A method for constructing a chimeric mutant of Serpin5 that produces highly active antimicrobial peptides in two-spotted crickets

Abstract

The present invention discloses a method for constructing a chimeric mutant of Serpin5 in the two-spotted cricket that produces highly active antimicrobial peptides, belonging to the field of genetic engineering technology. The present invention discloses an sgRNA for knocking out the Serpin5 gene, the nucleotide sequence of which is shown as SEQ ID NO. 25. The present invention utilizes CRISPR / Cas9 gene editing technology to knock out specific sites in the Serpin5 gene in the two-spotted cricket, thereby enhancing the antimicrobial activity of antimicrobial peptides in the cricket. This method provides theoretical support and technical ideas for improving the quality of antimicrobial peptides and promoting the industrialization of high-quality antimicrobial peptides.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a method for constructing a Serpin5 chimeric mutant cricket capable of producing highly active antimicrobial peptides. Background Art

[0002] In some countries and regions (especially developing countries), the lack of effective medical supervision often leads to patients misusing or overdosing on antibiotics. Similarly, in animal husbandry, antibiotics are often used as growth promoters and preventive drugs in feed, which not only leads to the proliferation of drug-resistant bacteria in animals but can also affect human health through the food chain. The above-mentioned misuse of antibiotics not only promotes bacterial resistance but also poses a serious threat to public health. The World Health Organization (WHO) has listed antibiotic resistance as one of the most pressing global public health threats, predicting that by 2050, 10 million people will die annually from drug-resistant infections. Therefore, strengthening the research and development of alternative therapies to antibiotics is one of the key measures to combat antibiotic resistance.

[0003] Antimicrobial peptides (AMPs), as novel antimicrobial agents, are emerging as effective alternatives to antibiotics due to their unique mechanism of action and broad spectrum of antimicrobial activity. AMPs exhibit potent antimicrobial activity against a wide range of pathogens, including bacteria, fungi, and certain viruses, potentially offering a wider range of applications than traditional antibiotics. In medicine, AMPs can be used to treat various infections, such as skin, respiratory, and intestinal infections. In the food industry, AMPs act as natural preservatives, extending the shelf life of foods and preventing the growth of pathogens, thereby meeting consumer demand for healthy and safe food. In agriculture, AMPs act as plant protectants, helping crops remain pathogen-free, reducing reliance on chemical pesticides and improving agricultural sustainability. In animal husbandry, AMPs can be used as feed additives to prevent and control infections in animal populations. AMPs also possess immunomodulatory properties, helping to boost overall immunity and enhance resistance to various diseases.

[0004] Antimicrobial peptides are a class of short peptides with antimicrobial activity that are widely found in nature, including animals, plants, insects, amphibians, and microorganisms. As a traditional insect, the two-spotted cricket (Cricketus striata) has increasingly demonstrated potential as a new insect resource due to its high nutritional value, robust growth and reproductive capacity, high environmental adaptability, and eco-friendly environment. Modifying the synthesis pathway of two-spotted cricket antimicrobial peptides through genetic engineering will significantly advance the industrialization of antimicrobial peptides. Summary of the Invention

[0005] The present invention aims to provide a method for constructing a chimeric mutant of Serpin5 in the two-spotted cricket (Serpin5) that produces highly active antimicrobial peptides, thereby addressing the aforementioned problems in the prior art. This method utilizes CRISPR / Cas9 gene editing technology to knock out specific sites in the Serpin5 gene in the two-spotted cricket, thereby enhancing the antimicrobial activity of the antimicrobial peptides in the cricket. This method provides theoretical support and technical ideas for improving the quality of antimicrobial peptides and promoting the industrialization of high-quality antimicrobial peptides.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an sgRNA for knocking out the Serpin5 gene, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO.25.

[0008] Preferably, the method for preparing the sgRNA comprises the following steps:

[0009] (1) Using the PAC-sgRNA-Cas9 plasmid as a template, amplify the primer pair shown in SEQ ID NO. 22-23 to obtain the crRNA / tracrRNA fragment;

[0010] (2) Using the crRNA / tracrRNA fragment as a template, amplify using the primer pair described in SEQ ID NO.23-24 and the primer pair shown in SEQ ID NO.16-17 to obtain a DNA fragment of gRNA;

[0011] (3) Using the DNA fragment of the gRNA as a template, in vitro transcription is performed to obtain the sgRNA.

[0012] The present invention also provides the use of the above-mentioned sgRNA in preparing a reagent for knocking out the Serpin5 gene or reducing the expression level of the Serpin5 gene.

[0013] The present invention also provides a reagent for knocking out the Serpin5 gene, which comprises the above-mentioned sgRNA and Cas9 protein.

[0014] Preferably, the mass ratio of the sgRNA to the Cas9 protein is 2:3.

[0015] The present invention also provides the use of the above reagent in improving the antibacterial activity of antimicrobial peptides in two-spotted crickets, or constructing a Serpin5 chimeric mutant two-spotted cricket that produces highly active antimicrobial peptides.

[0016] The present invention also provides a method for improving the antibacterial activity of antimicrobial peptides in Cricket bispot, comprising the following steps:

[0017] (a) injecting the above reagent into two-spotted cricket eggs, incubating and culturing them to obtain the G0 generation of two-spotted cricket mutants;

[0018] (b) The G0 generation of the two-spot cricket mutant is mated with a wild-type two-spot cricket, and after incubation, culture and identification, the G1 generation of the two-spot cricket chimeric mutant is screened. The antimicrobial activity of the antimicrobial peptide in the G1 generation of the two-spot cricket chimeric mutant is higher than that in the wild-type two-spot cricket.

[0019] The present invention also provides a method for constructing a Serpin5 chimeric mutant cricket that produces highly active antimicrobial peptides, comprising the following steps:

[0020] (i) injecting the above reagent into two-spotted cricket eggs, incubating and culturing them to obtain the G0 generation of two-spotted cricket mutants;

[0021] (ii) The G0 generation of the two-spotted cricket mutant is mated with the wild type, and after incubation, culture and identification, a Serpin5 chimeric mutant two-spotted cricket that produces highly active antimicrobial peptides is obtained.

[0022] The present invention discloses the following technical effects:

[0023] The production of antimicrobial peptides is regulated by the Toll signaling pathway of insect humoral immunity. The Toll pathway is activated by a cascade of serine proteases. Serpins, a family of serine protease inhibitors, inhibit serine proteases. Therefore, Serpins negatively regulate the Toll signaling pathway, inhibiting the production of antimicrobial peptides. Serpin5, a member of the Serpin family, also negatively regulates the production of antimicrobial peptides. This study utilizes CRISPR / Cas9 gene editing technology to knock out specific sites in the Serpin5 gene of the two-spotted cricket (Cricket cricket), achieving a knockout efficiency of 75%. This method can generate mutant chimeric Crickets and enhance the antimicrobial activity of their antimicrobial peptides. This approach provides theoretical support and technical insights for improving the quality of antimicrobial peptides and promoting the industrialization of high-quality antimicrobial peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 The exon structure of Serpin5 gene and the distribution of sgRNA-014 sites;

[0026] Figure 2 The results of in vitro site gene editing efficiency verification are shown, where A is a schematic diagram of the gene editing process at the sgRNA-014 site, and B is the in vitro enzyme digestion efficiency test results at each site;

[0027] Figure 3 The results are for optimizing the microinjection parameters of the two-spotted cricket.

[0028] Figure 4 Verify the gene editing efficiency at different sites in vivo and generate sequencing peaks;

[0029] Figure 5 Flowchart for the construction and screening of Serpin5 knockout chimeric mutants in the two-spotted cricket;

[0030] Figure 6 The inhibitory effects of different two-spot cricket enzymatic peptides on Escherichia coli and Staphylococcus aureus are shown in Figure 1, where A represents the inhibition zones of different two-spot cricket enzymatic peptides on Escherichia coli, and 1-6 represent pH=3, 1 mol / L sodium chloride solution, 0.05 mg / mL ampicillin solution, wild-type two-spot cricket female enzymatic peptide solution, wild-type two-spot cricket male enzymatic peptide solution, Serpin5 knockout chimeric mutant two-spot cricket female enzymatic peptide solution, and Serpin5 knockout chimeric mutant two-spot cricket male enzymatic peptide solution; B represents the inhibition zones of different two-spot cricket enzymatic peptides on Staphylococcus aureus, and 1-6 represent pH=3, 1 mol / L sodium chloride solution, 0.1 mg / mL ampicillin solution, wild-type female two-spot cricket enzymatic hydrolysis peptide solution, wild-type male two-spot cricket enzymatic hydrolysis peptide solution, Serpin5 knockout chimeric mutant female two-spot cricket enzymatic hydrolysis peptide solution, Serpin5 knockout chimeric mutant male two-spot cricket enzymatic hydrolysis peptide solution; C is the statistical results of the inhibition zone diameters of different two-spot cricket enzymatic hydrolysis peptides against Escherichia coli and Staphylococcus aureus. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Example 1

[0037] 1. sgRNA synthesis and verification of in vitro enzyme cleavage activity

[0038] (1) sgRNA site design

[0039] The mRNA sequence and whole genome sequence of Serpin5 of two-spotted cricket were obtained by Blast tool on NCBI official website, and sequence alignment analysis was performed using SnapGene to mark exons and introns. Analysis of the genome sequence of GbSerpin5 revealed that the gene is 11388930bp in length and contains 7 exons (see Figure 1 Log in to the E-CRISP website, use the 1-3 exon sequences of the Serpin5 gene as the design range, screen the sgRNA target sites online, and select the top three sequences with high scores and few off-target sites on the third exon as target sequences based on the comprehensive score. The target sequence information is shown in Table 1. Taking the sgRNA-014 site as an example, its distribution position is shown in Figure 1 .

[0040] Table 1 GbSerpin5 gene target sequence

[0041]

[0042] (2) Design of specific genome amplification primers containing target sequences

[0043] Based on the location of each sgRNA site in the genome, SnapGene analysis was used on the approximately 400 bp genomic sequence containing the sgRNA target sequence to design specific nested PCR amplification primers (primer sequence information is shown in Table 2) to amplify the genomic sequence containing the target sequence.

[0044] Table 2 Primers for nested PCR amplification of GbSerpin5 target gene sequence

[0045]

[0046] (3) Design of sgRNA synthesis primers

[0047] Forward and reverse oligonucleotides were designed and assembled by PCR with 80 bp crRNA / tracrRNA fragments to generate the gRNA DNA template. The sgRNA synthesis primer sequence information is shown in Table 3.

[0048] Table 3 sgRNA synthesis primers

[0049]

[0050] (4) Cas9 / sgRNA in vitro enzyme digestion verification

[0051] a. PCR amplification to obtain specific genomic fragments containing target sequences

[0052] The genome of the two-spotted cricket was extracted using a genome extraction kit. Using the genome as a template, the target gene fragments were amplified using the specific nested first-round primers F1 / R1 listed in Table 2 (see Table 4 for the first-round PCR system and Table 5 for the PCR program). The first-round amplification product was used as a template for the second-round amplification of the target gene fragments using the F2 / R2 primers listed in Table 2 (see Table 6 for the second-round PCR system and Table 7 for the PCR program). PCR products were analyzed on a 1% agarose gel to determine the size of the amplified bands. Once the band size matched the expected value, the target gene fragments amplified in the second round were recovered using a Gel Extraction Kit to obtain the target sequences for GbSerpin5-014, GbSerpin5-193, and GbSerpin5-261.

[0053] Table 4 PCR system for the first amplification of target gene sequences

[0054]

[0055] Table 5 Target gene amplification first amplification procedure

[0056]

[0057] Table 6 PCR system for the second amplification of target gene sequences

[0058]

[0059] Table 7 Target gene amplification second amplification program

[0060]

[0061] b. Amplification of 80 bp crRNA / tracrRNA fragments

[0062] Using the plasmid (PAC-sgRNA-Cas9) as a template, amplification was performed using the upstream primers crRNA / tracrRNA-F and the downstream primer Universal-R shown in Table 8. An 80 bp fragment was obtained, which was recovered using a gel extraction kit and, after quantification, diluted to 50 ng / μL for later use. The PCR amplification system and procedure are shown in Tables 9 and 10, respectively.

[0063] Table 8 Primer sequences

[0064]

[0065] Table 9 PCR amplification system

[0066]

[0067] Table 10 PCR amplification program

[0068]

[0069] c. Amplification of DNA fragments of gRNA

[0070] Using the 80 bp crRNA / tracrRNA fragment as a template, amplification was performed using the upstream primer Universal-F and downstream primer Universal-R shown in Table 11, as well as the sgRNA synthesis primers shown in Table 3. Before use, the sgRNA primers were diluted to 0.3 μM. A 50 μL reaction system (Table 12) was established in a 200 μL Eppendorf tube. Following the amplification protocol in Table 10, DNA fragments of gRNAs at the sgRNA-014, sgRNA-193, and sgRNA-261 sites were obtained, respectively. These fragments were purified using a gel extraction kit and quantified by electrophoresis to determine band size.

[0071] Table 11 Primer sequences

[0072]

[0073] Table 12 PCR amplification system

[0074]

[0075] d. In vitro transcription and purification of sgRNA

[0076] The T7 RiboMAX™ Express RNAi System kit was used to transcribe gRNA DNA fragments targeting the sgRNA-014, sgRNA-193, and sgRNA-261 sites in vitro. After transcription, 0.5 μL of the DNA fragments were run on a 2% agarose gel to verify single bands. sgRNA-014, sgRNA-193, and sgRNA-261 were purified using the GeneJET RNA Purification and Concentration Kit. The sequences of these fragments are shown in Table 13.

[0077] Table 13 sgRNA sequences

[0078]

[0079] e. In vitro enzyme digestion efficiency detection

[0080] The target sequences of GbSerpin5-014, GbSerpin5-193, and GbSerpin5-261 obtained in step (4) were used as DNA templates for in vitro enzyme digestion efficiency verification. The enzyme digestion reaction system is shown in Table 14. During the experiment, one experimental group (adding sgRNA and Cas9) and two control groups (adding sgRNA without adding Cas9 and adding Cas9 without adding sgRNA) need to be set up; incubate at 37°C for 2 h in a PCR instrument; and detect enzyme digestion efficiency on a 2% agarose gel. Taking the sgRNA-014 site as an example, the gene editing process is shown in the following figure. Figure 2 The results of in vitro enzyme digestion efficiency test at each site are shown in A. Figure 2 As shown in B, through grayscale value calculation, it was found that the in vitro cutting efficiency of the sgRNA-014 site was 0.99, the in vitro cutting efficiency of the sgRNA-193 site was 0.99, and the in vitro cutting efficiency of the sgRNA-261 site was 0.99.

[0081] Table 14 Cas9 / sgRNA in vitro enzyme digestion system

[0082]

[0083] 2. Verification of the in vivo knockout efficiency of Serpin5 in two-spotted crickets

[0084] (1) Optimization of microinjection parameters for Cricketus bimaculata

[0085] Microinjection was performed at injection pressures of 200, 300, and 400 hPa (PI), injection times of 0.5 and 1 s (TI), and reflux pressures of 10 and 20 hPa (PC). 100 eggs were injected for each parameter combination, and the number of hatched eggs was counted to calculate the survival rate. Figure 3 As shown in the figure (the horizontal axis is "injection pressure, injection time, and reflux pressure"), an injection pressure of 300 hPa, an injection time of 0.5 s, and a reflux pressure of 10 hPa were finally selected for microinjection of the two-spotted cricket.

[0086] (2) Acquisition of RNP complex

[0087] Mix 3 µL of Cas9 protein (300 ng / µL) with 2 µL of sgRNA (300 ng / µL) obtained in step (1) to obtain 5 µL of RNP complex for injection.

[0088] (3) Detection of in-ovo mutation efficiency

[0089] The optimal injection parameters were selected to inject the RNP complex into the eggs of the two-spotted cricket, and 90 two-spotted cricket eggs were injected for each sgRNA site. On the 5th day after the injection, 20 samples were selected from each site and divided into 4 groups, each containing 5 samples. The genome of the egg to be tested was extracted, and the extracted genome was used as a template. The target gene fragment was amplified by nested PCR according to the primers in Table 2 and the system and procedures in Tables 4-7. The mutation efficiency was analyzed by electrophoresis detection and sequencing. The results showed that there were 3 groups with overlapping peaks when the sgRNA-014 site was injected, and there was no impurity peak in 1 group, with a mutation efficiency of 75%; there were 2 groups with overlapping peaks when the sgRNA-193 site was injected, and there were no impurity peaks in 2 groups, with a mutation efficiency of 50%; there was 1 group with overlapping peaks when the sgRNA-261 site was injected, and there were no impurity peaks in 3 groups, with a mutation efficiency of 25% (as shown in Table 2). Figure 4 Therefore, the sgRNA-014 site was selected for subsequent experiments.

[0090] 3. Obtaining a Serpin5 knockout chimeric mutant in the two-spotted cricket

[0091] The remaining eggs injected with the sgRNA-014 locus were cultured until first-instar nymphs hatched. The hatched first-instar nymphs were collected and reared in disposable plastic boxes. When the nymphs reached the sixth instar, three were individually housed in culture cups. Antennae were cut into approximately 1 cm lengths using dissecting scissors and placed in 1.5 mL centrifuge tubes. The genome was obtained using alkaline lysis: 45 μL of 50 mM NaOH was added and lysed at 95°C for 5 minutes. Then, 5 μL of 1 M Tris-HCl (pH 9.5) was added. One μL of the template was used for nested PCR of the target gene fragment. The PCR product was sequenced by Sangon Biotech (Beijing) Co., Ltd. The sequencing primer sequence was 014-Serpin5-F2 (see Table 2).

[0092] According to the sequencing results, it was found that there were mutants of the G0 generation with base mutations and base deletions, and there were hybrid peaks at the target site. The G0 generation mutants were mated with wild-type, and the chimeric mutants of the G1 generation were identified and screened from their offspring. They were the two-spotted cricket Serpin5 knockout chimeric mutants. The process of screening the two-spotted cricket Serpin5 knockout chimeric mutants is shown in Figure 5 .

[0093] 4. Antibacterial activity detection of Serpin5 knockout chimeric mutant peptides from two-spotted crickets

[0094] (1) The peptide extraction process is as follows:

[0095] a. Sample pretreatment: Separate male and female G1 adults of the Serpin5 knockout chimeric mutant of the two-spotted cricket and freeze them at -80°C for 24 hours. Then, freeze-dry them in a vacuum freeze dryer for 24 hours. The resulting freeze-dried insects were crushed in a grinder. The resulting insect powder can be stored in a sealed refrigerator at -20°C.

[0096] b. Degreasing: Weigh 20 g of insect powder into a beaker, add 400 mL of anhydrous ethanol, seal with plastic wrap and pierce the container. Stir using a rotor and magnetic stirrer at room temperature for 1 hour. Centrifuge in a 50 mL centrifuge tube at 4000 rpm for 30 minutes at room temperature. Discard the supernatant. Repeat this three times. Remove all the precipitate from the centrifuge tube with a spoon, spread it flat on a Petri dish, and place it in a fume hood for 12 hours. After drying, remove it and store it in a refrigerator at -20°C.

[0097] c. Protein extraction: Take 1 g of defatted powder and add 20 mL of 1 mol / L NaCl solution. Stir in a 50°C water bath for 1 h. Centrifuge at 4000 rpm for 20 min. Filter the supernatant to obtain a protein solution, which can be stored in a 4°C refrigerator for short periods of time.

[0098] d. Adjust the pH of the protein solution to the optimal pH of 3 for enzymatic hydrolysis with 1 mol / L hydrochloric acid. Add 5000 U of acid protease and hydrolyze at the optimal temperature of 48°C for 1 hour. Then, inactivate the enzyme in a boiling water bath for 10 minutes. Cool to room temperature and centrifuge at 4000 rpm for 20 minutes. Pass the supernatant through a 0.45 μm filter to obtain the enzymatic polypeptide solution.

[0099] e. Prepare wild-type (WT) two-spotted cricket enzymatic peptide solution as a control group according to steps ad.

[0100] (2) The detection process of peptide yield is as follows:

[0101] Take 2 mL of enzymatic polypeptide solution, add an equal volume of 20% trichloroacetic acid solution, mix well and let it stand for 10 minutes. After the precipitate is precipitated, add 4 mL of biuret reagent, mix well and let it stand at room temperature for 30 minutes. Replace the supernatant with 1 mL of single distilled water and treat it in the same way as a blank control. Measure the absorbance at 540 nm. According to the standard curve, substitute △A determination (y, △A determination) into the formula to calculate the polypeptide concentration (x, mg / mL).

[0102] The peptide yield calculation formula is:

[0103]

[0104] Where:

[0105] W: polypeptide yield, in %; C1: polypeptide concentration, in mg / mL; V1: volume of the obtained enzymatic polypeptide solution, in mL; m: mass of the defatted powder raw material, in g; C2: protein concentration, in mg / mL; V2: volume of anhydrous ethanol added to the defatted powder raw material at a material-liquid ratio of 1:20, in mL; 100: conversion factor.

[0106] The results of peptide yield are shown in Table 15. There is no significant difference in the peptide yield of each sample.

[0107] Table 15 Yield of G1 generation chimeric mutant cricket polypeptides

[0108]

[0109] (3) The process of detecting peptide activity is as follows:

[0110] The antibacterial activity of the four enzymatic peptide solutions was tested, with Escherichia coli and Staphylococcus aureus as the test bacteria, and the concentration of each was 5×10 6CFU / mL; 11 mg / mL of four enzymatic hydrolyzed polypeptide solutions were used as drug solutions; 1 mol / L sodium chloride solution with the same pH (pH=3) was used as negative control; 0.05 mg / mL of ampicillin was used as positive control for Escherichia coli, and 0.1 mg / mL of ampicillin was used as positive control for Staphylococcus aureus. The antibacterial effect of the Serpin5 chimeric mutant enzymatic hydrolyzed polypeptide solution was then reflected by measuring the size of the inhibition zone and calculating the inhibition rate.

[0111] The results are shown in Table 16 and Figure 6 The four enzymatic hydrolyzed polypeptide liquids of two-spot crickets all had significant inhibitory effects on Escherichia coli and Staphylococcus aureus. Among them, the inhibitory effect of Serpin5 female two-spot cricket enzymatic hydrolyzed polypeptide on Escherichia coli was significantly better than that of the control group, and the inhibitory effect of Serpin5 two-spot cricket enzymatic hydrolyzed polypeptide on Staphylococcus aureus was significantly better than that of the control group, indicating that these enzymatic hydrolyzed polypeptide liquids had good inhibitory effects on Escherichia coli and Staphylococcus aureus.

[0112] Table 16 Inhibition zone diameter and inhibition rate of different enzymatic peptides against Escherichia coli and Staphylococcus aureus

[0113]

[0114] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A sgRNA for knocking out the Serpin5 gene, characterized in that The nucleotide sequence of the sgRNA is shown in SEQ ID NO.

25.

2. The sgRNA according to claim 1, characterized in that The method for preparing the sgRNA comprises the following steps: (1) Using the PAC-sgRNA-Cas9 plasmid as a template, amplify the primer pair shown in SEQ ID NO. 22-23 to obtain the crRNA / tracrRNA fragment; (2) Using the crRNA / tracrRNA fragment as a template, amplify using the primer pair described in SEQ ID NO.23-24 and the primer pair shown in SEQ ID NO.16-17 to obtain a DNA fragment of gRNA; (3) Using the DNA fragment of the gRNA as a template, in vitro transcription is performed to obtain the sgRNA.

3. Use of the sgRNA according to claim 1 or 2 in preparing a reagent for knocking out the Serpin5 gene or reducing the expression level of the Serpin5 gene.

4. A reagent for knocking out the Serpin5 gene, characterized in that The reagent comprises the sgRNA and Cas9 protein according to claim 1 or 2.

5. The reagent according to claim 4, characterized in that The mass ratio of the sgRNA to the Cas9 protein is 2:

3.

6. Use of the reagent according to claim 4 or 5 in improving the antimicrobial activity of antimicrobial peptides in two-spotted crickets, or in constructing a chimeric mutant Serpin5 of two-spotted crickets that produces highly active antimicrobial peptides.

7. A method for improving the antimicrobial activity of antimicrobial peptides in Cricket crickets, characterized in that: The steps include: (a) injecting the reagent according to claim 4 or 5 into two-spotted cricket eggs, incubating and culturing them to obtain two-spotted cricket mutants of the G0 generation; (b) The G0 generation of the two-spot cricket mutant is mated with a wild-type two-spot cricket, and after incubation, culture and identification, the G1 generation of the two-spot cricket chimeric mutant is screened. The antimicrobial activity of the antimicrobial peptide in the G1 generation of the two-spot cricket chimeric mutant is higher than that in the wild-type two-spot cricket.

8. A method for constructing a chimeric mutant of Serpin5 that produces highly active antimicrobial peptides in a two-spotted cricket, characterized in that: The steps include: (i) injecting the reagent according to claim 4 or 5 into two-spotted cricket eggs, incubating and culturing them to obtain two-spotted cricket mutants of the G0 generation; (ii) The G0 generation of the two-spotted cricket mutant is mated with the wild type, and after incubation, culture and identification, a Serpin5 chimeric mutant two-spotted cricket that produces highly active antimicrobial peptides is obtained.

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