利用改良的CRISPR/Cas9基因编辑系统构建HeLa细胞SND1基因敲除稳定株

目的:利用改良的CRISPR/Cas9基因编辑系统敲除HeLa细胞中SND1基因，构建HeLa细胞SND1基因敲除稳定株。方法:设计一对特异性识别SND1基因第二个启动子的上下游sgRNA，以PX462质粒为载体，构建出一对重组真核表达质粒。酶切和测序鉴定后，将一对重组质粒共同转染进入HeLa细胞中，使用嘌呤霉素进行阳性细胞筛选，挑取单克隆细胞进行培养。最后用Western Blot鉴定敲除效果。结果:sgRNA正确插入到PX462质粒载体中，转染并筛选单克隆后的细胞中没有SND1蛋白的表达。结论:成功构建出HeLa细胞SND1基因敲除稳定株。

Construction of HeLa SND1 knockout gene stable strain by using modified CRISPR/Cas9 gene editing system

Objective: To Apply modified CRISPR/Cas9 gene editing system to knock out the SND1 gene in HeLa cell and construct HeLa SND1 gene knockout stable strain. Methods: A pair of sgRNAs that could specially identify the upstream and downstream of SND1 gene second promoter was designed, then a recombinant eukaryotic expressional plasmid by the carrier of PX462 was constructed. After enzyme digestion and sequencing, a pair of recombinant plasmids into HeLa cell was co-transfected, then puromycin was used to screen positive cell and the monoclonal cell was developed. The knockout effect was measured by western blotting. Results: sgRNA was correctly inserted into the PX462 recombinant plasmid, and no SND1 protein was detected in HeLa cell after transfection and screening of monoclonal cell. Conclusion: HeLa SND1 gene knockout stable strain can be successfully built