稳定表达Cas9蛋白的SW620细胞株构建

摘要 目的：建立稳定表达Cas9蛋白的SW620人结肠癌细胞系的单克隆细胞株，提高基因编辑效率，为利用基于CRISPR/Cas9技术的高通量筛选结肠癌相关致病基因提供细胞工具。方法：用Cas9慢病毒侵染SW620细胞系，用致死剂量的puro筛选5-7天，通过有限稀释法获得单克隆细胞株。提取单克隆细胞基因组进行Sanger测序，筛选出含有Cas9基因序列的单克隆细胞株。利用基于SSA修复荧光素酶的报告系统检测单克隆细胞株中Cas9的编辑活性，并通过细胞增殖实验检测Cas9蛋白的表达是否影响细胞增殖。结果：获得了两个表达Cas9蛋白的SW620单克隆细胞株，并通过Sanger测序验证了Cas9序列；荧光素酶报告系统检测显示单克隆细胞株的Cas9蛋白有较高的编辑活性；细胞增殖实验显示Cas9蛋白的表达对SW620增殖活性影响不大。结论：本研究利用慢病毒感染的方式，构建了稳定表达Cas9蛋白的SW620单克隆细胞株，为后续大规模筛选与人结肠癌相关的基因突变提供了细胞工具。

Construction of Monoclonal SW620 Stably Expressing Cas9

ABSTRACT Objective: Establish monoclonal SW620 cell lines stably expressing Cas9 to improve gene editing efficacy and provide cellular tools for CRISPR/Cas9-based high-throughput screening. Methods: We used Cas9 lentivirus to infect the SW620 cell line and used lethal doses of puromycin to screend the SW620 cell line for 5-7 days. The genomes of monoclonals were extracted for Sanger sequencing to detect the Cas9 fragment. We determined the editing activity of Cas9 in monoclonals by the single strand annealing (SSA) based luciferase reporter assay, and used the cell proliferation assay to determine whether Cas9 expression affected cell proliferation. Results: We obtained two monoclonals of SW620 expressing Cas9 and verified the Cas9 fragment by Sanger sequencing; the SSA based reporter assay showed high editing activity of Cas9; and the cell proliferation assay showed that Cas9 expression had little effect on the proliferation of SW620. Conclusion: In this study, we constructed a monoclonals of SW620 stably expressing Cas9 using lentiviral infection, thus providing a cellular tool for subsequent large-scale screening of genetic mutations associated with human colon cancer.