中国实验动物学报2017，Vol.25Issue(6)：587-593,7.DOI:10.3969/j.issn.1005-4847.2017.06.001
基于CRISPR/Cas9系统定点编辑小鼠MAD2L1基因及其脱靶效应分析
Mouse MAD2L1 gene editing by CRISPR/Cas9 and analysis of its off-target effect
摘要
Abstract
Objective To establish a method for specific gene editing of the second exon of mouse MAD2L1 gene by CRISPR/Cas9, and analyze its off-target effect. Methods The gene editing site for MAD2L1 gene was designed by CHOP?CHOP, and the Cas9?MAD2L1 vector was constructed based on the designed editing site. Cas9?MAD2L1 was then transfected into NIH/3T3 cells and screened with puromycin, followed by observing GFP expression using fluorescence microscopy. The genomic DNA from transfected cells was extracted and a partial fragment of MAD2L1 gene was amplified by PCR. T7E1 analy?sis and Sangger sequencing were used for gene editing and off?target analysis. Results After Cas9?MAD2L1 transfection and puromycin screening, a large number of GFP?expressing cells were observed under the fluorescence microscope. Combined the PCR result with TE71 analysis, the amplified 228 bp PCR products can be digested into 166 bp and 62 bp fragments. The se?quencing result showed that the second exon of MAD2L1 gene was successfully edited, and the off?target effect was undetected in our system. Conclusions The method for specific gene editing of the second exon of mouse MAD2L1 gene by CRISPR/Cas9 is successfully established, and off?target effect of MAD2L1 gene is not detected.关键词
CRISPR/Cas9/小鼠/MAD2L1/脱靶效应Key words
CRISPR/Cas9/Mice/MAD2L1/Off-target effect分类
生物科学引用本文复制引用
陈丽香,彭秀华,谭丹,韩铖潇,赵锐,王超,李顺,周晓辉..基于CRISPR/Cas9系统定点编辑小鼠MAD2L1基因及其脱靶效应分析[J].中国实验动物学报,2017,25(6):587-593,7.基金项目
国家自然科学基金青年基金项目(No.31601908) （No.31601908）
上海市科技发展基金实验动物研究项目(No.15140904000) （No.15140904000）
上海市人才发展资金(No.2017043) （No.2017043）
上海市卫生和计划生育委员会科研课题项目(No.20154Y0075) （No.20154Y0075）
上海市公共卫生临床中心院内项目(No.2016-02,KY-GW-2017-04). （No.2016-02,KY-GW-2017-04）
