CN110684736A - Chicken Shp-2 gene knockout cell line based on CRISPR-Cas9 editing technology and construction method thereof - Google Patents
Chicken Shp-2 gene knockout cell line based on CRISPR-Cas9 editing technology and construction method thereof Download PDFInfo
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Abstract
The invention relates to a cell line for knocking out a chicken Shp-2 gene based on a CRISPR-Cas9 editing technology and a construction method thereof, the principle and the most central key technology of the invention are scientifically and reasonably constructing sgRNA of a target chicken Shp-2 gene, cloning the sgRNA to lentiCRISPR v2 plasmid with Cas9 gene and transfecting the plasmid into cells, achieving gene silencing by using a CRISPR-Cas9 system, and finally obtaining a positive monoclonal cell line by using a drug screening and subcloning method, thereby obtaining the chicken-derived Shp-2 knocked-out cell line. The invention aims to construct a method for targeted knockout of a chicken Shp-2 gene based on CRISPR-Cas9 and sgRNA of the targeted chicken Shp-2 gene so as to obtain a chicken-derived Shp-2 gene knockout cell model for research of related diseases. The construction of the cell line based on CRISPR-Cas9 targeted knockout of the chicken Shp-2 gene is not reported in related fields, and the cell line fills the blank of related technologies at home and abroad and has great application and research values.
Description
Technical Field
The invention relates to a cell line for knocking out a chicken Shp-2 gene based on a CRISPR-Cas9 editing technology and a construction method thereof, belonging to the technical field of genetic engineering.
Background
The CRISPR-Cas system is an acquired immune system widely existing in bacteria and archaea, and plays an important role in the aspects of resisting foreign germs and transforming plasmids of the bacteria and the like. The CRISPR-Cas is currently found to be of 6 types I-VI, and the CRISPR/Cas9 is formed by modifying a type II system. The CRISPR/Cas9 system consists of a Cas9 protein with nuclease activity, crRNA transcribed from CRISPR, and tracrRNA complementary to the trans-activated CRISPR repeat. The crRNA is combined with the tracrRNA through base pairing to form double-stranded RNA, the Cas9 protein is recruited to a target gene site to play endonuclease activity, the target double-stranded DNA is specifically cut at the upstream of a PAM sequence at the 3' end of a target sequence to cause DNA double-stranded break (DSB), so that a DNA damage repair mechanism in a cell is started, base deletion or insertion is caused after repair to cause frameshift mutation, and the purpose of gene knockout is finally achieved. At present, the CRISPR/Cas9 gene editing technology is widely applied to gene editing of prokaryotes and eukaryotes.
The protein tyrosine phosphatase Shp-2(SH2 domain-binding protein-tyrosinesphatase-2, Shp2) is an intracellular non-receptor type tyrosine phosphatase and is ubiquitously expressed in cells of various vertebrates. The Shp-2 can negatively regulate a downstream cell signal transduction pathway through the catalytic activity of phosphatase, and can also receive cytokines and extracellular stimulation factors to play a positive regulation role, thereby being widely involved in a plurality of important cell life activities such as cell differentiation and movement and related signal transduction processes. Numerous studies have shown that alterations in the expression and activity of Shp-2 are associated with hematopoietic cell malignancies, human Noonan syndrome, childhood leukemia, various human malignancies, autoimmune diseases, and invasion of partial viral infections, however, studies on the function of chicken-derived Shp-2 are rarely reported. The construction of a chicken-derived Shp-2 knockout cell line is very important for exploring the role of chicken-derived Shp-2 in avian diseases. The chicken liver cell line with the stable Shp-2 gene knockout function is successfully obtained by using a CRISPR/Cas9 technology and through methods such as drug screening and subcloning, and is used for researching the biological function of chicken-derived Shp-2 and the effect of the chicken-derived Shp-2 in related diseases.
Disclosure of Invention
The invention aims to construct a chicken-derived Shp-2 knockout cell line. The invention provides a cell line for knocking out a chicken Shp-2 gene based on a CRISPR-Cas9 editing technology and a construction method thereof, the principle and the most central key technology of the invention are scientifically and reasonably constructing sgRNA of a target chicken Shp-2 gene, cloning the sgRNA to lentiCRISPR v2 plasmid with a Cas9 gene and transfecting the plasmid into cells, achieving gene silencing by using a CRISPR-Cas9 system, and finally obtaining a positive monoclonal cell strain by using a drug screening and subcloning method, thereby obtaining the chicken-derived Shp-2 knocked-out cell line.
The invention aims to realize the CRISPR-Cas9 editing technology-based chicken Shp-2 gene knockout cell line and the construction method thereof, and is characterized by comprising the following steps:
step 1), constructing a sgRNA of a specific target chicken Shp-2 gene, wherein the sgRNA is positioned in a second exon region of the chicken Shp-2 gene, and a target sequence is unique;
step 2), preparing a CRISPR-Cas9 system for targeted knockout of chicken Shp-2 gene, wherein the CRISPR-Cas9 system contains Cas9 protein and sgRNA of the specific targeted knockout chicken Shp-2 gene, or contains a coding sequence carrying the Cas9 protein and a coding sequence coding the sgRNA; in a CRISPR-Cas9 system, a Cas9 protein gene and a sequence of sgRNA are positioned on the same vector, and the vector is a lentiCRISPR v2 plasmid;
step 3), cloning sgRNA of the specific target knockout chicken Shp-2 gene constructed in the step 1) to lentiCRISPR v2 plasmid with Cas9 gene, and transfecting the lentiCRISPR v2 plasmid into cells;
gene silencing is achieved by using a CRISPR-Cas9 system, and a positive monoclonal cell strain is finally obtained by a drug screening and subcloning method, so that a chicken-derived Shp-2 knockout cell line is obtained.
In the step 1), 5 sgRNAs are designed for the chicken Shp-2 gene, the target sites of the sgRNAs are positioned in the second exon of the Shp-2 gene, and the exon sequences are shown in SEQ ID NO. 1.
5 sgRNA coding strands and complementary strands of the specific targeting chicken Shp-2 gene, the sequences of which are shown in SEQ ID NO. 2.
In the step 1), the preparation method of sgRNA of the targeted chicken Shp-2 gene comprises the following steps:
the method comprises the steps of annealing a synthesized sgRNA coding strand and a complementary strand to form double-stranded DNA, then carrying out enzyme digestion on a vector by BsmBI restriction enzymes, and placing the double-stranded DNA under a T7 promoter to construct the double-stranded DNA.
In the step 3), the lentiCRISPR v2 plasmid is transfected into chicken liver cells to obtain a chicken-derived Shp-2 knockout cell line, and the cell line is the chicken liver cells for knocking out chicken Shp-2 genes.
The method is advanced and scientific, provides a cell line for knocking out the chicken Shp-2 gene based on a CRISPR-Cas9 editing technology and a construction method thereof, firstly provides sgRNA of a specific target chicken Shp-2 gene, the sgRNA is positioned in a second exon region of the chicken Shp-2 gene, the target sequence is unique, and the sgRNA is shown in a table 1. The sequence of the sgRNA is shown in SEQ ID NO.1 aiming at a target site of a chicken Shp-2 gene. A CRISPR-Cas9 system for targeted knockout of chicken Shp-2 gene contains Cas9 protein and sgRNA of the specific targeted chicken Shp-2 gene, or contains a coding sequence carrying the Cas9 protein and a coding sequence coding the sgRNA. Wherein, the coding sequence of the Cas9 protein and the coding sequence of the sgRNA are positioned on the same plasmid, and the plasmid is lentiCRISPR v 2.
The specific operation steps are as follows:
searching a chicken Shp-2 genome sequence ENSGALG00000004821.5 and Transcript ID: ENSGALT00000007704.4 by using an ensemblel online database, and designing a knockout target site in a second exon of the gene, wherein the sequence of the first exon is shown as SEQ ID NO. 1.
Construction of sgrnas: the designed sgRNA coding strand and complementary strand were diluted to a concentration of 50. mu. mol/L, respectively, as follows: the sgRNA primers for the positive strand and the negative strand were 5. mu.L each, 5. mu.L of 10 XNEB buffer, and 35. mu.L of high-pressure deionized water. The annealing program was 95 ℃ for 5min, then from 70 ℃ every 1min, 5 ℃ down to 25 ℃. In addition, plasmid lentiCRISPRv2 was digested using the BsmBI restriction enzyme line as follows: BsmBI restriction enzyme 1uL, lentiCRISPR v2 plasmid 1ug, 10 XNEB buffer3uL, the rest with sterile deionized water to make up to 30 uL. The digestion conditions were 37 ℃ for 2 h. After the enzyme digestion, enzyme digestion identification is carried out through agarose gel electrophoresis, and a lentiCRISPR v2 vector fragment containing a sticky end is obtained through recovery. Finally, the annealed and diluted double-stranded sgRNA and the linearized lenticrispsrv 2 vector were ligated as follows: 50ng of linearized lentiCRISPRV2 vector, 4 muL of 20-fold diluted double-stranded sgRNA, 4 muL of 10 XT 4 DNA ligasebuffer, 1 muL of T4 DNA ligase and 2 muL of sterilized deionized water. Finally, a plasmid with the sgRNA and the Cas9 protein gene positioned in the same vector is obtained and named lentiCRISPRv 2-sgRNA.
Obtaining of knockout cell line: the cells in the 6-well plate were transfected with the constructed lentiCRISPRV2-sgRNA, 4ug plasmid per well, and pcDNA3.1 plasmid was transfected with the negative control. And adding a culture medium containing puromycin after 48 hours for drug screening, changing the drug every 48 hours, changing the culture medium into a normal culture medium after all negative control cells die, collecting part of the cells for identification of the knockout effect after the viable cells grow to a proper density, subcloning the cells with good knockout effect into a 96-well plate by using a limiting dilution method, identifying when the cells grow to a sufficient number, and freezing and storing the positive cells.
The invention aims to construct a method for targeted knockout of a chicken Shp-2 gene based on CRISPR-Cas9 and sgRNA of the targeted chicken Shp-2 gene so as to obtain a chicken-derived Shp-2 gene knockout cell model for research of related diseases. The construction of the cell line based on CRISPR-Cas9 targeted knockout of the chicken Shp-2 gene is not reported in related fields, and the cell line fills the blank of related technologies at home and abroad and has great application and research values.
Drawings
FIG. 1 is the enzyme digestion of lentiCRISPR v2 vector plasmid;
lane M: super DNA marker; lane 1: carrying out enzyme digestion on the lentiCRISPR v2 vector plasmid;
FIG. 2 is a graph showing the effect of recombinant plasmid knockout;
lanes 1-5 are cytolytic samples collected from transfected lenticrisprrv 2-sgRNA 1-5; lane 6: negative LMH cells;
FIG. 3 is an identification of a monoclonal KO-Shp-2 cell line;
lanes 1-3: LMH-KO-Shp-2-3, LMH-KO-Shp-2-6, LMH-KO-Shp-2-11 cell lines; lane 4: normal LMH cells.
FIG. 4 shows the genome sequencing analysis of the LMH-KO-Shp-2 cell line.
Detailed Description
The invention will be described in detail below with reference to the accompanying drawings.
1. Searching a target gene: searching a chicken Shp-2 genome sequence ENSGALG00000004821.5 and Transcript ID: ENSGALT00000007704.4 by using an ensemblel online database, and designing a knockout target site in a second exon of the gene, wherein the sequence of the first exon is shown as SEQ ID NO. 1.
2. Design of sgRNA and synthesis of oligonucleotide chain: specific sgRNA scores were obtained by analysis using websites (http:// crispr-era. stanford. edu/index. jsp and http:// crispr. mit. edu), and 5 sgRNAs (sgRNA-1, sgRNA-2, sgRNA-3, sgRNA-4, sgRNA-5) among the 2 nd exons with the highest scores were selected as candidate sequences. Adding CACC at the 5' end of the sgRNA sense strand template; the 5 'end of the antisense chain template is added with C, the 3' end is added with AAAC, and is matched with the cohesive end after the lentiCRISPRV2 enzyme digestion, 5 sections of primers are designed in total and synthesized by Suzhou Jinwei Zhi company, and the specific primer sequence information is shown in Table 1.
TABLE 1 targeting 5 sgRNA sequences against the Shp-2 gene
3. Obtaining of double-stranded sgRNA: the designed sgRNA coding strand and complementary strand were diluted to a concentration of 50 μmol/L with sterile deionized water, respectively, as follows: the sgRNA primers for the positive and reverse strands were 5. mu.L each, 5. mu.L of 10 XNEB buffer, and 35. mu.L of sterilized deionized water. The annealing program was 95 ℃ for 5min, then from 70 ℃ every 1min, 5 ℃ down to 25 ℃.
4. And (3) enzyme digestion of the vector plasmid: plasmid lenticrisprrv 2 was digested using the BsmBI restriction enzyme line as follows: BsmBI restriction enzyme 1uL, lentiCRISPR v2 plasmid 1ug, 10 XNEB buffer3uL, the rest of ddw to 30uL enzyme cutting conditions for 2h at 37 ℃. After the completion of the digestion, the digestion was identified by agarose gel electrophoresis (see FIG. 1), and the digestion product containing the cohesive ends was recovered from the gel.
5. Construction of recombinant plasmid expressing sgRNA: diluting the annealed double-stranded sgRNA by 20 times, simultaneously diluting the linearized vector plasmid recovered from the gel to 50ng/uL, and performing ligation and recombination according to the following system: linearized lentiCRISPRV2 vector 2uL, double-stranded sgRNA 4uL, 10 XT 4 DNA ligase buffer 1uL, T4 ligase 1uL, ddw 2uL are connected at 16 ℃ overnight, then the connection products are transformed into Stbl3 competent cells, colonies are picked for PCR primary identification, and plasmids are extracted to be sequenced and identified again by companies.
6. And (3) verifying the knockout effect of the recombinant plasmid: LMH cells in a 6-well plate were prepared, and when the cell density reached about 90%, 6ug of recombinant sgRNA-expressing plasmids were transfected into each well, and a non-transfected group was set as a negative control. And (3) after 6h of transfection, replacing growth liquid containing 10% fetal calf serum, removing the original culture medium after 24h, replacing 10% culture medium containing 8 mug/mL puromycin for screening, replacing fresh 10% growth liquid containing 8 mug/mL puromycin every 48h until all cells of a negative control group die, replacing 10% growth liquid without drugs, collecting part of remaining viable cells for cell lysis, detecting the expression level of the Shp-2 protein by Western blot (the result is shown in figure 2), and continuously culturing the rest cells.
7. Screening and identification of stable Shp-2 knock-out LMH cell lines: and (3) subcloning the surviving cells corresponding to the sgRNA recombinant plasmids with good knockout effect, carrying out amplification culture when the subcloned cells grow to a sufficient density, collecting the cells, carrying out Western blot to detect the expression level of the Shp-2 protein (the result is shown in figure 3), and then extracting a genome from the obtained Shp-2 knockout monoclonal cell strain to carry out sequencing identification on the target sequence knockout site (the result is shown in figure 4).
SEQ ID NO.1
Second exon sequence of the chicken Shp-2 gene:
ATGGTTTCATCCAAATATCACTGGGGTGGAGGCAGAAAATCTACTGTTAACAAGAGGAG TTGATGGCAGTTTTCTGGCACGGCCCAGCAAAAGTAACCCAGGAGACTTCACACTCTCT GTTAG
SEQ ID NO.2
targeting 5 sgRNA sequences against the Shp-2 gene
Sequence listing
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tag 123
Claims (5)
1. A cell line for knocking out a chicken Shp-2 gene based on a CRISPR-Cas9 editing technology and a construction method thereof are characterized by comprising the following steps:
step 1), constructing a sgRNA of a specific target chicken Shp-2 gene, wherein the sgRNA is positioned in a second exon region of the chicken Shp-2 gene, and a target sequence is unique;
step 2), preparing a CRISPR-Cas9 system for targeted knockout of chicken Shp-2 gene, wherein the CRISPR-Cas9 system contains Cas9 protein and sgRNA of the specific targeted knockout chicken Shp-2 gene, or contains a coding sequence carrying the Cas9 protein and a coding sequence coding the sgRNA; in a CRISPR-Cas9 system, a Cas9 protein gene and a sequence of sgRNA are positioned on the same vector, and the vector is a lentiCRISPR v2 plasmid;
step 3), cloning sgRNA of the specific target knockout chicken Shp-2 gene constructed in the step 1) to lentiCRISPR v2 plasmid with Cas9 gene, and transfecting the lentiCRISPR v2 plasmid into cells;
gene silencing is achieved by using a CRISPR-Cas9 system, and a positive monoclonal cell strain is finally obtained by a drug screening and subcloning method, so that a chicken-derived Shp-2 knockout cell line is obtained.
2. The cell line for knocking out the chicken Shp-2 gene based on the CRISPR-Cas9 editing technology and the construction method thereof according to claim 1, characterized in that 5 sgRNAs are designed for the chicken Shp-2 gene in step 1), the target sites of the sgRNAs are located in the second exon of the Shp-2 gene, and the exon sequences are shown in SEQ ID No. 1.
3. The cell line for knocking out the chicken Shp-2 gene based on the CRISPR-Cas9 editing technology and the construction method thereof according to claim 2, characterized in that 5 sgRNA coding chains and complementary chains of the chicken Shp-2 gene are specifically targeted, and the sequences of the sgRNA coding chains and complementary chains are shown in SEQ ID No. 2.
4. The cell line for knocking out the chicken Shp-2 gene based on the CRISPR-Cas9 editing technology and the construction method thereof according to claim 1, wherein the preparation method of sgRNA targeting the chicken Shp-2 gene in the step 1) is as follows:
the method comprises the steps of annealing a synthesized sgRNA coding strand and a complementary strand to form double-stranded DNA, then carrying out enzyme digestion on a vector by BsmBI restriction enzymes, and placing the double-stranded DNA under a T7 promoter to construct the double-stranded DNA.
5. The chicken Shp-2 gene knockout cell line based on the CRISPR-Cas9 editing technology and the construction method thereof as claimed in claim 1, wherein in step 3), lentiCRISPR v2 plasmid is transfected into chicken liver cells to obtain chicken-derived Shp-2 knockout cell line, and the cell line is chicken liver cells with chicken Shp-2 gene knockout.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662907A (en) * | 2020-07-09 | 2020-09-15 | 成都华西海圻医药科技有限公司 | Method for knocking out NANS gene of induced pluripotent stem cell and application |
| CN113151277A (en) * | 2021-05-10 | 2021-07-23 | 安徽农业大学 | Construction method of chicken DF-1 cell IHH gene knockout stable cell strain and specific sgRNA thereof |
| CN113201561A (en) * | 2021-05-08 | 2021-08-03 | 扬州大学 | Cell line construction method for accurately knocking out W38 amino acid of chicken NHE1 gene |
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| CN113201561A (en) * | 2021-05-08 | 2021-08-03 | 扬州大学 | Cell line construction method for accurately knocking out W38 amino acid of chicken NHE1 gene |
| CN113151277A (en) * | 2021-05-10 | 2021-07-23 | 安徽农业大学 | Construction method of chicken DF-1 cell IHH gene knockout stable cell strain and specific sgRNA thereof |
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