摘要： 目的 通过常间回文重复序列丛集（CRISPR）/常间回文重复序列丛集关联蛋白9（Cas9）系统敲除技术靶向敲除人源结肠癌HCT116细胞中的SAMHD1基因，并初步研究敲除该基因后HCT116细胞增殖能力的变化。方法 利用单向导RNA（sgRNA）在线设计工具，针对SAMHD1设计sgRNA；利用pYSY-CMV-Cas9-EF1α-Puromycin质粒载体，构建pYSY-CMV-Cas9-EF1α-Puromycin-sgRNA-SAMHD1；转染HCT116细胞株，以嘌呤霉素进行筛选鉴定后，采用蛋白质印迹法检测HCT116细胞中SAMHD1蛋白表达水平；采用蛋白质印迹法检测靶向敲除SAMHD1基因后HCT116细胞株DNA损伤修复能力的变化；通过光学显微镜和CCK-8法比较Cas9-vector组（HCT116细胞中转染pYSY-CMV-Cas9-EF1α-Puromycin载体质粒，对照组）和Cas9-SAMHD1组（转染pYSY-CMV-Cas9-EF1α-Puromycin-sgRNA-SAMHD1靶向敲除SAMHD1质粒，实验组）的细胞数量和增殖能力水平。结果 通过酶切载体、退火产物结果和重组质粒的测序结果，验证重组质粒构建成功；蛋白质印迹法检测结果验证明利用CRISPR/Cas9系统技术靶向敲除SAMHD1的HCT116细胞株无SAMHD1蛋白表达，即Cas9-SAMHD1细胞株构建成功；蛋白质印迹法结果显示，Cas9-SAMHD1组与Cas9-vector组相比，在DNA损伤诱导剂阿霉素刺激作用下DNA损伤指标γH2AX降低；光学显微镜和CCK-8结果显示，Cas9-SAMHD1组与Cas9-vector组相比，细胞增殖能力显著增强（P<0.01）。结论 采用CRISPR/Cas9系统成功敲除人源结肠癌细胞SAMHD1基因后，结肠癌细胞的增殖能力和DNA损伤修复能力显著增强。

利用CRISPR/Cas9系统构建人源结肠癌SAMHD1基因敲除细胞株及其功能的初步研究

张洲, 操孙润, 武晋英, 马孟涛, 宋晓宇

中国医科大学转化医学研究院, 沈阳 110122

收稿日期:2019-04-01出版日期:2020-03-30发布日期:2020-03-19
通讯作者:宋晓宇E-mail:xysong@cmu.edu.cn
作者简介:张洲(1995-),男,硕士研究生.
基金资助:国家自然科学基金（31300963）；辽宁省高等学校基本科研项目（LFWK201725）；辽宁省重点研发计划指导计划项目（2018225083）；沈阳市科技计划（17-2311-83）


关键词: CRISPR/Cas9系统, 结肠癌, HCT116, SAMHD1, 细胞增殖
Abstract: Objective To specifically knockout SAMHD1 gene in HCT116 cells using CRISPR/Cas9 system,and to study the changes in the proliferation of the cells following the knockout. Methods The pYSY-CMV-Cas9-EF1α-Puromycin plasmid vector was used to construct pYSY-CMV-Cas9-EF1α-Puromycin-sgRNA-SAMHD1. Following transfection of HCT116 cells,the cells were selected using puromycin,and the expression of SAMHD1 protein was analyzed by Western blotting. Western blotting was used to detect the changes in DNA damage repair ability of HCT116 cells following knockout of SAMHD1. The changes in the number and the proliferative ability of the cells in the pYSY-CMV-Cas9-EF1α-Puromycin control (Cas9-vector) and pYSY-CMV-Cas9-EF1α-Puromycin-sgRNA-SAMHD1(Cas9-SAMHD1) groups were compared using optical microscopy and CCK-8 assay. Results The successful construction of the recombinant plasmid was verified using restriction enzyme digestion and sequencing. Western blotting analysis confirmed that the Cas9-SAMHD1 cells had no expression of SAMHD1 protein,indicating that the knockout strategy was successful. Following treatment with doxorubicin, Western blotting analysis showed that the expression of γH2AX was decreased in the Cas9-SAMHD1 group compared to the Cas9-vector group. Optical microscopy and CCK-8 assays showed that the proliferative ability of the cells in the Cas9-SAMHD1 group was significantly enhanced compared to those in the Cas9-vector group (P<0.01). Conclusion Knockout of the SAMHD1 gene using CRISPR/Cas9 system in HCT116 cells significantly enhanced their proliferation and resistance to DNA damage.
Key words: CRISPR/Cas9, colon cancer, HCT116, SAMHD1, cell proliferation
PDF全文下载地址:

https://journal.cmu.edu.cn/CN/article/downloadArticleFile.do?attachType=PDF&id=2452