基于CRISPR/Cas9系统高效编辑小鼠miR let-7a  

Efficient editing of mouse miR let-7a based on CRISPR/Cas9 system

作　　者：周岚 张生贵 胡祖梁 王添贤 岳鹏鹏 于鸿浩 ZHOU Lan;ZHANG Sheng-gui;HU Zu-liang;WANG Tian-xian;YUE Peng-peng;YU Hong-hao(Department of Cell and Genetics,School of Biotechnology,Guilin Medical University,Guilin 541104;Department of Radiology,the Second Affiliated Hospital,Guilin Medical University,Guilin 541199,China)

机构地区：[1]桂林医学院生物技术学院细胞与遗传学教研室,广西桂林541104 [2]桂林医学院第二附属医院放射科,广西桂林541199

出　　处：《基础医学与临床》2022年第6期857-863,共7页Basic and Clinical Medicine

基　　金：国家自然科学基金(31860302);广西自然科学基金(2018JJA140455);广西中青年教师能力提升项目(2018KY0399);广西科技计划项目(2019AC20329)。

摘　　要：目的构建靶向小鼠miR let-7a的高效CRISPR/Cas9系统。方法根据小鼠miR let-7a的两个编码位点let-7a-1和let-7a-2的编码序列各设计了两对dual-sgRNAs导向序列,通过sgRNA表达载体的构建、小鼠N2a细胞的共转染、药物筛选、阳性细胞PCR产物测序以及TA克隆测序分析等实验步骤完成了4对dual-sgRNAs导向序列的打靶效力分析。结果所构建的4对打靶载体在细胞中发挥了作用,打靶位点均出现了碱基插入或缺失,药物筛选阳性细胞打靶位点RCR产物Sanger法测序峰图在4个靶点位置均有套峰出现。TA克隆测序结果表明本研究设计的4对dual-sgRNAs导向序列的打靶效率分别为42.10%、62.50%、52.63%和47.37%。结论本研究所设计的4对dual-sgRNAs导向序列均可以有效介导Cas9蛋白切割小鼠miR let-7a,形成突变效应。为后续miR let-7a作用靶点的深入挖掘,阐明其发挥抑癌功能的作用机制提供了依据。Objective To establish effective CRISPR/Cas9 system targeting mouse miR let-7a.Methods Two pairs of dual-sgRNAs sequences were designed according to the coding sequences of two coding sites let-7a-1 and let-7a-2 of mouse let-7a respectively.The targeting potency of four pairs of sgRNA was analyzed through the construction of sgRNA expression vector,cotransfection of N2a cells,drug screening.Positive cells'PCR products sequencing and TA clone sequencing.Results The four pairs of targeting vectors played a role in the cells,and there were base insertions or deletions at the targeting sites,and the Sanger sequencing peaks of RCR products at the drug screening positive cells showed nested peaks at all four targeting sites.The results of TA cloning sequencing showed that the targeting efficiency of the four pairs of dual-sgRNAs targeting sequences designed in this study was 42.10%,62.50%,52.63%and 47.37%respectively.Conclusions The results showed that the four pairs of dual-sgRNAs-directed sequences designed in this study deavaged miR let-7a by inducing Cas9 protein effectively and thus generated a mutating effect in mice.Our study laid a foundation for further exploring of let-7a targets andelucidating the mechanism of its'anti-cancer function.

关 键 词：MIRS let-7a CRISPR/Cas9 基因编辑 肿瘤发生 

分 类 号：Q789[生物学—分子生物学]