Accurate editing method for gamma-PGA high-yield strain based on CRISPR-Cas9n

Abstract

The invention discloses a precise editing method of a gamma-PGA high-yield strain based on CRISPR-Cas9n, and relates to the technical field of high-yield strains. According to the invention, a CRISPR-Cas9n double-nickase system is adopted, and adjacent DNA nicks are generated through the synergistic effect of double sgRNA, so that the non-specific cutting risk is greatly reduced; the double sgRNA targeting design is combined with a homologous recombination template, so that directional editing of key genes is realized, the homologous recombination efficiency reaches 75 + / -5%, and target modification is ensured to be stably integrated into a genome; by virtue of a PEG-mediated protoplast conversion method, the limitation of low efficiency of traditional electrotransfection is overcome, and the CRISPR carrier is rapidly removed in combination with a temperature-sensitive plasmid elimination system, so that cytotoxicity caused by continuous expression is avoided; under optimized fermentation conditions, the gamma-PGA yield of the edited strain reaches 15 + / -2g / L and is increased by 200% compared with that of the original strain.

Description

Technical Field

[0001] This invention relates to the field of high-yield strain technology, specifically to a precise editing method for γ-PGA high-yield strains based on CRISPR-Cas9n. Background Technology

[0002] High-yield strains refer to microbial strains optimized or screened through specific technologies, whose yields of target products (such as antibiotics, enzymes, and flavor compounds) are significantly higher than those of wild-type or conventional strains. These strains have wide applications in industrial fermentation, agricultural production, and the pharmaceutical field. High-yield strains significantly increase the yield of target products through techniques such as genetic engineering, mutagenesis breeding, or metabolic engineering.

[0003] High-yield strain editing significantly enhances the metabolic efficiency and product yield of microorganisms through gene editing technology. Its main functions include: improving metabolic efficiency by enhancing the activity of metabolic pathways through editing key genes (such as promoters and enzyme genes). For example, replacing the PGPDA promoter can increase enzyme activity by 300%, and missense mutations can relieve lysine feedback inhibition, increasing L-AAA production by 150%. Relieving metabolic limitations: Some strains experience metabolic bottlenecks due to gene defects, which can be relieved through editing. For example, knocking out the phoR gene relieves phosphate inhibition, or partially deleting creA alleviates glucose repression. Targeted screening and optimization: Combining biosensor technology allows for rapid screening of high-yield strains. For example, integrating a whole-genome CRISPRi library with a sensor can be used to screen for high-yield D-lactic acid strains, improving screening efficiency. Enhanced delivery efficiency: Using delivery systems such as PEG-mediated protoplast transformation can improve the transformation efficiency of gene editing (e.g., reaching 80%), ensuring stable editing results.

[0004] Existing high-yield strain editing methods mainly fall into two categories: traditional mutagenesis breeding and modern gene editing technologies. Traditional methods, such as ultraviolet or chemical mutagenesis (e.g., EMS treatment), screen for high-yield strains by randomly inducing gene mutations. However, these methods suffer from drawbacks such as low efficiency, strong nonspecificity, and long screening cycles, and are prone to generating non-target mutations, leading to poor strain stability. In modern gene editing technologies, the CRISPR-Cas9 system is widely used. It uses sgRNA to guide Cas9 enzymes to cleave target genes, achieving gene knockout, insertion, or replacement, significantly improving editing precision. However, CRISPR-Cas9 is prone to off-target effects, affecting editing reliability. In addition, delivery systems such as electroporation or plasmid transformation are inefficient, with transformation rates often below 50%, limiting large-scale application. CRISPRi technology, combined with biosensors, can improve efficiency for high-throughput screening, but its regulatory precision is insufficient, making it difficult to achieve multi-gene collaborative editing. For high-yield γ-PGA strains, existing methods attempt to edit key genes (such as the pgsBCA operon), but yield fluctuations are often caused by cytotoxicity or metabolic interference. To this end, we propose a precise editing method for high-yield γ-PGA strains based on CRISPR-Cas9n. Summary of the Invention

[0005] The purpose of this invention is to provide a precise editing method for high-yield γ-PGA strains based on CRISPR-Cas9n, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention specifically adopts the following technical solution: A precise editing method for high-yield γ-PGA strains based on CRISPR-Cas9n includes the following steps: Step 1, Target gene sgRNA design and vector construction: Targeting key gene clusters for γ-PGA synthesis and metabolic regulatory genes, double sgRNA sequences were designed, and the double sgRNA and Cas9n-D10A mutant gene were cloned into the pET plasmid vector using the CRISPR-Cas9n double nickase system to form the pET-Cas9n-sgRNA1-sgRNA2 expression cassette. Step 2, Efficient Delivery and Genome Editing: Recombinant plasmids were introduced into Bacillus subtilis hosts via PEG-mediated protoplast transformation. Positive transformants were screened in LB medium containing erythromycin, and Cas9n expression was induced to generate targeted DNA double-strand cuts. Step 3, Source Recombination Repair and Editing Validation: The homology repair template was imported simultaneously, the CRISPR vector was removed using a temperature-sensitive plasmid elimination system, the target gene was amplified by PCR, and Sanger sequencing was used to confirm the insertion of the Pg32 promoter and the deletion of ΔcreA-152. Step 4: Screening and Fermentation Validation of High-Yield Strains: High-throughput screening of strains that enhance γ-PGA production was conducted using biosensors, and the fermentation process was optimized.

[0007] Furthermore, the dual sgRNA sequence in step 1 targets the promoter region of the pgsB gene and the coding region of the creA gene, has a length of 20 nt, and the GC content is controlled between 45-55%.

[0008] Furthermore, the transformation conditions in step 2 are as follows: 30% PEG8000, heat shock at 37°C for 5 minutes, transformation efficiency of 80±5%, and erythromycin in LB medium of 10μg / mL.

[0009] Furthermore, the host is Bacillus subtilis strain 168, whose genome has been pre-edited to remove endogenous restriction systems.

[0010] Furthermore, the template in step 3 includes: a strong promoter of the pgsB gene (Pg32 replacement) and a partially deleted sequence of the creA gene (ΔcreA-152).

[0011] Furthermore, the homology repair template in step 3 contains a 1000bp homology arm, and the homology recombination efficiency reaches 75±5%.

[0012] Furthermore, the optimized fermentation process in step 4 involves culturing in M9 medium containing 8% glucose at 37°C for 48 hours, followed by HPLC detection of γ-PGA yield.

[0013] Furthermore, the biosensor in step 4 is a γ-PGA-specific sensing system based on a luciferase reporter gene, with a detection threshold of 0.1 g / L.

[0014] Furthermore, the screening criterion for high-yielding strains in step 4 is that the γ-PGA yield reaches 15±2 g / L.

[0015] Furthermore, in step 4, the initial screening of high-yield strains is carried out in M9 medium containing 8% glucose and cultured at 37°C for 24 hours. Strains with a fluorescence intensity ≥5000 AU detected by a biosensor are determined to be positive in the initial screening.

[0016] The beneficial effects of this invention are as follows: 1. Significantly reduced off-target risk: Utilizing the CRISPR-Cas9n dual-cutting enzyme system (instead of the traditional Cas9), adjacent DNA nicks are generated through the synergistic action of two sgRNAs, significantly reducing the risk of non-specific cleavage. Experiments show that the off-target rate is reduced by more than 90% compared to the traditional Cas9 system, and editing specificity is significantly improved.

[0017] 2. Highly efficient and precise editing: Dual sgRNA targeted design combined with homologous recombination templates (containing 1000bp homologous arms) enables targeted editing of key genes (such as Pg32 promoter insertion and ΔcreA-152 deletion). Homologous recombination efficiency reaches 75±5%, ensuring stable integration of the target modification into the genome.

[0018] 3. Simple operation and high conversion rate: The PEG-mediated protoplast transformation method (conversion efficiency 80±5%) overcomes the limitation of low efficiency (<50%) of traditional electroporation; combined with a temperature-sensitive plasmid removal system, the CRISPR vector is quickly removed, avoiding cytotoxicity caused by continuous expression.

[0019] 4. High and stable yield: Under optimized fermentation conditions (8% glucose M9 medium, 37℃), the edited strain achieved a γ-PGA yield of 15±2 g / L, which is 200% higher than the original strain. After 5 generations of subculturing, the yield fluctuation rate was <5%, demonstrating excellent genetic stability.

[0020] 5. High-throughput screening advantage: Integrating a γ-PGA specific biosensor (detection threshold 0.1g / L), it can initially screen positive strains by fluorescence intensity (≥5000 AU) within 24 hours, with a screening efficiency 10 times higher than the traditional shake-flask fermentation method.

[0021] 6. Potential for Universality and Expansion: The CRISPR-Cas9n system, protoplast transformation, and template design strategy used in this method can be adapted to the precise metabolic engineering of other industrial Bacillus strains. Attached Figure Description

[0022] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0024] Please see Figure 1 This invention provides a method for precise editing of high-yield γ-PGA strains based on CRISPR-Cas9n, comprising the following steps: Step 1: Target Gene sgRNA Design and Vector Construction: Dual sgRNA sequences were designed targeting key gene clusters and metabolic regulatory genes involved in γ-PGA synthesis. Using the CRISPR-Cas9n dual-cutase system, the dual sgRNAs and the Cas9n-D10A mutant gene were cloned into the pET plasmid vector, forming the pET-Cas9n-sgRNA1-sgRNA2 expression cassette. The sgRNA design was optimized using the CRISPRdirect online tool, targeting conserved regions of the γ-PGA synthesis gene cluster, ensuring the PAM sequence was NGG and the off-target risk was less than 5%. The Cas9n-D10A mutant gene, derived from *Streptococcus pyogenes*, was amplified by PCR and cloned into the pET-28a(+) vector using the EcoRI / HindIII restriction endonuclease. The expression cassette contains a T7 promoter and a T7 terminator for easy IPTG-induced expression. After construction, Sanger sequencing was used to verify the accuracy of the vector sequence, ensuring the inserted fragments were correct.

[0025] Step 2, Efficient Delivery and Genome Editing: The recombinant plasmid was introduced into the Bacillus subtilis host via PEG-mediated protoplast transformation. Positive transformants were screened in LB medium containing erythromycin, inducing Cas9n expression and generating targeted DNA double-strand cuts. Double-strand breaks (DSBs) of approximately 20-50 bp were formed, activating the host cell's endogenous non-homologous end joining (NHEJ) or homologous directed repair (HDR) mechanisms. Homologous recombination repair templates (linear DNA fragments containing the expected mutation sequence and homologous arms) were used to achieve precise insertion, deletion, or point mutation of the target gene. After editing, plasmid elimination culture was performed in LB medium without erythromycin. The editing efficiency and genotype accuracy were verified by genomic PCR amplification of the target region and Sanger sequencing.

[0026] Step 3: Source Recombination Repair and Editing Validation: Homologous repair template was imported simultaneously, and the CRISPR vector was removed using a temperature-sensitive plasmid elimination system. The target gene was amplified by PCR, and Sanger sequencing confirmed the insertion of the Pg32 promoter and the deletion of ΔcreA-152. Temperature-sensitive plasmid elimination was achieved by culturing transformants at 42°C for 24 hours to inactivate replicons and remove the CRISPR vector. PCR amplification used high-fidelity DNA polymerase and specific primers (e.g., primer F: 5'-ATGCTAGCT-3', primer R: 5'-TCGAGCTAG-3') targeting the Pg32 promoter region and the creA gene site. After Sanger sequencing, sequence alignment was performed using BioEdit software to verify the precise insertion position of the Pg32 promoter and the range of base deletions in ΔcreA-152. Simultaneously, the functional changes of the edited strain were confirmed through plate screening or phenotypic analysis.

[0027] Step 4: Screening and Fermentation Validation of High-Yield Strains: High-throughput screening was performed using biosensors (such as γ-PGA-specific sensors based on fluorescence resonance energy transfer (FRET)). Edited strains were inoculated into 96-well plates, and after adding the inducer IPTG, the plates were cultured for 24 hours. The fluorescence signal intensity was detected, and positive strains with a γ-PGA yield increase of more than 50% were screened. The fermentation process was optimized, including adjusting the culture medium composition (e.g., glucose concentration to 20 g / L, monosodium glutamate to 40 g / L), controlling the temperature at 37°C and pH at 7.0, and optimizing the aeration rate (1.0 vvm) and stirring speed (300 rpm). Batch fermentation experiments were conducted in a 5L fermenter to validate the process. Samples were taken and the γ-PGA yield was analyzed (using high-performance liquid chromatography (HPLC) with a C18 column and acetonitrile-water gradient elution as the mobile phase). The product yield was calculated and the stability of the strain was evaluated. Batch fermentation was also repeated to confirm the reproducibility of the process.

[0028] The above steps address the issues of high off-target effects, unstable transformation efficiency, insufficient regulatory precision, and yield fluctuations mentioned in the background technology. Specifically, the CRISPR-Cas9n dual-nicking enzyme system is used to replace the traditional CRISPR-Cas9, significantly reducing the off-target risk to below 5%; the transformation efficiency is stably improved to 80±5% by optimizing the PEG-mediated protoplast transformation method, overcoming the transformation rate bottleneck of existing delivery systems; the simultaneous introduction of homologous repair templates (such as Pg32 promoter insertion and ΔcreA-152 deletion) ensures editing accuracy of 75±5%, effectively avoiding yield fluctuations caused by metabolic interference; high-throughput screening is performed using a biosensor based on luciferase reporter genes, with a detection threshold as low as 0.1 g / L, shortening the screening cycle by more than 50%, achieving efficient targeted optimization; finally, the edited strain achieves a stable increase in γ-PGA yield to 15±2 g / L in the optimized fermentation process, exhibits high genetic stability, and good reproducibility in repeated batch fermentations, completely solving the non-specific defects of traditional mutagenesis breeding, insufficient CRISPRi regulatory precision, and large-scale screening challenges in industrial applications in the background technology.

[0029] In this embodiment, preferably, the dual sgRNA sequences in step 1 target the promoter region of the pgsB gene and the coding region of the creA gene, with a length of 20 nt and a GC content controlled between 45-55%. sgRNAs are designed targeting the pgsB gene promoter region to insert into the strong promoter Pg32, enhancing the expression of γ-PGA synthase; while sgRNAs are designed targeting the creA gene coding region to delete the creA-152 fragment, relieving carbon metabolism repression. The sgRNA sequences are optimized using CRISPR design software (such as CRISPRdirect) to ensure target specificity, avoid binding to non-target sites, and their efficiency is verified through in vitro cleavage experiments.

[0030] In this embodiment, the preferred transformation conditions in step 2 are: 30% PEG8000, heat shock at 37°C for 5 minutes, achieving a transformation efficiency of 80±5%, and 10 μg / mL of erythromycin in LB medium. This operation can significantly improve the transformation efficiency to 80±5%, effectively promoting the stable introduction of recombinant plasmids and the survival rate of host cells, thereby laying a solid foundation for the subsequent expression of Cas9n protein and the generation of targeted DNA double-stranded nicks, ensuring the efficiency and reproducibility of the genome editing process, while minimizing non-specific damage and off-target effects.

[0031] In this embodiment, the preferred host is Bacillus subtilis strain 168, whose genome has been pre-edited to remove the endogenous restriction system. This operation can effectively eliminate the risk of degradation of the CRISPR vector by the host's endogenous restriction modification system, significantly improve the stability of exogenous DNA and the efficiency of homologous recombination, and reduce the probability of unexpected mutations, ensuring the accuracy and reproducibility of genome editing, and laying the foundation for the stable inheritance of high-yield phenotypes.

[0032] In this embodiment, preferably, the template in step 3 includes: a strong promoter of the pgsB gene (Pg32 replacement) and a partially deleted sequence of the creA gene (ΔcreA-152). This operation can precisely replace the pgsB gene promoter with Pg32 and delete the creA-152 fragment, thereby efficiently promoting the homologous recombination repair process, ensuring the accuracy of targeted editing, maximizing the enhancement effect of the γ-PGA synthesis pathway, and completely removing the carbon metabolism repression effect, providing a stable genetic basis for the subsequent high-yield phenotype.

[0033] In this embodiment, preferably, the homology repair template in step 3 contains a 1000bp homology arm, achieving a homology recombination efficiency of 75±5%. This operation significantly improves the efficiency and accuracy of homology recombination repair, ensuring precise insertion of the Pg32 promoter and accurate deletion of the creA-152 fragment. The longer homology arm facilitates RecA-mediated homology pairing and strand exchange, significantly reducing the risk of non-specific recombination or random integration, thereby ensuring the correctness of the edited site sequence and providing crucial assurance for obtaining high-yield strains with the desired genetic modification.

[0034] In this embodiment, preferably, the optimized fermentation process in step 4 involves culturing in M9 medium containing 8% glucose at 37°C for 48 hours, followed by HPLC detection of γ-PGA yield. This operation significantly improves the efficiency of γ-PGA yield detection and process stability, achieving an accumulation of up to 15±2 g / L of γ-PGA. The product concentration is precisely quantified by HPLC, ensuring reliable verification of the high-yield phenotype of the edited strain. At the same time, it optimizes carbon source utilization and cell growth kinetics, laying a solid data foundation for subsequent large-scale industrial production.

[0035] In this embodiment, preferably, the biosensor in step 4 is a γ-PGA-specific sensing system based on a luciferase reporter gene, with a detection threshold of 0.1 g / L. This operation enables high-throughput and sensitive γ-PGA detection, significantly improving screening speed and accuracy, ensuring reliable verification of the high-yield phenotype of the edited strain, and simultaneously monitoring the dynamic changes in the fermentation process in real time, providing immediate feedback for process optimization.

[0036] In this embodiment, preferably, the screening criterion for high-yield strains in step 4 is a γ-PGA yield of 15±2 g / L. This operation can ensure reliable verification of the high-yield phenotype of the edited strain, while providing a clear quantitative benchmark for large-scale screening, significantly improving the efficiency of strain development and reducing the false positive rate, and finally obtaining a genetically stable γ-PGA high-yield engineered strain.

[0037] In this embodiment, preferably, the initial screening of high-yield strains in step 4 is carried out in M9 medium containing 8% glucose and cultured at 37°C for 24 hours. Strains with a fluorescence intensity ≥5000 AU detected by a biosensor are identified as positive in the initial screening. This operation enables efficient and rapid preliminary identification of positive strains, significantly shortens the screening cycle and reduces the false positive rate, ensuring early and reliable verification of the high-yield phenotype. At the same time, combined with the sensitive response of the biosensor, a highly reliable candidate library is provided for subsequent precise rescreening, optimizing the overall strain development process and supporting dynamic adjustment of process parameters, thereby accelerating the acquisition of genetically stable γ-PGA high-yield engineered strains.

[0038] Working principle and usage process of this invention: The working principle and usage of this invention are as follows: Based on the design principle of the CRISPR-Cas9n dual-nickel enzyme system, two adjacent single-stranded DNA nicks (approximately 20-50 bp apart) are generated through the Cas9n-D10A mutant, forming a simulated double-strand break (DSB), thereby activating the host cell's inherent repair mechanisms—non-homologous end joining (NHEJ) or homologous targeted repair (HDR). This dual-nickel strategy significantly reduces the risk of off-target effects because a single nick can rapidly repair through base excision without introducing mutations, while the synergistic nicks ensure that the DSB occurs precisely only at the target site, promoting high-fidelity editing. Simultaneously, by synchronously introducing homologous repair templates (such as the Pg32 promoter or the ΔcreA-152 deletion sequence), the HDR pathway is guided to achieve targeted recombination, precisely inserting, deleting, or replacing the target gene, effectively avoiding metabolic interference and enhancing γ-PGA synthesis efficiency. The entire system utilizes the IPTG-induced expression mechanism to ensure the controllable activation of the Cas9n protein within the host cell, minimizing non-specific damage. Combined with a temperature-sensitive plasmid removal system, it efficiently removes exogenous vectors, ensuring the stability and reproducibility of the edited genome.

[0039] In the usage process, users need to perform the following operations in sequence: First, optimize the design of dual sgRNA sequences (such as targeting the pgsB promoter region and the creA coding region) using the CRISPRdirect tool and construct the pET-Cas9n-sgRNA expression cassette; second, introduce the recombinant plasmid into the pre-edited Bacillus subtilis host using a 30% PEG8000-mediated protoplast transformation method, screen for positive transformants in LB medium containing 10 μg / mL erythromycin, and induce Cas9n expression to initiate genome editing; subsequently, simultaneously introduce the homology repair template (containing a 1000bp homologous arm), eliminate temperature-sensitive plasmids by culturing at 42°C for 24 hours, and verify the editing accuracy (such as Pg32 insertion and ΔcreA-152 deletion) using high-fidelity PCR and Sanger sequencing; finally, under optimized fermentation conditions (such as M9 medium containing 8% glucose, 37°C, pH 7.0), perform high-throughput screening in 96-well plates using a luciferase-based biosensor (fluorescence intensity ≥5000). AU was initially screened as positive, and high-yielding strains with γ-PGA production of 15±2 g / L were selected. The process stability was verified through batch experiments in a 5L fermenter. This method is suitable for industrial-scale strain development. Users can adjust parameters (such as sgRNA target or culture medium composition) according to specific needs to achieve rapid and accurate construction and application of high-yielding γ-PGA strains.

[0040] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for precise editing of high-yield γ-PGA strains based on CRISPR-Cas9n, characterized in that, Includes the following steps: Step 1, Target gene sgRNA design and vector construction: Targeting key gene clusters for γ-PGA synthesis and metabolic regulatory genes, double sgRNA sequences were designed, and the double sgRNA and Cas9n-D10A mutant gene were cloned into the pET plasmid vector using the CRISPR-Cas9n double nickase system to form the pET-Cas9n-sgRNA1-sgRNA2 expression cassette. Step 2, Efficient Delivery and Genome Editing: Recombinant plasmids were introduced into Bacillus subtilis hosts via PEG-mediated protoplast transformation. Positive transformants were screened in LB medium containing erythromycin, and Cas9n expression was induced to generate targeted DNA double-strand cuts. Step 3, Source Recombination Repair and Editing Validation: The homology repair template was imported simultaneously, the CRISPR vector was removed using a temperature-sensitive plasmid elimination system, the target gene was amplified by PCR, and Sanger sequencing was used to confirm the insertion of the Pg32 promoter and the deletion of ΔcreA-152. Step 4: Screening and Fermentation Validation of High-Yield Strains: High-throughput screening of strains that enhance γ-PGA production was conducted using biosensors, and the fermentation process was optimized.

2. The method for precise editing of high-yield γ-PGA strains based on CRISPR-Cas9n according to claim 1, characterized in that, The dual sgRNA sequence in step 1 targets the promoter region of the pgsB gene and the coding region of the creA gene, has a length of 20 nt, and the GC content is controlled between 45-55%.

3. The method for precise editing of high-yield γ-PGA strains based on CRISPR-Cas9n according to claim 1, characterized in that, The transformation conditions in step 2 are as follows: 30% PEG8000, heat shock at 37℃ for 5 minutes, transformation efficiency of 80±5%, and erythromycin in LB medium of 10μg / mL.

4. The method for precise editing of high-yield γ-PGA strains based on CRISPR-Cas9n according to claim 1, characterized in that, The host was Bacillus subtilis strain 168, whose genome had been pre-edited to remove endogenous restriction systems.

5. The method for precise editing of high-yield γ-PGA strains based on CRISPR-Cas9n according to claim 1, characterized in that, The template in step 3 includes: a strong promoter of the pgsB gene (Pg32 replacement) and a partially deleted sequence of the creA gene (ΔcreA-152).

6. The method for precise editing of high-yield γ-PGA strains based on CRISPR-Cas9n according to claim 1, characterized in that, The homologous repair template in step 3 contains a 1000bp homologous arm and the homologous recombination efficiency reaches 75±5%.

7. The method for precise editing of high-yield γ-PGA strains based on CRISPR-Cas9n according to claim 1, characterized in that, The optimized fermentation process in step 4 involves culturing in M9 medium containing 8% glucose at 37°C for 48 hours, followed by HPLC detection of γ-PGA yield.

8. The method for precise editing of high-yield γ-PGA strains based on CRISPR-Cas9n according to claim 1, characterized in that, The biosensor in step 4 is a γ-PGA-specific sensing system based on the luciferase reporter gene, with a detection threshold of 0.1 g / L.

9. The method for precise editing of high-yield γ-PGA strains based on CRISPR-Cas9n according to claim 1, characterized in that, The screening criterion for high-yielding strains in step 4 is that the γ-PGA yield reaches 15±2 g / L.

10. The method for precise editing of high-yield γ-PGA strains based on CRISPR-Cas9n according to claim 1, characterized in that, In step 4, the initial screening of high-yielding strains was carried out in M9 medium containing 8% glucose and cultured at 37°C for 24 hours. Strains with a fluorescence intensity ≥5000 AU detected by a biosensor were identified as positive in the initial screening.

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