利用CRISPR/Cas9n double nick系统构建人DNAH2基因敲除的U2OS细胞株

Knocking out of human DNAH2 gene in U2OS cells by CRISPR/Cas9n double nick system

摘要:

基于CRISPR/Cas9n double nick技术构建人DNAH2 (Homo sapiens dynein, axonemal, heavy chain 2) 基因敲除的U2OS稳定细胞株，旨在研究DNAH2基因的生物学功能。首先设计并合成A、B两个sgRNA (Single guide RNA) 以及各自的互补链，退火连接形成DNAH2 sgRNA-A、B双链，再分别与带有BbsⅠ粘性末端的pX462线性载体相连，形成pX462-DNAH2-A、pX462-DNAH2-B重组真核表达质粒。质粒共转染至U2OS细胞后，加入嘌呤霉素，以有限稀释法获得阳性单克隆细胞株，再以蛋白印迹实验检测DNAH2蛋白的表达，最后通过PCR-基因测序技术分析突变特点。结果显示A、B sgRNA双链成功插入pX462载体，U2OS-DNAH2-KO单克隆细胞株中DNAH2蛋白不表达，DNAH2基因发生移码突变，从而证实利用CRISPR/Cas9n double nick系统成功构建人DNAH2基因敲除的U2OS稳定细胞株，为研究DNAH2基因提供有利工具。

Abstract:

To study the biological function of DNAH2 (Homo sapiens dynein, axonemal, heavy chain 2) gene, we constructed human stable U2OS cell line of DNAH2 gene knockout through CRISPR/Cas9n double nick system. The A, B sgRNAs (Single guide RNA) and complementary strands were designed and synthesized. The double-stranded structures were formed during annealing, and connected with BbsⅠ cohesive ends-containing pX462 linear vector to construct the recombinant eukaryotic expression plasmids, including pX462-DNAH2-A and pX462-DNAH2-B. After the co-transfection of the two plasmids into U2OS cells, the addition of puromycin and limiting dilution method were used to obtain positive monoclonal cell line. Western blotting assay was then performed to detect the expression of DNAH2 protein, and PCR-sequencing technology was finally utilized to analyze the mutation feature. The results showed that A, B sgRNAs duplex was successfully inserted into pX462 vector, and DNAH2 protein was not expressed and DNAH2 gene suffered from the frame-shift mutation in U2OS-DNAH2-KO monoclonal cell line. These demonstrated that DNAH2 knockout U2OS stable cell line was successfully constructed through CRISPR/Cas9n double nick system, which providing a useful tool for the study of DNAH2 gene.