| 基于CRISPR/Cas9系统定点编辑小鼠MAD2L1基因及其脱靶效应分析 |
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| 引用本文： | 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]. 实验动物与比较医学, 2017, 25(6): 587-593 |
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| 作者姓名： | 陈丽香 彭秀华 谭丹 韩铖潇 赵锐 王超 李顺 周晓辉 |
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| 作者单位： | 上海市公共卫生临床中心, 上海 201508,上海市公共卫生临床中心, 上海 201508,上海市公共卫生临床中心, 上海 201508,上海交通大学农业与生物学院, 上海 200240,上海市公共卫生临床中心, 上海 201508,上海市公共卫生临床中心, 上海 201508,上海市公共卫生临床中心, 上海 201508,上海市公共卫生临床中心, 上海 201508 |
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| 基金项目： | 国家自然科学基金青年基金项目（No.31601908）;上海市科技发展基金实验动物研究项目（No.15140904000）;上海市人才发展资金（No.2017043）;上海市卫生和计划生育委员会科研课题项目（No.20154Y0075）;上海市公共卫生临床中心院内项目（No.2016-02,KY-GW-2017-04）。 |
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| 摘 要： | 目的 建立利用CRISPR/Cas9系统对小鼠MAD2L1基因第二外显子基因编辑的方法体系,并分析CRISPR/Cas9对MAD2L1基因编辑的脱靶效应。方法 通过CHOPCHOP网站设计位于小鼠MAD2L1基因第二外显子的靶点序列,并构建Cas9-MAD2L1载体,将该载体转染到NIH/3T3细胞中,通过嘌呤霉素筛选并结合荧光显微镜观察GFP后,提取细胞转染后的基因组DNA,利用PCR方法,结合T7E1分析和桑格尔测序,鉴定对小鼠MAD2L1基因编辑情况,并对转染后的NIH/3T3细胞进行CRISPR/Cas9脱靶效应分析。结果 将Cas9-MAD2L1载体转染到NIH/3T3细胞并进行嘌呤霉素筛选后,荧光显微镜下观察到大量表达GFP的细胞,PCR结合T7E1结果显示,以转染后的细胞DNA为模板扩增出的228 bp MAD2L1 PCR产物,可被酶切成166 bp和62 bp片段,测序结果显示,成功对MAD2L1基因第二外显子靶点处进行基因编辑,脱靶效应分析未检测到CRISPR/Cas9有对脱靶位点进行基因编辑。结论 成功建立对小鼠MAD2L1基因第二外显子进行基因编辑的方法体系,设计的对MAD2L1基因编辑靶点未检测到脱靶效应的发生。
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| 关 键 词： | CRISPR/Cas9 小鼠 MAD2L1 脱靶效应 |
| 收稿时间： | 2017-05-22 |
Mouse MAD2L1 gene editing by CRISPR/Cas9 and analysis of its off-target effect |
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| CHEN Li-xiang,PENG Xiu-hu,TAN Dan,HAN Cheng-xiao,ZHAO Rui,WANG Chao,LI Shun and ZHOU Xiao-hui. Mouse MAD2L1 gene editing by CRISPR/Cas9 and analysis of its off-target effect[J]. Laboratory Animal and Comparative Medicine, 2017, 25(6): 587-593 |
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| Authors: | CHEN Li-xiang PENG Xiu-hu TAN Dan HAN Cheng-xiao ZHAO Rui WANG Chao LI Shun ZHOU Xiao-hui |
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| Affiliation: | Shanghai Public Health Clinical Center, Shanghai 201508, China,Shanghai Public Health Clinical Center, Shanghai 201508, China,Shanghai Public Health Clinical Center, Shanghai 201508, China,School of Agricalture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China,Shanghai Public Health Clinical Center, Shanghai 201508, China,Shanghai Public Health Clinical Center, Shanghai 201508, China,Shanghai Public Health Clinical Center, Shanghai 201508, China and Shanghai Public Health Clinical Center, Shanghai 201508, China |
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| 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 CHOPCHOP,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 analysis 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 sequencing 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. |
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| Keywords: | CRISPR/Cas9 Mice MAD2L1 Off-target effect |
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