基于episomal CRISPR/Cas9载体的基因敲入方法  

An Episomal Vector-based CRISPR/Cas9 System for Gene Knockin

作　　者：邹征伟 赖兴强[1] 李伟强[1] ZOU Zheng-wei;LAI Xing-qiang;LI Wei-qiang(Center for Stem Cell Biology and Tissue Engineering,Zhongshan School of Medicine,Sun Yat-sen University,Guangzhou 510080,China)

机构地区：[1]中山大学中山医学院干细胞与组织工程研究中心,广东广州510080

出　　处：《中山大学学报（医学版）》2018年第5期660-668,共9页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(81570487);中山大学青年教师重点培育项目(17ykzd07)

摘　　要：【目的】研究一种在哺乳动物细胞中高效的基因敲入方法,比较带有oriP-EBNA1序列的episomal CRISPR/Cas9载体与普通CRISPR/Cas9载体的基因敲入效率是否有差别。【方法】首先利用慢病毒载体构建稳定表达红色荧光蛋白DsRedE2的人胚肾细胞系(293FT)与人诱导多能干细胞系(hiPS);利用CRISPR/Cas9序列设计网站,设计和合成靶向DsRedE2的向导RNA(sgRNA),分别克隆至带有oriP-EBNA1的episomal CRISPR/Cas9敲除载体与普通CRISPR/Cas9敲除载体中,测序筛选插入正确片段的克隆;然后设计靶向DsRedE2的同源臂,把同源臂连接在绿色荧光蛋白(GFP)的两端,构建GFP敲入片段的载体。把两种敲除载体分别导入到DsRedE2-293FT与DsRedE2-hiPS细胞系中,在不同时间点通过荧光显微镜观察以及流式细胞分析比较细胞的敲除效率,同时利用PCR检测靶基因的敲除情况;在筛选获得有效的sgRNA片段后,把两种敲除载体和同源臂同时导入到DsRedE2-293FT与DsRedE2-hiPS细胞系中,在不同时间通过荧光显微镜观察以及流式细胞分析比较细胞的敲入效率,同时利用PCR检测DsRedE2序列中GFP的敲入情况。【结果】成功构建表达DsRedE2的293FT与hiPS细胞系;获得靶向DsRedE2的episomal CRISPR/Cas9敲除载体、普通CRISPR/Cas9敲除载体及带有GFP的同源臂载体;荧光显微镜观察与流式细胞分析结果显示,转染两种CRISPR/Cas9质粒均可以使DsRedE2阳性细胞明显减少,而且episomal CRISPR/Cas9质粒组的DsRedE2阳性细胞数量显著低于转染普通CRISPR/Cas9质粒组。同时,敲入实验证实episomal CRISPR/Cas9组的GFP阳性细胞数量比普通CRISPR/Cas9组更多;TA克隆测序证实打靶位点有敲除与敲入片段。【结论】episomal CRISPR/Cas9载体与普通CRISPR/Cas9载体在细胞上都能实现基因的敲除与敲入;带有oriP-EBNA1序列的episomal CRISPR/Cas9载体敲除和敲入的效率均比普通CRISPR/Cas9载体高。以上结果为利用episomal CRISPR/Cas9载体进�【Objective】To seek an efficient method for gene knockin in mammalian cells and compare the gene knockin efficiency of episomal CRISPR/Cas9 vector carrying orip-EBNA1 sequence with traditional CRISPR/Cas9 vector.【Methods】Firstly,the DsRedE2-expressing human embryonic kidney cell line(DsRedE2-293FT)and human induced pluripotent stem cells(DsRedE2-hiPS)cell lines were constructed by lentiviral vector transduction.Then we designed and synthesized the guide RNAs(single-guide RNA,sgRNA)for targeting DsRedE2.And the sgRNAs were cloned into the episomal CRISPR/Cas9 vector and traditional CRISPR/Cas9 vector,respectively.Then the homologous arms of DsRedE2 were designed,amplified by PCR and connected to the two ends of green fluorescent protein(GFP)to con-struct the DsRedE2-specific knockin fragment.Two kinds of CRISPR/Cas9 vectors,guide RNA vector,with or without homologous arms were transfected into the DsRedE2-293FT and DsRedE2-hiPS cell lines respectively,and the knockout/knockin efficiency was confirmed and measured by PCR,fluorescence microscopy and flow cytometry at different time points post transfection.【Results】293FT and hiPS cell lines expressing DsRedE2 were successfully constructed;The episomal CRISPR/Cas9 knockout vector,traditional CRISPR/Cas9 knockout vector and the homologous arm with GFP for targeting DsRedE2 were also obtained respectively.The results of fluorescence microscopy and flow cytometry showed that the number of DsRedE2 positive cells in episomal group was significantly lower than that in traditional CRISPR/Cas9 plasmid transfection group.Meanwhile,the number of GFP positive cells(successful GFP knockin)in the episomal CRISPR/Cas9 group was statistically higher than that of the traditional CRISPR/Cas9 group.TA cloning sequencing confirmed that the target site has knock-out and knock-in fragments.【Conclusions】Both type of CRISPR/Cas9 vectors are functional in gene knockout and knockin in mammalian cells and episomal CRISPR/Cas9 vector with orip-EBNA1 sequence is more ef-ficient t

关 键 词：CRISPR/Cas9 episomal载体 敲除 敲入 

分 类 号：R31[医药卫生—基础医学]