应用CRISPR/Cas9系统构建MTHFD1基因敲除的HEK-293细胞系

Construction of MTHFD1 Gene Knockout HEK-293 Cell Line by CRISPR/Cas9 Technology

目的:利用成簇的、规律间隔的短回文重复序列/Cas9核酸酶(CRISPR/Cas9)基因编辑技术构建亚甲基四氢叶酸脱氢酶1(methylenetetrahydrofolate dehydrogenase 1, MTHFD1))基因敲除人胚肾(HEK-293)稳定细胞系。方法:利用在线软件筛选出评分最高的3条针对MTHFD1基因的单向导RNA (sg RNA),然后合成sg RNA序列并将其插入到含有GFP标签的质粒中;重组质粒转染HEK-293细胞后通过流式细胞仪分选出已被转入sg RNA的单细胞,通过测序确认单克隆细胞系中MTHFD1的DNA序列突变状态;最后应用实时荧光定量多聚核苷酸链式反应(real-time quantitative Polymerase Chain Reaction, RT-q PCR)和蛋白质印迹(Western blot)方法检测单克隆细胞中MTHFD1的m RNA和蛋白表达水平。结果:重组载体中含有正确的sg RNA序列;测序结果显示该细胞系中MTHFD1基因发生了单个碱基插入突变和6个碱基的缺失突变;RT-qPCR结果显示单克隆细胞系中MTHFD1在m RNA水平显著降低;Western blot检测成功构建MTHFD1蛋白缺失的HEK-293细胞。结论:本研究利用CRISPR/Cas9技术成功构建的MTHFD1敲除HEK-293细胞系。

Objective: To construct a stable methylenetetrahydrofolate dehydrogenase 1(MTHFD1) gene knockout HEK-293 cell line by clustered regularly interspersed short palindromic repeats(CRISPR)/CRISPR associated protein 9(Cas9) technique. Methods:Three high grade single-guided RNAs(sg RNAs) targeting MTHFD1 gene were screened by online software, then synthesized and inserted into plasmids containing GFP tags. After transfection of recombinant plasmids into HEK-293 cells, monoclonal cells were obtained by flow cytometry. The mutation status of MTHFD1 DNA sequence in monoclonal cell lines was confirmed by sequence analysis. The m RNA and protein expressing levels of MTHFD1 in monoclonal cells were conducted by real-time quantitative Polymerase Chain Reaction(RT-q PCR) and Western blot, respectively. Results: The expected sg RNA sequence of the recombinant vector was obtained in the HEK-293 cell. Sequencing results showed that MTHFD1 gene had a single base insertion and six base deletion mutations in the cell line. RT-q PCR and Western blot results showed that the MTHFD1 m RNA expression level was significantly reduced and its protein expression level wasn’t detected in the cell line, respectively. Conclusion: The MTHFD1 gene knockout HEK-293 cell line was successfully constructed by CRISPR/Cas9.