CRISPR/Cas9 targeted knockout of the human TCab1 gene and its specific gRNA
A specific, genetic technology, applied in the field of molecular biology and biomedicine, can solve the problems of cancer cell death, cancer cell apoptosis, telomere formation and maintenance limitation, etc.
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Embodiment 1
[0017] Example 1 Synthesis of gRNA targeting human TCAB1 gene and construction of vector
[0018] 1. Selection and design of gRNA targeting human TCAB1 gene
[0019] The sequence of human TCAB1 gene was found in Genebank, and potential target sites were designed in the exon region of human TCAB1 gene. Through the online design tool (http: / / crispr.mit.edu / ) and the design principles of gRNA, the target site with high score on the human TCAB1 gene sequence is evaluated to design gRNA, and the target site sequence is SEQ ID NO.1-SEQ ID NO. ID NO.6, and design the corresponding oligonucleotide.
[0020] 2. Synthesis of gRNA oligonucleotide sequence targeting human TCAB1 gene and construction of eukaryotic expression vector
[0021] The pSpCas9(BB)-2A-GFP(PX458) plasmid (Addgene plasmid ID: 48138, hereinafter referred to as pSpCas9(BB)) was digested with BbSI, and after 1 hour in a water bath at 37°C, 1% agarose electrophoresis was used to recover the digestion. product (TAKARA ...
Embodiment 2
[0034] Example 2 Lipotransfection of HepG2 cells
[0035] Three days before transfection, revive human hepatoma cells (HepG2 cells, Shanghai Cell Bank, Chinese Academy of Sciences), put the cells into culture flasks with 10% FBS+DMEM, and store them at 37°C in 5% CO. 2 Cultured in an incubator, the day before transfection, subculture the recovered cells.
[0036]Aspirate the medium in the T75 flask for culturing HepG2 cells, add 2 ml of 0.25% trypsin taken out of the refrigerator at 4°C to evenly cover the bottom of the flask, place it in a 37°C incubator for 3-5 minutes, take it out, and shake to find that the cells are The bottom is detached, shake it all off, add 3ml of 10% DMEM preheated in a 37°C water bath, pipette with a 10ml pipette, pipette 6-8 times, without leaving dead corners, the bottle mouth is difficult to pipette and pipette The tube is aligned with the opening, and the medium is poured out with a small force to cover the cells close to the opening of the bot...
Embodiment 3
[0047] Example 3 PCR product cloning and sample sequencing detection
[0048] The PCR reaction was carried out according to the method of Example 2, and the PCR product was purified with the TAKARA kit and then connected to the PMD18-T carrier. The connection system was:
[0049]
[0050] Ligation for 2 hours at 16°C. Take the competent cell DH5α, put it on ice to thaw for 5min, add 10ul of the ligation product, blow evenly, and place it on ice for 20min. Heat shock at 42°C for 90s, quickly transfer to an ice bath and let stand for 3min, add 500ul of LB liquid medium, place in a shaker, 37°C 180rpm for 1h. Take 100ul of bacterial liquid and spread it evenly on LB solid medium (containing 1 / 1000AMP), and culture at 37°C overnight.
[0051] Pick 3 single colonies and put them into 3ml LB liquid medium (containing 3ul AMP) respectively, at 37°C and 200rpm for 12h. PCR identification was carried out with 1ul bacterial solution as a template, and all were positive. Bacteria ...
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