p53基因及NHEJ相关基因表达水平对基因组编辑效率的影响.doc

国际医药卫生导报 201年第25卷第9期 IMHGN May 2019 , Vol. 25 No. 9-科研课题・p53基因及NHEJ相关基因表达水平 对基因组编辑效率的影响于洋 孔明圣 雷颖首 徐畅 张亚倩 张一帆 张文峰 沈晗 邵红伟 广东药科大学生命科学与生物制药学院 广东省生物技术候选药物研究重点实验室，广州510006通信作者：邵红伟， Email : shaohongwei@.【摘要】 目的 研究CRISPR/Cas9基因组编辑系统在不同细胞中编辑效率的差异及相关的影响因 素方法 基于靶向人 TCRa链C区(TRA0基因的 CRISPR/Cas9重组载体分别对 293T、HEK-293、 HeLa、SW480 Jurkat、K56等细胞进行基因组编辑，检测编辑效率的差异同时检测 p53基因在不同细胞中的突变情况，通过 RT-qPCR分析参与非同源末端连接( NHEJ)通路的17个主要基因的表达情况结果 流式检测各细胞的转染效率： 29为30.50% , HEK-293为29.30% , Hela为17.70% , SW480为21.90% , Jurkat为13.08% , K562为18.51%。

根据克隆测序结果计算出各种细胞中基因组的编辑率： 293T 为 74.70%、HEK-293 为 68.00%、Hela为 13.47%、SW480为 32.70%、Jurkat为 65.52%、K562为 77.18%测序结果表明293T、Jurkat、K562胞的p53基因发生了突变，而 HEK-293、Hela和SW480的 p53基因为野生型qPCR结果显示，在编辑效率较高的细胞系中，大部分NHEJ相关基因的表达水平也较高， 且其表达水平在p53功能异常细胞系中要比在 p53功能正常细胞系中的高 结论 本研究发现p53的异常程度以及NHEJ相关基因的表达水平与基因组编辑效率存在相关性关键词】 CRISPR/Cas9 ;非同源重组末端连接； 基因突变；人类基因编辑； NH修复相关基因基金项目 国家自然科学基金(3110064 , 31300737 , 314000149 )广东省自然科学基金(2014A030313586 , 1051022401000024;广东省科技计划项目( 2016A020215157 广东省高等教育“创新强校工程"项目( 2017KZDXM049, 2017KCXTD020)DOI: 10.3760/cma.j.issn.1007-1245.2019.09.006Effect of expressions of p53 gene and NHEJ-related gene on genome editing efficiency Yu Yang, Kong Mingsheng, Lei Yingshou, Xu Chang, Zhang Yaqian, Zhang Yifan, Zhang Wenfeng, Shen Han, Shao HongweiCollege of Life Science and Biological Drug Preparation, Guangdong Pharmaceutical University, Guangzhou 510006, ChinaCorresponding author: Shao Hongwei, Email: shaohongwei@【Abstract 】 Objective To investigate the genome editing efficiency of CRISPR/Cas9 system in different cells and the influencing factors. Methods The recombinant pX458-TCRaC-S2 plasmid targeting human TCR alpha chain C region (TRAC) was transfected into 293T, HEK-293, HeLa, SW480, Jurkat, and K562 cells,respectively, and the genome editing efficiencies in different cells were detected. The mutation of p53 gene in each cell line was investigated by sequencing. The expression levels of 17 genes participated in non-homologous end joining (NHEJ) pathway were detected by RT-qPCR. Results The transfection efficiencies of 293T, HEK- 293, Hela, SW480, Jurkat, and K562 were 30.50%, 29.30%, 17.70%, 21.90%, 13.08%, and 18.51%, respectively. Based on the clone sequencing results, the genome editing efficiencies of 293T, HEK-293, Hela, SW480, Jurkat, and K562 were 74.70%, 68.00%, 13.47%, 32.70%, 65.52%, and 77.18%, respectively. The p53 gene was mutated in 293T, Jurkat, and K562 cells, but not in HeLa, SW480, and HEK-293 cells. The qPCR results showed that the1365。