CN110684736A - A cell line knocking out chicken Shp-2 gene based on CRISPR-Cas9 editing technology and its construction method - Google Patents

Abstract

The present invention relates to a cell line for knocking out chicken Shp-2 gene based on CRISPR-Cas9 editing technology and a construction method thereof. Gene sgRNA, then clone the sgRNA into the lentiCRISPR v2 plasmid with the Cas9 gene and transfect the plasmid into the cells, use the CRISPR-Cas9 system to achieve gene silencing, and finally obtain a positive monoclonal cell line through drug screening and subcloning. , thereby obtaining a chicken-derived Shp-2 knockout cell line. The purpose of the present invention is to construct a method for knocking out chicken Shp-2 gene based on CRISPR-Cas9, and the sgRNA targeting chicken Shp-2 gene, in order to obtain the cell model of chicken Shp-2 gene knockout, using research on related diseases. The invention based on the CRISPR-Cas9 targeted knockout of the chicken Shp-2 gene cell line construction has not been reported in the related field, the invention will fill the gap of related technologies at home and abroad, and has great application research value.

Description

A cell line knocking out chicken Shp-2 gene based on CRISPR-Cas9 editing technology and how to build it

technical field

The invention relates to a cell line for knocking out chicken Shp-2 gene based on CRISPR-Cas9 editing technology and a construction method thereof, belonging to the technical field of genetic engineering.

Background technique

The CRISPR-Cas system is an acquired immune system that exists widely in bacteria and archaea, and plays an important role in bacterial resistance to foreign pathogens and plasmid transformation. At present, it has been found that there are 6 types of CRISPR-Cas I-VI, and CRISPR/Cas9 is transformed from the type II system. The CRISPR/Cas9 system consists of Cas9 protein with nuclease activity, crRNA transcribed from CRISPR, and tracrRNA complementary to the transactivated CRISPR repeat region. Among them, crRNA combines with tracrRNA through base pairing to form double-stranded RNA, recruits Cas9 protein to the target gene site to exert endonuclease activity, and specifically cuts the target double-stranded DNA upstream of the PAM sequence at the 3' end of the target sequence, resulting in DNA double-stranded Doublestrand break (DSB, doublestrand break), thereby starting the DNA damage repair mechanism in the cell, resulting in the occurrence of base deletion or insertion after repair, resulting in frameshift mutation, and finally achieving the purpose of gene knockout. At present, CRISPR/Cas9 gene editing technology has been widely used in prokaryotic and eukaryotic gene editing.

Protein tyrosine phosphatase Shp-2 (SH2 domain-containing protein-tyrosinephosphatase-2, Shp2) is an intracellular non-receptor tyrosine phosphatase that is ubiquitously expressed in various vertebrate cells. Shp-2 can negatively regulate downstream cell signaling pathways through the catalytic activity of phosphatase, and can also accept cytokines and extracellular stimuli to play a positive regulatory role, thus widely participating in many important cell life activities such as cell differentiation and migration. related signal transduction processes. Numerous studies have shown that changes in the expression and activity of Shp-2 are related to malignant proliferation of hematopoietic cells, human Noonan syndrome, childhood leukemia, a variety of human malignant tumors, autoimmune diseases, and the invasion of some virus infections. Few studies have been reported on the source Shp-2 function. The construction of a chicken-derived Shp-2 knockout cell line is very important to explore the role of chicken-derived Shp-2 in avian diseases. In this study, using CRISPR/Cas9 technology, through drug screening, subcloning and other methods, a chicken liver cell line with stable knockout of Shp-2 gene was successfully obtained, which was used to study the biological function of chicken-derived Shp-2 and its role in related diseases. effect.

SUMMARY OF THE INVENTION

The purpose of the present invention is to construct a chicken-derived Shp-2 knockout cell line. A cell line for knocking out chicken Shp-2 gene based on CRISPR-Cas9 editing technology and its construction method are provided. The principle and the most core key technology of the present invention are the scientific and reasonable construction of sgRNA targeting chicken Shp-2 gene. , and then clone the sgRNA into the lentiCRISPR v2 plasmid with Cas9 gene and transfect the plasmid into the cells, use the CRISPR-Cas9 system to achieve gene silencing, and finally obtain a positive monoclonal cell line through drug screening and subcloning, thereby obtaining Chicken-derived Shp-2 knockout cell line.

The object of the present invention is achieved in this way, a cell line based on CRISPR-Cas9 editing technology knocking out chicken Shp-2 gene and its construction method, are characterized in that, comprise the following steps:

Step 1), construct a kind of sgRNA of specific targeting chicken Shp-2 gene, described sgRNA is positioned at the second exon region of chicken Shp-2 gene, and the target sequence is unique;

Step 2), prepare a CRISPR-Cas9 system for targeted knockout of chicken Shp-2 gene, the CRISPR-Cas9 system contains Cas9 protein and the above-mentioned sgRNA specifically targeted to knock out chicken Shp-2 gene, or contains Carry the coding sequence encoding the Cas9 protein and the coding sequence encoding the sgRNA; in the CRISPR-Cas9 system, the Cas9 protein gene and the sequence of the sgRNA are located on the same vector, and the vector is the lentiCRISPR v2 plasmid;

Step 3), clone the sgRNA constructed in step 1) with the specific targeting knockout chicken Shp-2 gene into the lentiCRISPR v2 plasmid with the Cas9 gene, and transfect the lentiCRISPR v2 plasmid into the cell;

The CRISPR-Cas9 system was used to achieve gene silencing, and a positive monoclonal cell line was finally obtained through drug screening and subcloning, thereby obtaining a chicken-derived Shp-2 knockout cell line.

In step 1), five sgRNAs were designed for the chicken Shp-2 gene, and the target site of the sgRNAs was located in the second exon of the Shp-2 gene, and the exon sequence was shown in SEQ ID NO.1.

Five sgRNA coding strands and complementary strands specifically targeting chicken Shp-2 gene, the sequences of which are shown in SEQ ID NO.2.

In step 1), the sgRNA preparation method of targeting chicken Shp-2 gene is:

Including annealing the synthetic sgRNA coding strand and complementary strand to form double-stranded DNA, and then cutting the vector with BsmBI restriction endonuclease, and placing the double-stranded DNA under the T7 promoter to construct and obtain.

In step 3), the lentiCRISPR v2 plasmid was transfected into chicken hepatocytes to obtain a chicken-derived Shp-2 knockout cell line, and the cell line was a chicken hepatocyte knockout chicken Shp-2 gene.

The method of the invention is advanced and scientific, and provides a chicken Shp-2 gene knockout cell line based on CRISPR-Cas9 editing technology and a construction method thereof. First, an sgRNA specifically targeting the chicken Shp-2 gene is provided. The sgRNA described is located in the second exon region of the chicken Shp-2 gene, and has a unique target sequence. The sgRNA is shown in Table 1. The sgRNA targets the target site of the chicken Shp-2 gene, and its sequence is shown in SEQ ID NO.1. A CRISPR-Cas9 system for targeted knockout of chicken Shp-2 gene, which contains Cas9 protein and the above-mentioned sgRNA specifically targeting chicken Shp-2 gene in the system, or contains a code carrying a coding Cas9 protein Sequences and coding sequences encoding sgRNAs. Wherein, the coding sequence of Cas9 protein and the coding sequence of sgRNA are located on the same plasmid, and the plasmid is lentiCRISPR v2.

The specific operation steps are as follows:

Use the ensembl online database to find the chicken Shp-2 genome sequence ENSGALG00000004821.5, Transcript ID: ENSGALT00000007704.4, and design the knockout target site in the second exon of the gene. The sequence of the first exon is as SEQ ID NO.1 is shown.

Construction of sgRNA: Dilute the designed sgRNA coding strand and complementary strand to a concentration of 50 μmol/L respectively. The system is as follows: take 5 μL of each of the forward and reverse sgRNA primers, 5 μL of 10×NEB buffer, and 35 μL of high-pressure deionized water. The annealing program was 95°C for 5 min, then decreased by 5°C every 1 min from 70°C until 25°C. In addition, the plasmid lentiCRISPRv2 was digested using the BsmBI restriction endonuclease line. The restriction enzyme digestion system was as follows: BsmBI restriction endonuclease 1uL, lentiCRISPR v2 plasmid 1ug, 10×NEB buffer 3uL, and the rest were supplemented with sterilized deionized water to 30uL. The digestion conditions were 37°C for 2 h. After the end of the digestion, the digestion identification was carried out by agarose gel electrophoresis, and the lentiCRISPR v2 vector fragment containing the sticky ends was recovered and obtained. Finally, connect the annealed and diluted double-stranded sgRNA to the linearized lentiCRISPRv2 vector. The ligation system is as follows: 50 ng of linearized lentiCRISPRv2 vector, 4 μL of 20-fold diluted double-stranded sgRNA, 4 μL of 10×T4 DNA ligasebuffer, and 1 μL of T4 DNA ligase , 2uL of sterilized deionized water. Finally, a plasmid containing sgRNA and Cas9 protein gene in the same vector was obtained, named lentiCRISPRv2-sgRNA.

Obtainment of knockout cell line: cells in 6-well plate were transfected with constructed lentiCRISPRv2-sgRNA, 4ug plasmid per well, and pcDNA3.1 plasmid was transfected as negative control. After 48 hours, the medium containing puromycin was added for drug screening, and the dressing was changed every 48 hours. After all the negative control cells died, the medium was replaced with normal medium. After the surviving cells grew to an appropriate density, some cells were collected for knockout effect. For identification, the cells with good knockout effect were subcloned into 96-well plates by limiting dilution method, and the identification was performed when the cells grew to a sufficient number, and the positive cells were cryopreserved.

The purpose of the present invention is to construct a method for knocking out chicken Shp-2 gene based on CRISPR-Cas9, and its sgRNA targeting chicken Shp-2 gene, so as to obtain a cell model of chicken Shp-2 gene knockout. research on related diseases. The invention based on CRISPR-Cas9 targeted knockout of chicken Shp-2 gene cell line construction has not been reported in the related field, the invention will fill the gap of related technologies at home and abroad, and has great application research value.

Description of drawings

Fig. 1 is the restriction enzyme digestion of lentiCRISPR v2 vector plasmid;

Lane M: super DNA marker; Lane 1: lentiCRISPR v2 vector plasmid after digestion;

Fig. 2 is the verification of the knockout effect of the recombinant plasmid;

Lanes 1-5 are cell lysate samples collected by transfection of lentiCRISPRv2-sgRNA1-5; Lane 6: negative LMH cells;

Figure 3 is the identification of the 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.

Figure 4 shows the genome sequencing analysis of LMH-KO-Shp-2 cell line.

Detailed ways

The present invention will be described in detail below with reference to the accompanying drawings.

1. Find the target gene: Use the ensembl online database to find the chicken Shp-2 genome sequence ENSGALG00000004821.5, Transcript ID: ENSGALT00000007704.4, and design the knockout target site in the second exon of the gene, and the first exon The sequence of the sub is shown in SEQ ID NO.1.

2. Design of sgRNA and synthesis of oligonucleotide chain: use the website (http://crispr-era.stanford.edu/index.jsp and http://crispr.mit.edu) to analyze the specific sgRNA score , 5 sgRNAs (sgRNA-1, sgRNA-2, sgRNA-3, sgRNA-4, sgRNA-5) in the second exon with the highest score were selected as candidate sequences. CACC was added to the 5' end of the sgRNA sense strand template; C was added to the 5' end of the antisense strand template, and AAAC was added to the 3' end, which matched the sticky ends after lentiCRISPRv2 digestion. A total of 5 primers were designed, which were designed by Suzhou Gold. Synthesized by Weizhi Company, the specific primer sequence information is shown in Table 1.

Table 1 5 sgRNA sequences targeting the Shp-2 gene

3. Obtaining double-stranded sgRNA: Dilute the designed sgRNA coding strand and complementary strand with sterile deionized water to a concentration of 50 μmol/L, respectively. , 35 μL of sterilized deionized water. The annealing program was 95°C for 5 min, then decreased by 5°C every 1 min from 70°C until 25°C.

4. Restriction of the vector plasmid: Use the BsmBI restriction endonuclease line to digest the plasmid lentiCRISPRv2. The restriction system is as follows: BsmBI restriction endonuclease 1uL, lentiCRISPR v2 plasmid 1ug, 10×NEB buffer 3uL, and the rest are supplemented with ddw 30uL digestion conditions were 37℃ for 2h. After the digestion was completed, the digestion was identified by agarose gel electrophoresis (see Figure 1), and the digestion products containing sticky ends were recovered from the gel.

5. Construction of recombinant plasmid expressing sgRNA: Dilute the annealed double-stranded sgRNA by 20 times, and at the same time dilute the linearized vector plasmid recovered from the gel to 50ng/uL, and perform ligation and recombination according to the following system: linearized lentiCRISPRv2 vector 2uL, double-stranded 4uL of sgRNA, 1uL of 10×T4 DNA ligase buffer, 1uL of T4 ligase, 2uL of ddw, ligated overnight at 16°C, then transformed the ligation product into Stbl3 competent cells, picked colonies for preliminary identification by PCR, and then extracted the plasmid and sent it to the company Sequencing and re-identification.

6. Verification of the knockout effect of the recombinant plasmid: prepare a 6-well plate of LMH cells, and when the cell density reaches about 90%, transfect each well with 6ug of the recombinant plasmid expressing sgRNA, and set the non-transfection group as negative control. After 6 hours of transfection, it was changed to growth medium containing 10% fetal bovine serum. After 24 hours, the original medium was discarded and replaced with 10% medium containing 8 μg/mL puromycin for screening. The 10% growth medium of puromycin was changed to 10% growth medium without drug after all the cells in the negative control group died, and some of the remaining viable cells were collected for cell lysis, and Western blot was used to detect the expression level of Shp-2 protein (see Figure 2), the rest of the cells continue to be cultured.

7. Screening and identification of LMH cell lines that stably knocked out Shp-2: Subcloning the surviving cells corresponding to the sgRNA recombinant plasmid with better knockout effect, and expanding the culture when the subcloned cells grow to a sufficient density, The cells were collected for Western blot to detect the expression level of Shp-2 protein (see Figure 3 for the results), and then the genomes of the obtained knockout Shp-2 monoclonal cell lines were extracted for sequencing and identification of the knockout target sequence (see Figure 4 for the results).

SEQ ID NO.1

Second exon sequence of chicken Shp-2 gene:

ATGGTTTCATCCAAATATCACTGGGGTGGAGGCAGAAAATCTACTGTTAACAAGAGGAGTTGATGCAGTTTTCTGGCACGGCCCAGCAAAAGTAACCCAGGAGACTTCACACTCTCT GTTAG

SEQ ID NO.2

5 sgRNA sequences targeting the Shp-2 gene

sequence listing

<110> Yangzhou University

<120> A kind of chicken Shp-2 gene knockout cell line based on CRISPR-Cas9 editing technology and its construction method

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 1

caccgtggtt tcatccaaat atcac 25

<210> 2

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 2

aaacgtgata tttggatgaa accac 25

<210> 3

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 3

caccgggttt catccaaata tcact 25

<210> 4

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 4

aaacagtgat atttggatga aaccc 25

<210> 5

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 5

caccggtttc atccaaatat cactg 25

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 6

aaaccagtga tatttggatg aaacc 25

<210> 7

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 7

caccgtcatc caaatatcac tgggg 25

<210> 8

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 8

aaacccccag tgatatttgg atgac 25

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 9

caccggcctc caccccagtg atatt 25

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 10

aaacaatatc actggggtgg aggcc 25

<210> 11

<211> 123

<212> DNA

<213> Artificial Sequence

<400> 11

atggtttcat ccaaatatca ctggggtgga ggcagaaaat ctactgttaa caagaggagt 60

tgatggcagt tttctggcac ggcccagcaa aagtaaccca ggagacttca cactctctgt 120

tag 123

Claims (5)

1. a cell line based on CRISPR-Cas9 editing technology knockout chicken Shp-2 gene and its construction method, is characterized in that, comprises the following steps: Step 1), construct a kind of sgRNA of specific targeting chicken Shp-2 gene, described sgRNA is positioned at the second exon region of chicken Shp-2 gene, and the target sequence is unique; Step 2), prepare a CRISPR-Cas9 system for targeted knockout of chicken Shp-2 gene, the CRISPR-Cas9 system contains Cas9 protein and the above-mentioned sgRNA specifically targeted to knock out chicken Shp-2 gene, or contains Carry the coding sequence encoding the Cas9 protein and the coding sequence encoding the sgRNA; in the CRISPR-Cas9 system, the Cas9 protein gene and the sequence of the sgRNA are located on the same vector, and the vector is the lentiCRISPR v2 plasmid; Step 3), clone the sgRNA constructed in step 1) with the specific targeting knockout chicken Shp-2 gene into the lentiCRISPR v2 plasmid with the Cas9 gene, and transfect the lentiCRISPR v2 plasmid into the cell; The CRISPR-Cas9 system was used to achieve gene silencing, and a positive monoclonal cell line was finally obtained through drug screening and subcloning, thereby obtaining a chicken-derived Shp-2 knockout cell line. 2. a kind of cell line of knockout chicken Shp-2 gene based on CRISPR-Cas9 editing technology according to claim 1 and its construction method, it is characterized in that, in step 1), chicken Shp-2 gene is designed. Five sgRNAs, the target site of sgRNA is located in the second exon of Shp-2 gene, and the exon sequence is shown in SEQ ID NO.1. 3. a kind of cell line of knockout chicken Shp-2 gene based on CRISPR-Cas9 editing technology according to claim 2 and its construction method, it is characterized in that, 5 sgRNAs of specific targeting chicken Shp-2 gene The sequences of the coding strand and complementary strand are shown in SEQ ID NO.2. 4. a kind of cell line of knockout chicken Shp-2 gene based on CRISPR-Cas9 editing technology according to claim 1 and its construction method, it is characterized in that, in step 1), targeting chicken Shp-2 gene. The sgRNA preparation method is: Including annealing the synthetic sgRNA coding strand and complementary strand to form double-stranded DNA, and then cutting the vector with BsmBI restriction endonuclease, and placing the double-stranded DNA under the T7 promoter to construct and obtain. 5. a kind of cell line based on CRISPR-Cas9 editing technology knockout chicken Shp-2 gene and construction method thereof according to claim 1, it is characterized in that, step 3) in, the lentiCRISPR v2 plasmid is transfected into chicken In the hepatocytes, a chicken-derived Shp-2 knockout cell line was obtained, and the cell line was chicken hepatocytes knocked out of the chicken Shp-2 gene.

Images