CN117737063A

CN117737063A - sgRNA of targeted knockout STAT1 gene and pig kidney cell line of knockout STAT1 gene and application thereof

Info

Publication number: CN117737063A
Application number: CN202311761534.1A
Authority: CN
Inventors: 罗廷荣; 张丽媛; 李晓宁; 闵开骏
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2023-12-20
Filing date: 2023-12-20
Publication date: 2024-03-22

Abstract

The invention relates to the technical field of biology, and provides sgRNA (short guided RNA) targeted for knocking out STAT1 genes, a pig kidney cell line for knocking out STAT1 genes and application thereof, wherein the STAT1 gene knockout efficiency is up to 82.4%. The nucleotide sequence of the sgRNA of the STAT1 gene is shown as SEQ ID NO.1 and SEQ ID NO.2, the invention obtains the pig kidney cell line with the STAT1 gene knocked out for the first time, and the CRISPR-Cas9 system is utilized to knock out the STAT1 gene from pig kidney cells, thereby solving the problems of low transfection efficiency, low targeting efficiency and low monoclonal purity in the process of constructing the knocked-out cell line, and obtaining the editing cell line with simple operation and longer fragment deletion. The related cell line provides a better research tool for deeply excavating the biological functions of the STAT1 gene.

Description

sgRNA of targeted knockout STAT1 gene and pig kidney cell line of knockout STAT1 gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a pig kidney cell line for targeted knockout of sgRNA of STAT1 gene and knockout of STAT1 gene and application thereof.

Background

CRISPR/Cas is an adaptive immune system against invasion by bacteria that recognizes invading nucleic acids from phage or plasmids and cleaves RNA or DNA by Cas9 nuclease to eliminate infection. CRISPR/Cas9 is a simple and fast tool that can efficiently modify endogenous genes of various species and cell types. sgRNA (shortguided RNA) is an important component of the CRISPR/Cas9 system, consisting of two parts, CRISPR-derived RNA (crRNA) and transactivation crRNA (tracrRNA). Part of the crRNA sequence is homologous to the tracrRNA, and the crRNA can bind to the tracrRNA by base pairing to form a tracrRNA/crRNA complex. This complex directs the Cas9 nuclease to localize near the DNA sequence to be edited. Cas9 nuclease, a RNA-guided dsRNA binding protein, is the first known unifying factor capable of co-locating RNA, DNA, and protein, showing great potential for creation. CRISPR/Cas9 systems have been successfully applied to a variety of species, including zebra fish, mice and rats. In addition, the technology has no limitation on animal species, and breaks through plant gene modification aspects such as soybean, tobacco, rice, wheat and the like. However, sgRNA off-targeting is the primary reason for the inefficiency of affecting CRISPR/Cas knockdown, so finding a specific, efficient target is critical to technical success.

STAT1 (Signal Transducers And Activators Of Transcriptionare 1) is an important member of the STATs family, usually induced by type I IFNs in immune cells. The biological function of STAT may play a role in the response of cytokines, as well as regulate the activation of growth factors. By means of gene knockout mouse model, it is found that STAT1 is absent, IFN signal can not be transmitted, IFN alpha can not be produced, and only low-level IFN gamma can be produced. STAT1-STAT2 combined defects can lead to female infertility, smaller body type and enlarged spleen. Most of the current research on STAT1 genes utilizes inhibitors to discover the functions of the inhibitors in human and mice, has few reports on swine STAT1 function research, and the specificity and effectiveness of drug blocking are difficult to ensure, the gene functions cannot be well explained, and a certain risk exists. In addition, the methods of inhibitor, silencing, knocking down, interference and the like in the prior art have weak specificity, incomplete silencing or incapacitation of silencing STAT1 gene expression, so that it is necessary to develop a pig STAT1 deletion cell strain which can realize thorough silencing and can be stably cultured in vitro for a long time, and lay a foundation for deeply researching the effect of STAT1 in a swine fever infection pathogenic mechanism.

Disclosure of Invention

The invention aims to provide the sgRNA for targeted knockout of the STAT1 gene with high efficiency, easy operation and strong repeatability, solves the problems that the methods such as inhibitor, silencing, knocking down, interference and the like in the prior art are not strong in specificity, incomplete in silencing or incapable of silencing gene expression, simultaneously obtains the STAT1 gene-deleted pig kidney cell line for the first time, is an ideal PK-15 cell model for knockout of the STAT1 gene, and lays a foundation for further researching the functions of the STAT1 protein and the effects in the pathogenic mechanism of classical swine fever virus infection.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a targeting knock-out STAT1 gene sgRNA, and the nucleotide sequence of the sgRNA is shown as SEQ ID NO.1 and SEQ ID NO. 2.

The invention also provides application of the sgRNA in knocking out STAT1 genes.

The invention also provides an expression cassette containing the sgRNA.

The invention also provides an expression vector containing the sgRNA.

The invention also provides a construction method of the expression vector, which comprises the following steps:

(1) Adding an enzyme cutting site with a nucleotide sequence of CACC and a G at the 5' end of SEQ ID NO.1 to obtain SEQ ID NO.3;

(2) Adding an enzyme cutting site with a nucleotide sequence of AAAC at the 5 'end of SEQ ID NO.2 and adding C at the 3' end to obtain SEQ ID NO.4;

(3) Annealing SEQ ID NO.3 and SEQ ID NO.4 to form a double stranded DNA molecule;

(4) And (3) carrying out enzyme digestion on the PX459 vector, and then connecting the PX459 vector with the double-stranded DNA molecule to obtain an expression vector PX459-STAT1.

The invention also provides application of the sgRNA in construction of STAT1 gene deletion cell lines.

The invention also provides a construction method of the STAT1 gene deletion cell line, which comprises the following steps: when the cells are cultured to 70-80% fusion degree, the mixed solution containing the expression vector PX459-STAT1 is transfected into the cells and cultured.

Preferably, the temperature of the culture is 35-39 ℃ and the time of the culture is 12-16 h.

The invention also provides a STAT1 gene deletion cell line.

The invention also provides a STAT1 gene knockout kit, which comprises any one of the following (1) to (4):

(1) The nucleotide sequence of the sgRNA of claim 1;

(2) The expression cassette of claim 3;

(3) The expression vector of claim 4;

(4) The STAT1 gene-deleted cell line of claim 9.

The invention provides the sgRNA of the targeted knockout STAT1 gene with high efficiency, strong specificity and simple operation and the application thereof. The nucleotide sequence of the sgRNA of the STAT1 gene is shown as SEQ ID NO.1 and SEQ ID NO.2, and the STAT1 gene knockout efficiency reaches 82.4%. According to the invention, the pig kidney cell line with the STAT1 gene knocked out is obtained for the first time, the CRISPR-Cas9 system is utilized to knock out the STAT1 gene from pig kidney cells, the defects of weak specificity, incomplete silencing or incapacitation of silencing gene expression of methods such as inhibitor, silencing, knocking-down, interference and the like are effectively improved, and the knocking-out effect is more thorough. The gene modification is carried out under the condition of not introducing any exogenous gene, the obtained gene knockout cell line can be used as a model for researching the pathogenic mechanism of the swine fever virus in vitro, and simultaneously, a foundation is provided for deeply excavating the action research of STAT1 genes in the replication and transcription processes of the swine fever virus.

Drawings

FIG. 1 is a schematic diagram of the sgRNA sequence and primer design (sgRNA target design at exon 15 of STAT 1), where: uppercase italics are BbSI cleavage sites. Adding "C" to the 3' end to allow the U6 promoter to function effectively, the "G" being reverse complementary to "C";

FIG. 2 is a schematic diagram of a PX459 building block, wherein the red box is the cloning site position;

FIG. 3 is a graph of the sequencing result of PX459-STAT1 plasmid (sgRNA sequence in red);

FIG. 4 is a graph of transfection efficiency of PX459-STAT1 plasmid, wherein A is the result of transfection of PK15 cells with PX459-STAT1 plasmid IFA, and green fluorescence is a positive cell; b is flow cytometry analysis of PK15 cell transfection PX459-STAT1 plasmid efficiency;

FIG. 5 shows the efficiency of sgRNA editing assay, wherein T7E1 enzyme recognizes mismatched bases and cleaves, mismatched DNA edited by crispr/cas9 can be cleaved into two bands, and wild type DNA has only one band;

FIG. 6 is PK-15 ^STAT1-/- Monoclonal culturing of the cell strain;

FIG. 7 is a Westernblot verification knockout efficiency;

FIG. 8 is a graph of RT-qRCR verification knockout efficiency;

FIG. 9 is PK15 ^STAT1-/- Cell line sequencing validation, wherein the blue-marked part is the sgRNA sequence. And (5) the sleeve peak appears near the sgRNA target area and in the subsequent sequence, namely the target is effective.

Detailed Description

The invention also provides an expression cassette containing the sgRNA.

The invention also provides an expression vector containing the sgRNA.

In the present invention, the temperature of the culture is preferably 35 to 39 ℃, more preferably 37 ℃, and the time of the culture is preferably 12 to 16 hours, more preferably 14 hours.

The invention also provides the STAT1 gene deletion cell line.

(1) The nucleotide sequence of the sgRNA of claim 1;

(2) The expression cassette of claim 3;

(3) The expression vector of claim 4;

(4) The STAT1 gene-deleted cell line of claim 9.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

1. Sequence design of STAT1 gene sgRNA

The 15 th exon of STAT1 gene is designed with a knockout target site, 4 targets are designed in total, and the sgRNA with high score is selected according to software to be the sgRNA with high editing efficiency, and finally, the preferable sgRNA target sequence is: 5'-CAGCCAGCTCCCGAGCGGTT-3' (SEQ ID NO. 1) according to BbsI restriction endonuclease (cat# ER1011, manufacturer: thermo Scientific) ^TM The reagent is called BpiI (BbSI) (10U/. Mu.l) -LBID, ruleThe grid is: 200 units) designing enzyme cutting sites at two ends of the sgRNA, adding CACCG at the 5' end of the sgRNA to form SEQ ID NO.3, adding AAAC at the 5' end of the reverse complementary sequence, adding C at the 3' end as shown in figure 1 to form SEQ ID NO.4, and finally sending the designed sequence to biological company (Huada gene) for synthesis.

2. Construction of PX459 plasmid expression vector

(1) The PX459 vector (fully designated pSpCas9 (BB) -2A-Puro (PX 459) V2.0) backbone is shown in FIG. 2. Preparing an enzyme digestion system according to table 1, and placing the system into a water bath kettle at 37 ℃ for enzyme digestion for 1h after the preparation is completed. After completion of the cleavage, 1% agarose gel electrophoresis was performed, and the gel was collected according to the gel recovery kit (Thermo Scientific) ^TM Manufacturer, reagent name GeneJET Gel Extraction Kit, cat No. K0692, 250 preps) recovered and purified sticky end containing cleavage product.

TABLE 1PX459 enzyme digestion System reagent preparation Table

(2) Oligo annealing systems were prepared according to the actual amounts shown in Table 2 and placed in a PCR apparatus. Setting a PCR instrument program, firstly carrying out denaturation at 95 ℃ for 5min, and finally cooling to 25 ℃ at a speed of 5 ℃/one cycle for 15 cycles, thus obtaining the annealed product double-stranded DNA molecule.

TABLE 2 Oligo annealing System

Reagent(s)	Volume of
		Primer 1 (SEQ ID NO. 3))	4μL
Primer 2 (SEQ ID NO. 4)	4μL
		10×T4Ligastion buffer (T4 DNA Ligase kit)	2μL
Total volume of	10μL

(3) The ligation system of the annealed product and the digested PX459 plasmid was prepared and incubated at 22℃for 1h according to Table 3.

Table 3 connection system

(4) The recombinant vector is transformed into DH5 alpha escherichia coli competent cells (the product number is CD201, the manufacturer is that the reagent of Beijing full type gold biotechnology Co., ltd is Trans5 alpha Chemically Competent Cell, the specification is that the reagent of Beijing full type gold biotechnology Co., ltd is 20 multiplied by 100 mu L), the mixture is uniformly mixed and then subjected to ice bath for 30min, then the mixture is subjected to heat shock conversion at 42 ℃ for 90s, then subjected to ice bath for 2min, added with 600 mu L of sterile LB culture medium (1% tryptone, 0.5% yeast extract and 1% sodium chloride) which is incubated at 37 ℃, subjected to shake culture at 37 ℃ for 1h under the condition of 180rpm/min, 100 mu L of bacterial liquid is uniformly coated on ampicillin (the product number is A8180, the manufacturer is that the reagent of Beijing Soxhaustobao, the specification is that the ampicillin sodium, the specification is 25 g) resistant solid LB culture medium (containing 100 mu g/mL ampicillin), and the incubator is subjected to overnight inversion culture at 37 ℃.

(5) Sequencing: single colonies were taken and cultured in 1mL of liquid LB medium containing 100. Mu.g/mL ampicillin, shaking at 150rpm until turbidity, and the bacterial liquid was sent to sequencing company (Huada Gene), and the forward primer of the U6 promoter was used for sequencing, and the sequence was: 5'-GAGGGCCTATTTCCCATGATTCC-3' (SEQ ID NO. 5).

(6) And (3) preserving bacteria: as shown in FIG. 3, the colony with correct connection was selected, designated as PX459-STAT1, cultured in an expanded culture, and then 50% glycerol was added to the culture and stored in a refrigerator at-80 ℃.

3. Transfection efficiency assay

When the pig kidney cells in the six-hole plate grow to 70-80% confluence, carrying out liposome transfection on 1.0 mug PX459-STAT1 plasmid, wherein the specific process of liposome transfection is as follows: (1) The day before transfection, cells were trypsinized and counted, and cells plated in 12 well plates to a density of 90% on the day of transfection. Cells were plated in normal growth medium with 5% serum and no antibiotics. (2) For each well of cells, 300. Mu.L of OPTI-MEMI medium was diluted with 1.0. Mu.g of PX459-STAT1 plasmid. (3) mu.L Lipofectamine 2000 reagent was diluted with 300. Mu.L OPTI-MEM I medium per well of cells. Lipofectamine 2000 was diluted and incubated for 5 minutes and mixed with the diluted PX459-STAT1 plasmid solution over 30 minutes. (4) The diluted PX459-STAT1 plasmid solution (step 2) and the diluted Lipofectamine 2000 (step 3) were mixed. Incubate for 20 minutes at room temperature. (5) The complex was added directly to each well, the plate was shaken and gently mixed. At 37℃5% CO ₂ After 5 hours of medium temperature preservation, the growth medium is replaced.

A portion of the transfected cells was used for IFA assay (fig. 4A) at 48h, the specific procedure for IFA assay was: (1) Log grown cells were taken and digested with 0.25% trypsin to make a single cell suspension. Cells were inoculated into a 12-well plate, cultured in a carbon dioxide incubator for 36 hours, and after the cells were grown to nearly a monolayer, PX459-STAT1 plasmid was transfected, taken out of the incubator 24 hours after transfection, old medium was discarded, washed 2 times with PBS (0.01 mol/L, pH 7.4), and 95% acetone was selected to fix the cells for 30 minutes. (2) The fixative was discarded, washed 2 times with PBS (0.01 mol/L, pH 7.4), and 1:1000 diluted Flag-tagged antibodies were placed in a wet box, 4 degrees refrigerator overnight. (3) The next day the wet cartridge was removed, the primary antibody was discarded, washed 2 times with PBS (0.01 mol/L, pH 7.4), and 1:1000 diluted green fluorescent secondary antibodies are placed in a wet box and placed in an oven at 37 ℃ for 1 hour. (4) The secondary antibody was discarded, washed 2 times with PBS (0.01 mol/L, pH 7.4), and observed under a fluorescence microscope.

A portion was used for 48h to measure transfection efficiency by flow cytometry (FIG. 4B), and the procedure was as follows: (1) Log grown cells were taken and digested with 0.25% trypsin to make a single cell suspension. Cells were inoculated into 12-well plates and incubated in a carbon dioxide incubator for 36 hours until the cells nearly grew into a monolayer, and the PX459-STAT1 plasmid was transfected. (2) 24 hours after transfection, the old medium was removed from the incubator, washed 2 times with PBS (0.01 mol/L, pH 7.4), and digested with 0.25% trypsin to prepare a single cell suspension. (3) adding to the cell sample the nucleic acid of 1:200 dilution of Flag-RFP labeled antibody, which makes it to make specific binding with target molecule in sample. (4) The cells incubated with the antibodies were screened through a cell sieve to remove impurities such as cell debris greater than 0.22 μm to obtain single cells. (5) The flow cytometry is used for analysis, a sample is sprayed into a flow tube of the cytometry, and parameters such as fluorescence intensity, scattering degree and the like of marked cells in the sample are measured under a laser beam. The percentage of positive cells was calculated.

4. sgRNA editing efficiency detection

(1) When the pig kidney cells in the six-hole plate grow to 70-80% confluence, carrying out liposome transfection, wherein the specific process of the transfection is as follows: 1) The day before transfection, cells were trypsinized and counted, and cells plated in 12 well plates to a density of 90% on the day of transfection. Cells were plated in normal growth medium with 5% serum and no antibiotics. 2) For each well of cells, 300. Mu.L of OPTI-MEMI medium was diluted with 1.0. Mu.g of PX459-STAT1 plasmid. 3) mu.L Lipofectamine 2000 reagent was diluted with 300. Mu.L OPTI-MEM I medium per well of cells. Lipofectamine 2000 was diluted and incubated for 5 minutes and mixed with the diluted PX459-STAT1 plasmid solution over 30 minutes. 4) The diluted PX459-STAT1 plasmid solution (step 2) and the diluted Lipofectamine 2000 (step 3) were mixed. Incubate for 20 minutes at room temperature. 5) The complex was added directly to each well, the plate was shaken and gently mixed. At 37℃5% CO ₂ After 5 hours of medium temperature preservation, the growth medium is replaced.

After 24h of cell culture after transfection, cell extract media was collectedFor the group, wild type cells and PX459-STAT1 plasmid transfected cells were subjected to PCR amplification with STAT1-F as the amplification primer: 5'-ACAAAAAACAAACAAGCGTT-3' (SEQ ID NO. 6). STAT1-R:5'-GTCAGGGCCAGCAGTGGGAC-3' (SEQ ID NO. 7). PCR reaction system: 1. Mu.L of forward primer, 1. Mu.L of reverse primer, 3. Mu.L of DNA, 12.5. Mu.L of 2 XTaqPCRMastermix II, ddH ₂ O7.5. Mu.L, 25. Mu.L total. PCR reaction procedure: pre-denaturation at 94℃for 2min; denaturation at 98℃for 10s, annealing at 58℃for 30s, and cycling for 34 times; the temperature is kept at 72 ℃ for 5min. The 720bp product obtained was hybridized with the wild-type PCR product transfected with PX459-STAT1 plasmid PCR product according to the system of Table 4 and placed in a PCR instrument. The PCR procedure was as follows: denaturation at 95 ℃ for 2min, cooling to 85 ℃ at a speed of 2 ℃/one cycle, and cooling to 25 ℃ at a speed of 0.1 ℃/one cycle, thus obtaining the hybridization product.

TABLE 4 hybridization System

Reagent(s)	Volume of
		Wild-type PCR products	2.5μL
Transfection set PCR products	2.5μL
		10×T7E1 buffer	1.1μL
ddH ₂ O	4.4μL
		Total volume of	10.5μL

The hybridized product was digested with T7E1 restriction enzyme (product No. E3321, manufacturer No. NEB (NEW England BioLabs) reagent: T7 endonucleolytic I, specification No. 250 units), and the digestion system was as shown in Table 5.

Table 5 enzyme digestion System

Reagent(s)	Volume of
		10 XNEB#2 buffer (the buffer is matched with T7E1 enzyme)	2μL
Hybridization PCR products	10μL
		T7E1 enzyme	0.25μL
ddH ₂ O	7.75μL
		Total volume of	20μL

(3) The result of gel running is shown in FIG. 5, and the T7E1 enzyme can cut the DNA fragment, and the knockout efficiency reaches 82.4% through imageJ analysis, so that the method can be used for subsequent experiments.

5. Screening of Positive monoclonal cells

(1) When the pig kidney cells grow to 70-80% confluence, preparing a mixed solution containing an expression vector PX459-STAT1 and 2 mug puromycin (product number is P8230, manufacturer is Soy pal reagent full name: puromycin, specification is 25 mg) resistant plasmid, and using Lip2000 liposome for transfection.

(2) After 6h of transfection, the cells are changed into growth medium containing 10% fetal bovine serum (product number is 04-400-1A, manufacturer is the full name of Israel Bioind reagent: foetal Bovine Serum, specification is 500 mL), the cells in a 6-hole plate are transferred to 15 culture dishes of 10cm after 24h of digestion and separation, puromycin of 2 mug/mL is added for screening, liquid is changed once every 5 days, and monoclonal cell clusters can be obtained after about 3 times of change.

(3) Monoclonal cells with regular morphology and good state were selected and transferred to 48-well plates with cloning rings for further culture, as shown in fig. 6.

(4) After the number of the cells is enough, the cells are transferred to a 24-well plate for culture, and after the cells grow fully, one part of the cells are continuously passaged to a 12-well plate for cryopreservation after the cells grow fully, and the other part of the cells extract genome DNA.

2. Identification of Positive monoclonal cells

And when the pig kidney cells in the six-hole plate grow to 70-80% of confluence, carrying out liposome transfection, culturing the transfected cells for 24 hours, collecting cell extraction genome, carrying out PCR amplification, and sending the obtained 720bp product to a company for sequencing so as to detect knockout efficiency. The upstream primer and the downstream primer used for PCR identification are respectively: F-5'-ACAAAAAACAAACAAGCGTT-3' (SEQ ID NO. 8) and R-5'-GTCAGGGCCAGCAGTGGGAC-3' (SEQ ID NO. 9). The PCR amplification used a 20. Mu.L reaction system: 10 μL 2×PowerTaqPCRMastermix,8 μL ddH ₂ O, 0.5. Mu.L of each of the upstream and downstream primers, and 1. Mu.L of the DNA template; PCR reaction conditions: pre-denaturation at 94℃for 5min;30 cycles: denaturation at 94℃for 30s, annealing at 58℃for 15s, extension at 72℃for 10s; finally, the extension is carried out for 5min at 72 ℃.

The sequencing result is shown in FIG. 9, the 720bp PCR product is sequenced, and compared with the wild PCR reference sequence, the STAT1 knockout cell line can be cut near the target site of the pig kidney cell genome, so that the STAT1 knockout is proved to be successful.

Meanwhile, the knockout effect is detected by Westernblot and RT-qPCR, as shown in figures 7-8, and STAT1 knockout is proved to be successful.

The Westernblot specific process comprises the following steps: (1) PK-15 cells were cultured in 12-well plates to about 1.0X10 ⁵ Cells were stimulated with 100IU/mL IFN alpha, lysed 48 hours after infection with RIPA lysate containing protease inhibitor, centrifuged at 12000rpm at 4deg.C, and the supernatant was sub-packaged and stored at-80deg.C for use. (2) STAT1 protein was separated from total protein by 10% sds-PAGE and the transferred PVDF membrane was blocked overnight at 4 ℃ in 5% skim milk. (3) STAT 1-specific antibodies via 1: after incubation for 1h at 37℃after 1000 dilution, the PVDF membrane was washed three times with TBS, each for 20min. (4) Incubation with corresponding alkaline phosphatase-labeled secondary antibodies for 1h at 37℃at a secondary antibody dilution ratio of 1:500. (5) After the secondary antibody incubation, the PVDF membrane was washed three times with TBS for 20min each time, and finally developed. The color development step is carried out by referring to an alkaline phosphatase color development kit of Biyun institute of biotechnology under the condition of avoiding light until the target protein specific band appears. (6) scanning and saving the image with an Odyssey imaging system.

RT-qPCR specific procedure: (1) PK-15 cells were cultured in 24-well plates to 1.0X10 ⁵ Cells were stimulated with 100IU/mL IFN alpha, lysed 48 hours after infection with Tirzol, blown several times and stored in a-80℃freezer. (2) extraction of total RNA: RNA of each sample was extracted by referring to the method of the total Tiangen RNA extraction kit, and the RNA concentration of each sample was measured. 1.0. Mu.g of RNA was taken from each RNA sample and reverse transcribed according to the following system:

TABLE 6 reverse transcription System

Total RNA of cells	1μg
		Oligo dT(25μM)	1μL
dNTP(10mM)	2μL
		M-MLV reverse transcriptase	1μL
5 Xreverse transcriptase buffer	5μL
		RNase inhibitors	1μL
dd H ₂ O	Make up to 25. Mu.L

The reverse transcription reaction was carried out at 37℃for 1.5 h.

TABLE 7 RT-qPCR reaction System

Upstream primer (25. Mu.M)	0.18μL
		Downstream primer (25. Mu.M)	0.18μL
cDNA	3μL
		H ₂ O	6.64μL
SYBR Green mix	10μL

RT-qPCR reaction conditions: pre-denaturation at 95 ℃ for 15min, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 20s,40 cycles, fluorescence signal acquisition, and dissolution curve analysis at 60-95 ℃.

Each set of experiments was performed in triplicate. The Cq value of STAT1 detected is referenced to the Cq value of GAPDH, with a Mock sample set of 1. By 2 ^-ΔΔCT The method is used for carrying out relative quantitative analysis on the experimental results. The results of the three experiments were averaged and T-checked using SPSS17.0 software (P<0.05 Differences between groups were analyzed.

From the above examples, the invention provides the sgRNA for targeted knockout of STAT1 gene, which has high efficiency, strong specificity and simple operation, and the application thereof, wherein the efficiency of the STAT1 gene knockout reaches 82.4%. The nucleotide sequence of the sgRNA of the knock-out STAT1 gene is shown as SEQ ID NO.1 and SEQ ID NO. 2. According to the invention, the pig kidney cell line with the STAT1 gene knocked out is obtained for the first time, the CRISPR/Cas9 system is utilized to knock out the STAT1 gene from pig kidney cells, the defects of weak specificity, incomplete silencing or incapacitation of silencing gene expression of methods such as inhibitor, silencing, knocking-down, interference and the like are effectively improved, and the knocking-out effect is more thorough. The gene modification is carried out under the condition of not introducing any exogenous gene, the obtained gene knockout cell line can be used as a model for researching the pathogenic mechanism of the swine fever virus in vitro, and simultaneously, a foundation is provided for deeply excavating the action research of STAT1 genes in the replication and transcription processes of the swine fever virus.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The sgRNA of the target knock-out STAT1 gene is characterized in that the nucleotide sequence of the sgRNA is shown as SEQ ID NO.1 and SEQ ID NO. 2.

2. Use of the sgRNA of claim 1 for knocking out STAT1 gene.

3. An expression cassette comprising the sgRNA of claim 1.

4. An expression vector comprising the sgRNA of claim 1.

5. A method for constructing the expression vector according to claim 4, comprising the steps of:

6. Use of the sgRNA of claim 1 for constructing STAT1 gene-deleted cell lines.

7. A method for constructing a STAT1 gene-deleted cell line, comprising the steps of: when the cells are cultured to 70-80% fusion degree, the mixed solution containing the expression vector PX459-STAT1 is transfected into the cells and cultured.

8. The method according to claim 7, wherein the culturing is carried out at a temperature of 35 to 39℃for a period of 12 to 16 hours.

9. A STAT1 gene deleted cell line, characterized in that it is obtained by the construction method according to any one of claims 7 to 8.

10. A kit for STAT1 gene knockout, comprising any one of the following (1) to (4):

(1) The nucleotide sequence of the sgRNA of claim 1;

(2) The expression cassette of claim 3;

(3) The expression vector of claim 4;

(4) The STAT1 gene-deleted cell line of claim 9.