentis, characterized in that, Includes the following steps: Construct the *Stipa lunata* gene editing vector as described in any one of claims 1-3; The gene-editing vector of *Stibulobacterium crescentis* was transformed into competent *Stibulobacterium crescentis* cells, cultured, and screened to obtain the edited strain of *Stibulobacterium crescentis*.

5. The gene editing method according to claim 4, characterized in that, The steps for constructing the *Stipa crescentis* gene editing vector include: assembling each of the gene elements using Gibson technology to obtain the *Stipa crescentis* gene editing vector.

6. The gene editing method according to claim 4, characterized in that, The steps of transforming the *Stipa crescentis* gene-editing vector into *Stipa crescentis* competent cells, culturing, and screening include: The gene-editing vector of *Stylos lunulae* was transformed into competent *Stylos lunulae* cells, cultured on resistant agar plates, and then observed under blue light to see if single colonies emitted green fluorescence. The edited strain of *Stylosus crescentis* was obtained by verifying the monoclonal colonies that fluoresced green under blue light.

7. The gene editing method according to claim 6, characterized in that, After the step of verifying the monoclonal colonies that fluoresce green under blue light, the method further includes the following steps: transferring the verified Crestedia crescentis edited strain to antibiotic-free PYE liquid medium for overnight culture, then serially diluting and plating it onto antibiotic-free PYE solid medium for culture, and then performing PCR identification to obtain the Crestedia crescentis edited strain without the gene editing vector.

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