Construction of Stable HEK293T Cell with Cyr61 Knocked Down Based on CRISPR/Cas9 Technology and Its Biological Function Detection
WANG Junjie1, JIANG Yan2, Soulixay Senouthai1, FU Dongdong1, YOU Yanwu1*
To investigate the biological function of Cyr61 (Cysteine-rich protein 61, Cyr61), HEK293T cell stable strain which knocked down Cyr61 was constructed by the technology of CRISPR/Cas9, detected the effect of Cyr61 on the proliferation and apoptosis in vitro in present study. According to the principle of CRISPR/Cas9 target design, three pairs of gRNA of Cyr61 were designed online at http://crispr.mit.edu/. LentiCRISPRv2 was used as the vector to construct lenticrisprv2-gRNA recombinant plasmid and transformed into Stbl3. Then the recombinant plasmids were screened and packaged as Cyr61 CRISPR/Cas9 KO plasmid. Transfected the HEK293T cells by CRISPR/Cas9 KO plasmid and HDR plasmid and selected by puromycin (8 μg/mL). HEK293T knockeddown Cyr61 cell strain was identified by quantitative PCR and Western blot. The HEK293T cells were cultured normally, then detected the proliferation by cell counting kit (CCK8), and detected the apoptosis by flow cytometry. Cyr61 knocked down HEK293T cell strain was successfully constructed. Compared with the control group, the proliferation of Cyr61 knocked down cell strain was significantly increased (P<0.05), and the apoptosis rate was significantly decreased (P<0.05). The present study demonstrated that Cyr61 knocked down HEK293T stable cell strain was successfully constructed through CRISPR/Cas9 gene editing system, which providing a useful tool for the study of Cyr61 gene.
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