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

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。

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

ABSTRACT 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 (sgRNAs) 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 mRNA and protein expressing levels of MTHFD1 in monoclonal cells were conducted by real-time quantitative Polymerase Chain Reaction (RT-qPCR) and Western blot, respectively. Results: The expected sgRNA 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-qPCR and Western blot results showed that the MTHFD1 mRNA 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.