利用基于CRISPR-Cas9 的基因编辑技术对miR-29a在GT1-7 细胞中进 行基因敲除

目的:本文利用CRISPR-Cas9技术在GT1-7细胞中对miR-29a基因进行基因编辑,用于构建miR-29a基因敲除GT1-7细胞模型。方法:通过构建Cas9稳转的GT1-7细胞株并转染sgRNA质粒用于在靶向miR-29a基因区域引发突变。然后构建EGFP与sgRNA共表达质粒并转染Cas9稳转GT1-7细胞,利用流式细胞仪富集表达绿色荧光蛋白的阳性细胞和分选阳性单克隆细胞。最后利用实时荧光定量PCR(realtimefluorescencequantitativePCR)对富集细胞和单克隆细胞进行miR-29a表达量检测。结果:T7E1检测结果显示CRISPR-Cas9系统有效地在miR-29a基因区域引发了突变。荧光定量PCR结果显示,与对照组相比,富集后阳性细胞miR-29a的表达量整体下降了50%左右(P0.05)。此外,通过流式筛选获得了一个纯合miR-29a基因敲除细胞克隆,与对照组相比,其miR-29a的表达量下降了75%左右(P0.05)。结论:本文建立了一种有效编辑GT1-7细胞基因的方法,并采用该方法构建了miR-29a稳定敲除细胞模型。

The Gene Knockout of miR-29a in GT1-7 Cells based on Gene Editing by CRISPR-Cas9 System

Objective:CRISPR-Cas9 technology was used to edit miR-29a gene in GT1-7 cells and construct miR-29a knockout GT1-7 cell model.Methods:To induce mutation on miR-29a loci, Cas9 steadily expressed GT1-7 cells were constructed and transfected by sgRNA plasmids. Then a plasmid co-expressing EGFP and sgRNA was constructed and transfected into Cas9 expressing cells, flow cytometry was applied to enrich EGFP positive cells and test positive Monoclonal cell. Finally RT-PCR was used to detect the expression levels of miR-29a in enriched cells and Monoclonal cell.Results:Mutation could be efficiently induced on miR-29a loci by CRISPR-Cas9 systemdetected by T7E1 assays. Compared with control group, the expression of miR-29a in enriched positive cells totally decreased by 50 percent (P<0.05) using RT-PCR. Furthermore, one homogeneous miR-29a gene knockout cell clone was obtained by flow screening. The miR-29a expression of this cell clone decreased by 75 percent (P<0.05)compared with control group.Conclusion:An effective gene edit- ing method in GT1-7 cells was established, which constructed a miR-29a steadily knockout cell model.