CN114908101B - Aag2 cell line with beta-1,3-glucan binding protein gene knocked out, construction method and application thereof - Google Patents

Abstract

The application discloses an Aag2 cell line with beta-1,3-glucan binding protein gene knocked out, a construction method and application thereof. The construction method comprises the following steps: editing a beta-1,3-glucan binding protein gene in a mosquito cell based on a CRISPR/Cas9 system, so that the function of the beta-1,3-glucan binding protein gene is lost, and the mosquito cell with the beta-1,3-glucan binding protein gene knocked out is obtained; the nucleotide sequence of the beta-1,3-glucan binding protein gene is shown as a sequence 9 in a sequence table. The Aag2 cell line with the beta-1,3-glucan binding protein gene knocked out can be used as an in-vitro research cell model for research of interaction between mosquitoes and viruses and metabolism of the mosquitoes, and meanwhile, the success of gene editing of the mosquito cell line by the application also enables the gene editing of more non-model biological cells to be possible.

Description

Aag2 cell line with beta-1,3-glucan binding protein gene knocked out, construction method and application thereof

Technical Field

The application belongs to the technical field of biology, and particularly relates to an Aag2 cell line for knocking out a beta-1,3-glucan binding protein gene, a construction method and application thereof, and in particular relates to an Aag2 cell line for knocking out a beta-1,3-glucan binding protein gene constructed based on a CRISPR/Cas9 gene editing technology, a construction method and application thereof.

Background

Mosquito-borne diseases (mosquito-borne infections) are natural epidemic diseases transmitted by disease-borne mosquitoes, and with the unprecedented spread of mosquito-borne infections dengue and chikungunya fever history, outbreaks of yellow fever and the prevalence of village-card disease in latin america in 2015, the risk of insect-borne viruses of aedes is rapidly increasing, creating an increasingly serious challenge to global hygiene, threatening more than 40% of the world population. In order to cope with the increasingly serious problem, various innovative mosquito medium control technologies are being developed at home and abroad, and the disease medium control intervention measures play an increasingly important role.

The gene modification mainly adopts 2 technologies of ZFN (zinc finger endonuclease) and transcription activator-like effector nuclease based on eukaryotic transcription factors, but the 2 technologies are relatively limited due to the defects of complex design, lower efficiency and the like, and cannot be widely implemented. The CRISPR/Cas9 system is an adaptive immune system formed by bacteria and archaea during long-term evolution, and can form a specific defense mechanism against phage infection, plasmid binding and gene transfer caused by transformation. CRISPR technology has emerged from the 90 s of the 20 th century, and its first use in biochemical experiments was 7 years later, immediately after which it became the most widely used gene editing tool in many fields (e.g., microbiology, agriculture, human biology, etc.). Since 2012, the bacterial type II CRISPR-Cas9 system was modified into a genetic engineering tool, greatly expanding the ability to modify genomes, has been applied to various fields, and will play an increasingly important role in mosquito vector control. Functional genetic studies in mosquito cells are critical to understanding the viral and antiviral mosquito host factors and potential mosquito control strategies. However, there are few mutations in the genes of mosquito compared to model organisms.

Beta-1,3-glucan binding protein (βgbp) as a typical pattern recognition receptor is capable of recognizing and binding to pattern-associated molecules on the surface of pathogenic microorganisms, thereby activating the natural immune defense system of insects. Natural immunity of insects includes cellular immunity and humoral immunity. Cellular immunity mainly relies on pattern recognition receptors on the cell surface to recognize and combine pattern related molecules on the surface of pathogenic microorganisms, so that a serine protease cascade activation system is activated, and further a prophenoloxidase system is activated to induce blood cells to degranulate, melanin is generated, and encapsulation, nodule, phagocytosis and the like are carried out to remove pathogens. The humoral immunity mainly depends on the recognition and combination of the pattern recognition receptor and the pattern related molecules to activate three signal paths, thereby generating antibacterial peptide, active oxygen, nitric oxide and other effector molecules and achieving the purpose of eliminating the pathogens.

Disclosure of Invention

The application aims to provide an application of beta-1,3-glucan binding protein and related biological materials thereof in regulating and controlling mosquito or mosquito cell virus infection level, and provides an Aag2 cell line with beta-1,3-glucan binding protein gene knocked out constructed based on a CRISPR/Cas9 gene editing technology, and a construction method and application thereof.

In a first aspect, the application provides a method for preparing a mosquito cell with a beta-1,3-glucan-binding protein gene knocked out.

The preparation method of the beta-1,3-glucan binding protein gene knockout mosquito cell comprises the following steps: editing a beta-1,3-glucan binding protein gene in a mosquito cell based on a CRISPR/Cas9 system, so that the function of the beta-1,3-glucan binding protein gene is lost, and the mosquito cell with the beta-1,3-glucan binding protein gene knocked out is obtained; the nucleotide sequence of the beta-1,3-glucan binding protein gene is shown as a sequence 9 in a sequence table.

In the above method, the CRISPR/Cas9 system comprises an sgRNA targeting the β -1,3-glucan-binding protein gene;

the sgrnas include sgRNA1 and sgRNA2;

the target sequence identified by the sgRNA1 is shown as a sequence 2 in a sequence table;

the target sequence identified by the sgRNA2 is shown as a sequence 3 in a sequence table.

Further, the nucleotide sequence of the sgRNA1 is shown as a sequence 4 in a sequence table; wherein, the nucleotide sequence of the sgRNA1 is subjected to oxymethyl modification and thio modification from the first three nucleotides from the 5 'end to the last three nucleotides from the 3' end.

The nucleotide sequence of the sgRNA2 is shown as a sequence 5 in a sequence table; wherein, the nucleotide sequence of the sgRNA2 is subjected to oxymethyl modification and thio modification from the first three nucleotides from the 5 'end to the last three nucleotides from the 3' end.

Still further, the CRISPR/Cas9 system further comprises a Cas9 nuclease. The Cas9 nuclease utilizes the recognition and positioning of the sgRNA to complete the cutting of the beta-1,3-glucan binding protein gene, so that the beta-1,3-glucan binding protein gene in mosquito cells is broken, homologous recombination repair or non-homologous end connection occurs, and further, base frame shift mutation is caused to achieve the purpose of gene knockout.

The method further comprises the step of electrotransfecting the sgRNA and the Cas9 nuclease into a mosquito cell.

The ratio of the sgRNA1, the sgRNA2, and the Cas9 nuclease may specifically be 1.2nm:1.2nm:160 μg.

Furthermore, the amino acid sequence of the Cas9 nuclease is shown as a sequence 7 in a sequence table.

The mosquito cells may be aedes aegypti cells, in particular aedes aegypti cells, such as Aag2 cells.

In a specific embodiment of the present application, the method may specifically comprise the steps of: electrotransfer the mixed reagent containing the sgRNA and the Cas9 nuclease into mosquito cells to obtain electrotransferred cells; culturing the cells after electrotransformation to a proper state, screening monoclonal and subculturing; detecting the mutation condition of the beta-1,3-glucan binding protein gene in the monoclonal cell to obtain the mosquito cell with the beta-1,3-glucan binding protein gene knocked out. The beta-1,3-glucan binding protein gene knockdown has an increased level of viral infection (viral load) in the mosquito cell compared to the mosquito cell prior to editing. The virus is in particular dengue virus or Zika virus.

In a second aspect, the application provides a method of preparing a transgenic cell having an increased level of viral infection.

The preparation method of the transgenic cell with the improved virus infection level, which is protected by the application, comprises the following steps: knocking out DNA molecules shown in 247-746 positions corresponding to sequence 1 in beta-1,3-glucan binding protein genes in mosquito cells to obtain transgenic cells with the increased virus infection level.

Further, the increased level of viral infection may specifically be an increased viral load.

Further, the virus is in particular dengue virus or Zika virus.

The mosquito cells may be aedes aegypti cells, in particular aedes aegypti cells, such as Aag2 cells.

In a third aspect, the application provides a cell prepared according to any one of the methods described above.

In a fourth aspect, the application protects any of the following applications x 1) -x 7):

use of x 1) a beta-1,3-glucan-binding protein or a related biological material for modulating mosquito or mosquito cell virus infection levels;

x 2) use of a substance that inhibits β -1,3-glucan binding protein or a gene encoding the same for increasing the level of infection by a mosquito or mosquito cell virus;

x 3) use of a substance that inhibits β -1,3-glucan-binding protein or a gene encoding the same in the preparation of a mosquito cell or a transgenic cell with increased levels of viral infection that has been knocked out by β -1,3-glucan-binding protein;

x 4) use of the above method for the preparation of transgenic cells with increased levels of viral infection;

x 5) use of the above method for preparing a cell model for studying mosquito metabolism or mosquito-virus interaction or pesticide resistance;

x 6) use of cells prepared according to the above method as a cell model for studying mosquito metabolism or mosquito-virus interaction or pesticide resistance;

application of x 7) beta-1,3-glucan binding protein or a coding gene thereof as a target in preparing mosquito vector control products.

In the above application, the β -1,3-glucan-binding protein is a protein as described in any one of the following (a 1) to (a 4):

(a1) Protein shown in a sequence 8 in a sequence table;

(a2) A fusion protein obtained by connecting a tag to the N-terminal or/and the C-terminal of the protein of (a 1);

(a3) A protein with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues in the step (a 1);

(a4) A protein having 98% or more of identity with (a 1) and having the same function.

The related biological material is a nucleic acid molecule encoding beta-1,3-glucan binding protein or an expression cassette or a recombinant vector containing the nucleic acid molecule. The nucleic acid molecule for encoding the beta-1,3-glucan binding protein is shown as a sequence 9 in a sequence table.

The regulation and control of the infection level of the mosquito or mosquito cell virus is to inhibit the infection level of the mosquito or mosquito cell virus, and is specifically shown as follows: the mosquito or mosquito cell virus infection level is increased when the content and/or activity of the beta-1,3-glucan binding protein in the mosquito or mosquito cell is deleted, or the beta-1,3-glucan binding protein gene is knocked out.

In the above application, the substance that inhibits the β -1,3-glucan binding protein or the gene encoding the same may be an sgRNA or CRISPR/Cas9 system for editing the β -1,3-glucan binding protein gene;

the sgrnas for editing the β -1,3-glucan binding protein gene include sgRNA1 and sgRNA2; the target sequence identified by the sgRNA1 is shown as a sequence 2 in a sequence table; the target sequence identified by the sgRNA2 is shown as a sequence 3 in a sequence table;

the CRISPR/Cas9 system for editing a β -1,3-glucan binding protein gene comprises the sgRNA and Cas9 nuclease for editing a β -1,3-glucan binding protein gene.

In the above application, the level of viral infection may specifically be viral load.

In the above application, the virus is in particular dengue virus or Zika virus.

In the above application, the mosquito may be aedes aegypti, and in particular, aedes aegypti.

The mosquito cells may be aedes aegypti cells, in particular aedes aegypti cells, such as Aag2 cells.

The beneficial effects of the application are as follows:

1. the application constructs an Aag2 cell line with the beta-1,3-glucan binding protein gene knocked out by using the CRISPR/Cas9 technology for the first time, and the obtained beta-1,3-glucan binding protein gene knocked out line is helpful for researching intracellular genes, proteins or other functions, can be used as a cell model for mosquito metabolism research and pesticide resistance, can also provide a cell model for mosquito infection resistance pathogenic microorganisms, and can also provide a cell model for interaction of pathogenic microorganisms and mosquito vector hosts, which are influenced by related genes regulated by the beta-1,3-glucan binding protein gene.

2. Compared with the technical means such as gene silencing, interference and the like, the CRISPR/Cas9 gene knockout technology adopted by the application is more effective, and the technology is one of the gene therapy technologies with the most clinical and application prospects due to the advantages of strong operability, low cost, wide application range and the like.

3. The application utilizes two gRNAs to precisely and efficiently realize the knockout of the beta-1,3-glucan binding protein gene.

4. The electroporation transfection method adopted by the application is more suitable for gene editing of insect cells, a large number of gene knockout cell strains can be obtained by one-time electrotransformation, a large number of knockout cells can be obtained in a short time, the method has the advantages of high knockout rate and simple and easy operation, in-vitro research can be carried out by using the cells instead of mosquitoes, and the problems of long breeding period and the like are solved.

5. The monoclonal screening method adopted by the application is more efficient than the traditional dilution method, and can separate more monoclonal strains at one time.

The application firstly utilizes CRISPR/Cas9 technology to knock out a pattern recognition receptor, namely beta-1,3-glucan binding protein gene, in an inherent immune pathway of mosquito in an Aag2 cell to obtain an Aag2 cell line with the beta-1,3-glucan binding protein gene knocked out, and a subsequent functional verification experiment shows that: compared with a wild type Aag2 cell line, the Aag2 cell line with the beta-1,3-glucan binding protein knocked out has improved viral load after virus infection, which shows that the beta-1,3-glucan binding protein has the effect of inhibiting virus infection. The Aag2 cell line with the beta-1,3-glucan binding protein gene knocked out can be used as an in-vitro research cell model for research of interaction between mosquitoes and viruses and metabolism of the mosquitoes, and meanwhile, the success of gene editing of the mosquito cell line by the application also enables the gene editing of more non-model biological cells to be possible.

Drawings

FIG. 1 is a schematic diagram of monoclonal cell screening.

FIG. 2 shows the electrophoresis verification of all cell lines obtained by the present application.

FIG. 3 is a β -1,3-glucan-binding protein gene knockout strategy.

FIG. 4 shows the verification of a beta-1,3-glucan-binding protein gene knockout strain in Aag2 cells, namely a sequencing peak diagram of the knockout strain and comparison with a wild type sequence.

FIG. 5 is a flow chart of functional verification of the beta-1,3-glucan-binding protein gene.

FIG. 6 shows the change in virus levels after infection of a beta-1,3-glucan-binding protein gene knockout cell line with virus from a wild-type cell line. Wherein A is the change in virus level after dengue virus infection; b is the virus difference level of the Day3-Day6 wild strain and the knockout strain; c is the change of virus infection level after the Zika virus is infected; d is the virus difference level of the Day4-Day7 wild strain and the knockout strain.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The Aag2 cell line in the examples described below is described in document "Transcriptome analysis of Aedes aegypti Aag2 cells in response to dengue virus-2 in.

The amino acid sequence of the beta-1,3-glucan binding protein in the following examples is shown as a sequence 8 in a sequence table, and the gene sequence of the beta-1,3-glucan binding protein is shown as a sequence 9 in the sequence table.

Example 1 construction method of Aag2 cell line with beta-1,3-glucan-binding protein Gene knockout

1. sgRNA preparation

1. Design of beta-1,3-glucan binding protein gene amplification primers

The DNA sequence of the Aedes aegypti beta-1,3-glucan binding protein Gene (GNGBP 6; gene ID: 5568698) was obtained according to the NCBI database (https:// www.ncbi.nlm.nih.gov /), and a pair of beta-1,3-glucan binding protein Gene primers were designed according to the DNA sequence, and the primer sequences were as follows:

F1：5’-AGGTGCCAATTGACCAAACA-3’；

R1：5’-AACTGAATCCGCCAAGTCTC-3’。

2. PCR amplification of beta-1,3-glucan-binding protein gene

Extracting genomic DNA of a wild type Aag2 cell line, and carrying out PCR amplification by adopting the primers F1 and R1 designed in the step 1 to obtain a PCR product. The PCR reaction system is shown in Table 1, and the PCR reaction conditions are shown in Table 2.

TABLE 1 PCR reaction System

Component (A)	Dosage of
		PCR Mix (full gold)	12.5μl
F1	0.5μl
		R1	0.5μl
DNA	2μl
		dd H 2 O	9.5μl
Total volume of	25μl

TABLE 2 PCR reaction conditions

Sequencing the PCR product with clear and bright bands to obtain the Aag2 cell beta-1,3-glucan binding protein gene sequence.

3. Design of sgRNA target

Sequencing the amplified product, and comparing and adjusting the amplified product with the beta-1,3-glucan binding protein gene sequence in NCBI database to avoid the situation that the designed sgRNA sequence is not combined with the genome due to individual base mutation of cells in long-term passage. The two sgRNA guide sequences were finally selected by designing the sgRNA guide sequences on the first two exons using the bench site (http:// bench. Com /), and named sgRNA1 and sgRNA2, respectively, with the following target sequences:

sgRNA1 target sequence: 5'-GCCAAACATTTCTGTCCAGT-3' (SEQ ID NO: 2);

sgRNA2 target sequence: 5'-ATTGGTTGTGGCCTGCTCTG-3' (SEQ ID NO: 3).

4. Synthesis of sgRNA sequences

Artificially synthesizing the sgRNA1 and the sgRNA2, wherein the sgRNA1 and the sgRNA2 are specifically as follows:

sgRNA1：

mA mC mU mA mC mU GGACAGAAUGUUUUUUGGGUUUAGAUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUACAACUUUGAAAGUGGCACCGGAGUCGGUGGUGCumU mU (SEQ ID NO: 4). Wherein m is an oxymethyl modification and x is a thio modification.

sgRNA2：

mC mG mC mG AGCAGGCCACCAAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUAUCAACUUUUGAAAGUGGCACCGGAGUCGGUGGUGCumU mU (SEQ ID NO: 5). Wherein m is an oxymethyl modification and x is a thio modification.

2. Preparation of Aag2 cell line with beta-1,3-glucan-binding protein Gene knockout

1. Preparation before electric power conversion

1) Preparation of RNP Complex

RNP complex 1 (20 μl): is prepared by mixing 8 mu L Guide-it ^TM Recombinant Cas9 (10 μg/. Mu.L) (available from TAKARA Co., ltd., cat. No. 632678) and 12. Mu.LThe sgRNA1 solution (solvent, water, concentration, 0.1 nM/. Mu.L) was mixed well.

RNP complex 2 (20 μl): is prepared by mixing 8 mu L Guide-it ^TM Recombinant Cas9 (10. Mu.g/. Mu.L) was mixed with 12. Mu.L of sgRNA2 solution (water as solvent at a concentration of 0.1 nM/. Mu.L).

After mixing each of the RNP complex 1 and the RNP complex 2 for 10 minutes, both were thoroughly mixed for 5 to 10 minutes to obtain an RNP complex (40. Mu.L).

2) Preparation of cells to be electrotransferred

Wild type Aag2 cells were washed once with PBS and cell pellet was collected for electrotransfection with a cell number of 5X 10 ⁴ -1×10 ⁶ And each.

3) Preparation of an electrotransport buffer

Electrotransport buffer (total volume 40. Mu.L) was prepared by adding 32.8. Mu. L P3 to the Primary CellSolution and Supplement 1.2. Mu.L were mixed. P3 Primary Cell->Both Solution and supply 1 were from the Lonza electrotransformation kit P3 Primary Cell 4D-nucleic acid selector ^TM X Kit S, product number is V4XP-3032.

2. Electric rotating device

And (3) mixing the prepared electrotransfer buffer with the RNP compound, then re-suspending the cells to be electrotransferred, placing the cells in an electrotransfer cup of a Lonza electrotransfer instrument, and obtaining the cells after electrotransfer after electric shock for 5 minutes by adopting a K562 electrotransfer program in the electrotransfer instrument. Transferring and inoculating the electrotransformed cells into a small dish to be cultured by using SDM culture medium containing 10% FBS (bovine serum albumin), wherein the FBS is Fetal Bovine Serum of Gibco, and the product number is 10099-141C; SDM medium was Gibco Schneider's Drosophila Medium medium, product number 21720024.

3. Picking monoclonal cell strain (96-well plate single sorting)

When the cells grow to be full of 80% -90% of the bottom, picking single cells into a 96-well plate under a microscope, picking single clones as far as possible from each well, and detecting whether the clones are single or not later.

4. Expansion culture

And (4) digesting the cells when the cells in the 96-well plate grow to be over 80% of the bottom of the plate, transferring the cells into a 24-well plate for expansion culture, and periodically replacing fresh culture medium during the expansion culture.

Digesting the cells when the cells in the 24-well plate grow to be more than 80% of the bottom of the 24-well plate, and extracting and sequencing half of the cells to verify the DNA; the other half of the cells are transferred into a 12-well plate for expansion culture.

And (4) digesting the cells when the cells in the 12-well plate are fully paved at the bottom 80%, and transferring the cells into a 6-well plate for expansion culture.

Transferring into a cell culture bottle after the cells in the 6-hole plate are paved at more than 80% of the bottom, and freezing the cells after the number is enough. A schematic of monoclonal cell selection is shown in FIG. 1.

3. Sequencing verification of beta-1,3-glucan binding protein gene knockout Aag2 cell line

Extracting cell DNA, detecting mutation condition of beta-1,3-glucan binding protein gene sequence in monoclonal cells by PCR amplification, amplifying the primer and the reaction system by the PCR, carrying out the same steps 1 and 2 with the same conditions, and sequencing the PCR product. And comparing the sequencing result with the beta GBP gene sequence, so as to judge whether the single cell clone is a beta GBP gene knockout cell strain.

The results show that: the application selects 74 cell strains, which are respectively named as No. 1-74, and the specific mutation conditions are as follows: no. 63, no. 64, no. 65, no. 66, no. 68, no. 69, 6 cell lines were sequenced without signals; 29 cell lines of 2, 8, 9, 10, 16, 17, 18, 23, 25, 26, 27, 31, 39, 44, 45, 46, 47, 48, 51, 52, 53, 54, 56, 57, 58, 59, 61, 70 and 73 are multicellular mixed clones; the total 9 cell strains of No. 33, no. 37, no. 38, no. 40, no. 42, no. 50, no. 55, no. 60 and No. 71 are monoclonal cell strains, but the sequencing sequences of the cell strains can only be partially matched with the target gene sequences; the total 3 cell strains of No. 4, no. 15 and No. 21 are wild monoclonal cell strains; 10 cell lines of No. 1, no. 7, no. 12, no. 19, no. 22, no. 34, no. 41, no. 43, no. 62 and No. 74 are monoclonal cell lines, and the base is deleted by 499bp, and the base 381bp deleted on the exon is a multiple of 3, so that the 10 cell lines do not cause frame shift mutation although the base is deleted; 13 cell lines of No. 3, no. 5, no. 6, no. 13, no. 14, no. 20, no. 28, no. 29, no. 32, no. 35, no. 49, no. 67 and No. 72 lack 500bp of base, and the 13 cell lines are monoclonal cell lines which successfully cause frame shift mutation because the base 382bp deleted on an exon is a multiple of non-3; in addition, the cell strain No. 30 respectively lacks 12bp and 3bp near two target points; the No. 36 monoclonal cell strain is deleted by 41bp and 12bp near two target points respectively; the 11 # monoclonal cell line inserts 513bp of base and lacks 501bp of base; the 24 # monoclonal cell line inserts base 9bp and lacks base 501bp. The electrophoresis verification of all cell lines obtained by the application is shown in FIG. 2. The deletion or insertion of a base was occurred in 27 of the total of 74 cell lines, and the editing efficiency (number of cell lines changed by a base/total cell line number) was 36.5%.

Wherein, compared with the wild type Aag2 cell, the monoclonal cell strain No. 3 (designated as a knockout strain) differs only in that a 500bp base deletion occurs on the βGBP gene, and the deletion bases are located at positions 247 to 746 of the sequence 1. The mutated beta GBP gene sequence in the knocked-out strain is shown as a sequence 6 in a sequence table. The knockout strategy and sequence alignment of the knockout strain are shown in FIG. 3.

Example 2 detection of Virus level after infection of the Virus with the Aag2 cell line with the beta-1,3-glucan-binding protein Gene knockout

Test cells: wild type Aag2 cells, beta-1,3-glucan-binding protein gene knockout strain constructed in example 1.

The experimental method comprises the following steps: after the test cells were cultured to a sufficient amount with SDM medium containing 10% fetal bovine serum, they were cultured in a 1X 10 medium ⁶ Each was added to T25 cell flasks at 5ml per flask and cultured in a 28 ℃ cell incubator for 12h to infect dengue virus at moi=0.01 or to infect zika virus at moi=0.001, each set was provided with 4 biological replicates. Cell supernatants were taken 200 μl daily after infection for viral load detection. Dengue hands-free kit and village card hands-free kit of Beijing Mei-Leibo company are used for the viral load of the supernatantAnd (5) detecting. The reaction system is shown in Table 3, and the reaction conditions are shown in Table 4.

TABLE 3 reaction System

2 Xdengue II or Zika amplification reaction solution	10μL
		Dengue II or Zika primer probe mixed solution	1μL
RT-PCR enzyme mixed solution	1μL
		ddH 2 O	4μL
Cell supernatant to be examined	4μL

TABLE 4 reaction conditions

The results are shown in FIG. 6. After dengue virus infection, the wild strain and the knockout strain have the same virus replication trend, the virus copy number is gradually increased, and the virus copy number of the knockout strain is higher than that of the wild-type cell virus on the 3 rd day to the 5 th day, and the specific difference is shown in fig. 6A and 6B. After the Zika virus infection, the wild strain and the knockout strain have the same virus replication trend, the virus copy number is gradually increased, and the virus copy number of the knockout strain is higher than that of the wild cell virus on the 4 th day to the 6 th day, and the specific difference is shown in fig. 6C and 6D.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<110> military medical institute of the military academy of China's civil liberation army

<120> Aag2 cell line with beta-1,3-glucan binding protein gene knocked out, construction method and application thereof

<160> 9

<170> PatentIn version 3.5

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ccgactggac agaaatgttt ggcagcatga aaattcgctt ggtggtggtg gcgtaagtta 300

tatttcaata tctaccacga atatttagca caatcaaggt tttatcatca taagaacaac 360

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acggttgcta tcgacgagga agtccagatc gtattctgaa cccagttcga agtgcacgac 660

tccgaacggt gaattcattc gccttcaaat atggaaaggt ggaaatcaat gctaagttgc 720

cacaaggaga ttggttgtgg cctgctctgt ggttgctccc aaaaggggat acctacggat 780

attggcccaa gtcaggtgaa gtggatctga tggagtcacg tggcaatcgg aatttcgtac 840

agaacaacga gaagatcgga atccagaagg tgtcatcgtg tctacatttc ggagacaacc 900

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acagatacca acttacttgg acgaaaaatg tcattcaatt tggagttaac gataggatat 1020

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agagtgaatt ttactgtagt gatgatacaa actcagaaaa gagtcgttca aaccgttggg 60

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ggattcagag ctccgaaagg tgaacatttg tagtacgaga cctgtgtaca tttacctcca 180

gtttattcat tgcataggtc gcatatgttc tggacagcta atttttcaag ataatttcaa 240

ccgactctgt ggttgctccc aaaaggggat acctacggat attggcccaa gtcaggtgaa 300

gtggatctga tggagtcacg tggcaatcgg aatttcgtac agaacaacga gaagatcgga 360

atccagaagg tgtcatcgtg tctacatttc ggagacaacc cgaatgtgcg cagttcccag 420

tgtggttctg ttagtggtaa tctttttggt gcaatgttta acagatacca acttacttgg 480

acgaaaaatg tcattcaatt tggagttaac gataggatat ttcgtactgt taccccttat 540

gagggtttct ggagacttgg cggattcagt t 571

<210> 7

<211> 1368

<212> PRT

<213> Artificial Sequence

<400> 7

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala

1010 1015 1020

Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe

1025 1030 1035

Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1040 1045 1050

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu

1055 1060 1065

Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val

1070 1075 1080

Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr

1085 1090 1095

Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys

1100 1105 1110

Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

1115 1120 1125

Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val

1130 1135 1140

Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys

1145 1150 1155

Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser

1160 1165 1170

Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys

1175 1180 1185

Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu

1190 1195 1200

Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly

1205 1210 1215

Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys

1250 1255 1260

His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys

1265 1270 1275

Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1280 1285 1290

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn

1295 1300 1305

Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala

1310 1315 1320

Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser

1325 1330 1335

Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1340 1345 1350

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1355 1360 1365

<210> 8

<211> 371

<212> PRT

<213> Artificial Sequence

<400> 8

Met Ile Gln Thr Gln Lys Arg Val Val Gly Thr Ile Gly Leu Leu Phe

1 5 10 15

Cys Val Val Gly Leu Ile Ser Gly Cys Lys Arg Ser Pro Thr Thr Ala

20 25 30

Ser Gly Phe Arg Ala Pro Lys Gly Arg Ile Cys Ser Gly Gln Leu Ile

35 40 45

Phe Gln Asp Asn Phe Asn Arg Leu Asp Arg Asn Val Trp Gln His Glu

50 55 60

Asn Ser Leu Gly Gly Gly Gly Asn Asn Glu Phe Gln Trp Tyr Ser Gly

65 70 75 80

Ser Gly Arg Asn Ser Tyr Ile Lys Asn Asn His Leu Tyr Ile Arg Pro

85 90 95

Thr Leu Thr Ser Asp Glu Tyr Gly Glu Ala Phe Leu Lys Ser Gly Val

100 105 110

Ile Asn Leu Asn Glu Gly Pro Gln Ser Gln Arg Cys Thr Asp Ala Pro

115 120 125

Gly Trp Ala Glu Gln Ile His Gly Cys Tyr Arg Arg Gly Ser Pro Asp

130 135 140

Arg Ile Leu Asn Pro Val Arg Ser Ala Arg Leu Arg Thr Val Asn Ser

145 150 155 160

Phe Ala Phe Lys Tyr Gly Lys Val Glu Ile Asn Ala Lys Leu Pro Gln

165 170 175

Gly Asp Trp Leu Trp Pro Ala Leu Trp Leu Leu Pro Lys Gly Asp Thr

180 185 190

Tyr Gly Tyr Trp Pro Lys Ser Gly Glu Val Asp Leu Met Glu Ser Arg

195 200 205

Gly Asn Arg Asn Leu Val Gln Asn Asn Glu Lys Ile Gly Ile Gln Lys

210 215 220

Val Ser Ser Cys Leu His Phe Gly Asp Asn Pro Asn Val Arg Ser Ser

225 230 235 240

Gln Cys Gly Ser Val Ser Gly Asn Leu Phe Gly Ala Met Phe Asn Arg

245 250 255

Tyr Gln Leu Thr Trp Thr Lys Asn Val Ile Gln Phe Gly Ile Asn Asp

260 265 270

Arg Ile Phe Arg Thr Val Thr Pro Tyr Glu Gly Phe Trp Arg Leu Gly

275 280 285

Gly Phe Ser Phe Asn Pro Trp Pro Lys Gly Ser Lys Met Ala Pro Phe

290 295 300

Asp Lys Glu Phe Tyr Ile Val Met Asn Val Ala Val Gly Gly Asp Tyr

305 310 315 320

Phe Pro Asp Asn Ala Trp Asn Pro His Pro Lys Pro Trp Arg Gln Gly

325 330 335

Asn Pro Ser Ala Met Thr Asp Phe Tyr Lys Ala Lys Ser Asn Trp Tyr

340 345 350

Ser Thr Trp Gly Asp Ala Ala Ala Leu Glu Val Asp Trp Val Lys Val

355 360 365

Trp Ala Ala

370

<210> 9

<211> 1425

<212> DNA

<213> Artificial Sequence

<400> 9

agagtgaatt ttactgtagt gatgatacaa actcagaaaa gagtcgttgg aaccattggg 60

ttgctgttct gcgttgttgg tttgatctca ggatgtaagc gctcaccgac cactgcttca 120

ggattcagag ctccgaaagg tgaacatttg tagtacgaga cctgtgtaca ttgacctcca 180

gcttattcat tgcataggtc gcatatgttc tggacagcta atttttcaag ataatttcaa 240

ccgactggac agaaatgttt ggcagcatga aaattcgctt ggtggtggtg gtgtaagtca 300

aatttcaata actaccacga atatttaaca caatcaaggt tttatcatca taagaacaac 360

gagttccaat ggtattctgg atccggacga aattcgtaca taaaaaacaa tcatctatac 420

atccgcccaa cattgacatc ggatgaatat ggtgaagcgt ttctgaaaag cggagtaatc 480

aatctcaacg agggtcccca gagtcaaagg taaattaaat tgtgcaacgt aatatctcaa 540

accataaatt gaccactatt gcagatgcac tgatgcaccg ggttgggcag aacagatcca 600

cggatgctat cgacgaggaa gtccagatcg tatcctgaac ccagttcgaa gtgcacgact 660

ccgaacggtg aattcattcg ctttcaaata tggaaaggtg gaaatcaatg ctaagttgcc 720

acaaggagat tggttgtggc ctgctctgtg gttgctccca aaaggggata cctacggata 780

ttggcccaag tcaggtgaag tggatctgat ggagtcacgt ggcaatcgaa acctcgtaca 840

gaacaacgag aagatcggaa tccagaaggt gtcatcgtgt ctacatttcg gagacaaccc 900

gaatgtgcgc agttcccagt gtggttctgt tagtggtaat ctttttggtg caatgtttaa 960

cagataccaa cttacttgga cgaaaaatgt cattcaattt ggaattaacg ataggatatt 1020

tcgtactgtt accccttatg agggtttctg gagacttggc ggattcagtt tcaacccatg 1080

gcctaaggga tcgaagatgg ctccattcga caaggagttt tatatagtca tgaatgttgc 1140

agtcggtgga gactactttc cggataatgc gtggaatcca cacccgaaac cctggagaca 1200

aggaaatccg agtgcgatga ccgacttcta caaagccaaa tccaattggt acagtacttg 1260

gggtgatgct gcggcattag aagttgactg ggtgaaagtt tgggctgcat agatatttaa 1320

ttttccaact ttggtatact aaaaatttat gattatgttg actgcaatga taacaccagc 1380

tattgcgttt cgtgaatgga atatatctaa cctaattgaa gaagc 1425

Claims (8)

1. A method for preparing transgenic mosquito cells with improved Zika virus infection level, comprising the following steps: knocking out DNA molecules shown in 247-746 positions corresponding to sequence 1 in beta-1,3-glucan binding protein genes in mosquito cells to obtain transgenic mosquito cells with improved Zika virus infection level; the mosquito cells are aedes aegypti Aag2 cell lines; the nucleotide sequence of the beta-1,3-glucan binding protein gene is shown as a sequence 9 in a sequence table.

2. Use of the method of claim 1 or the method of preparation of mosquito cells with β -1,3-glucan binding protein gene knockdown as follows 1) or 2):

1) Preparing transgenic mosquito cells with increased Zika virus infection level;

2) Preparing a mosquito cell model for researching interaction of aedes aegypti and Zika virus;

the preparation method of the beta-1,3-glucan binding protein gene knockout mosquito cell comprises the following steps: editing beta-1,3-glucan binding protein genes in mosquito cells based on CRISPR/Cas9 system, so that functions of the beta-1,3-glucan binding protein genes are lost，Obtaining the mosquito cells from which the beta-1,3-glucan binding protein gene is knocked out; the nucleotide sequence of the beta-1,3-glucan binding protein gene is shown as a sequence 9 in a sequence table;

the CRISPR/Cas9 system comprises an sgRNA targeting the β -1,3-glucan binding protein gene;

the sgrnas include sgRNA1 and sgRNA2;

the target sequence identified by the sgRNA1 is shown as a sequence 2 in a sequence table;

the target sequence identified by the sgRNA2 is shown as a sequence 3 in a sequence table;

the mosquito cell is an aedes aegypti Aag2 cell line.

3. The use according to claim 2, characterized in that:

the nucleotide sequence of the sgRNA1 is shown as a sequence 4 in a sequence table;

the nucleotide sequence of the sgRNA2 is shown as a sequence 5 in a sequence table.

4. The use according to claim 2, characterized in that: the CRISPR/Cas9 system further comprises a Cas9 nuclease; the method further comprises the step of electrotransfecting the sgRNA and the Cas9 nuclease into a mosquito cell.

5. The use according to claim 4, characterized in that: the ratio of the sgRNA1 to the sgRNA2 to the Cas9 nuclease is 1.2nM to 160 mug.

6. Use of a substance that inhibits β -1,3-glucan binding protein or a gene encoding the same for increasing the level of infection by aedes aegypti or aedes aegypti Aag2 cell line-based zika virus;

the amino acid sequence of the beta-1,3-glucan binding protein is shown as a sequence 8 in a sequence table;

the nucleotide sequence of the coding gene of the beta-1,3-glucan binding protein is shown as a sequence 9 in a sequence table.

7. Use of a substance that inhibits β -1,3-glucan-binding protein or a gene encoding the same in the preparation of transgenic mosquito cells with increased levels of zika virus infection; the mosquito cells are aedes aegypti Aag2 cell lines;

the amino acid sequence of the beta-1,3-glucan binding protein is shown as a sequence 8 in a sequence table;

the nucleotide sequence of the coding gene of the beta-1,3-glucan binding protein is shown as a sequence 9 in a sequence table.

8. Use according to claim 6 or 7, characterized in that: the substance for inhibiting the beta-1,3-glucan binding protein or the encoding gene thereof is an sgRNA or CRISPR/Cas9 system for editing the beta-1,3-glucan binding protein gene;

the sgrnas for editing the β -1,3-glucan binding protein gene include sgRNA1 and sgRNA2; the target sequence identified by the sgRNA1 is shown as a sequence 2 in a sequence table; the target sequence identified by the sgRNA2 is shown as a sequence 3 in a sequence table;

the CRISPR/Cas9 system for editing a β -1,3-glucan binding protein gene comprises the sgRNA and Cas9 nuclease for editing a β -1,3-glucan binding protein gene.