南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (03): 320-.doi: 10.12122/j.issn.1673-4254.2019.03.10

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利用CRISPR/Cas9 技术构建敲除G6PD基因c.392G>T（p.131G>V）突变位点的HEK293T稳定细胞株

周燕霞，胡韦维，张虹洋，邹琳，张鹏辉   

  • 出版日期:2019-03-20 发布日期:2019-03-20

Establishment of a stable HEK293T cell line with c.392G>T (p.131G>V) mutation site knockout in G6PD gene using CRISPR/Cas9 technique

  • Online:2019-03-20 Published:2019-03-20

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摘要： 目的利用CRISPR/Cas9技术构建敲除G6PD基因常见突变位点——c.392G>T（p.131G>V）突变位点的HEK293T细胞， 为后期研究其基因修复提供可靠的细胞模型。方法针对G6PD基因c.392G>T（p.131G>V）突变位点靶向设计4对向导RNA （sgRNA），构建表达Cas9-sgRNA的外源PX458质粒，将其转染至HEK293T细胞内，通过流式细胞术分选表达GFP荧光蛋白的 细胞进行培养，利用T7核酸内切酶1（T7E1）酶切验证CRISPR/Cas9的剪切效率，有限稀释法筛选单克隆细胞，测序鉴定并检测 G6PD mRNA、蛋白表达和细胞功能的改变。结果成功构建基于G6PD基因c.392G>T（p.131G>V）突变位点的Cas9-sgRNA外 源PX458 质粒，T7E1 酶切检测四对sgRNA 的编辑效率分别为6.74%、12.36%、12.54%、2.94%。测序鉴定敲除G6PD 基因 c.392G>T（p.131G>V）突变位点的细胞株构建成功，细胞G6PDmRNA、蛋白表达和G6PD酶活性降低，细胞增殖能力下降，维生 素K3诱导细胞死亡增加。结论本研究成功构建敲除G6PD基因c.392G>T（p.131G>V）突变位点的HEK293T稳定细胞模型， 为后期研究基因的修复奠定了基础。

Abstract: Objective To establish a stable HEK293T cell line with c.392G>T (p.131G>V) mutation site knockout in G6PD gene using CRISPR/Cas9 technique. Methods We designed 4 pairs of small guide RNA (sgRNA) for G6PD c.392G>T(p.131G>V) mutation site, and constructed exogenous PX458 plasmids expressing Cas9-sgRNA. The plasmids were transfected into HEK293T cells, and the cells expressing GFP fluorescent protein were separated by flow cytometry for further culture. After verification of the knockout efficiency using T7 endonuclease Ⅰ, the monoclonal cells were screened by limiting dilution and DNA sequencing to confirm the knockout. We detected the expressions of G6PD mRNA and protein and examined functional changes of the genetically modified cells. Results We successfully constructed the Cas9-sgRNA exogenous PX458 plasmid based on the c.392G>T(p.131G>V) mutation site of G6PD gene. The editing efficiency of the 4 pairs of sgRNA, as detected by T7E1 enzyme digestion, was 6.74%, 12.36%, 12.54% and 2.94%. Sanger sequencing confirmed that the HEK293T cell line with stable knockout of G6PD c.392G>T(p.131G>V) was successfully constructed. The genetically modified cells expressed lower levels of G6PD mRNA and G6PD protein and showed reduced G6PD enzyme activity and proliferative capacity and increased apoptosis in response to vitamin K3 treatment. Conclusion We successfully constructed a stable HEK293T cell model with G6PD gene c.392G>T(p.131G>V) mutation site knockout to facilitate future study of gene repair.