基于CRISPR/Cas9技术构建Cyr61敲低HEK293T稳定细胞株及其生物学功能检测

Construction of Stable HEK293T Cell with Cyr61 Knocked Down Based on CRISPR/Cas9 Technology and Its Biological Function Detection

为了探讨Cyr61的生物学功能,该研究利用CRISPR/Cas9基因编辑技术构建Cyr61敲低的HEK293T稳定细胞株,并在体外研究Cyr61对HEK293T细胞增殖和凋亡的影响。根据CRISPR/Cas9靶点设计原则,应用http://crispr.mit.edu/在线设计3条Cyr61的导向RNA(guide RNA,gRNA),用LentiCRISPRv2作为载体构建LentiCRISPRv2-gRNA重组质粒并转化至感受态的Stbl3菌体中,经筛选后包装成Cyr61 CRISPR/Cas9 KO质粒,利用Cyr61 CRISPR/Cas9 KO质粒和HDR质粒转染HEK293T细胞,加入嘌呤霉素(8μg/mL)进行筛选,通过实时荧光定量PCR技术及蛋白质免疫印迹(Western blot)鉴定出HEK293T敲低Cyr61细胞株;常规培养细胞,利用细胞计数检测试剂盒(CCK8)检测细胞增殖情况,流式细胞技术检测细胞凋亡情况。该研究成功构建出Cyr61敲低HEK293T细胞株,与对照组相比,Cyr61敲低细胞株的增殖明显升高(P<0.05),凋亡率明显减少(P<0.05)。通过CRISPR/Cas9基因编辑系统成功构建Cyr61敲低HEK293T稳定细胞株,为研究Cyr61基因提供了有效的工具。

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(8μg/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.