利用CRISPR/Cas9技术敲除小鼠ES细胞H2-K1基因

The H2-K1 gene knockout of mouse ES cells through CRISPR/Cas9 technology

利用CRISPR/Cas9和ES细胞技术,在小鼠ES细胞株上敲除小鼠H2-K1基因,为研发MHCⅠ类基因人源化小鼠打下基础。设计了两个单向导RNA(single guide RNA,sgRNA),分别靶向H2-K1基因的外显子2和外显子3。以p X330质粒为骨架构建表达sgRNA的打靶载体。用电穿孔转染法将构建好的质粒以及p SUPER-puro共同导入小鼠ES细胞中,实现基因敲除。在嘌呤霉素筛选后,利用PCR初步检测靶基因的敲除情况,再通过测序以及流式细胞分析确定敲除H2-K1基因的小鼠ES细胞。结果显示:利用CRISPR/Cas9技术,小鼠ES细胞的H2-K1基因被成功敲除,通过PCR检测到4个克隆为H2-K1单等位基因敲除(19.0%),2个克隆为H2-K1双等位基因敲除(9.5%)。经过测序以及流式细胞分析,2株小鼠ES细胞被确认为H2-K1双等位基因敲除。本研究利用CRISPR/Cas9技术得到H2-K1基因敲除的小鼠ES细胞株,为MHCⅠ类基因的敲除和置换提供了参考。

The CRISPR/Cas9 technology was used to achieve the H2-K1 gene knockout of mouse ES cells,which will be useful for generation of MHC class Ⅰ gene humanized mice. In this study,two sgRNAs were designed,which are targeting to the Exon2 and Exon3 of H2-K1,respectively. The plasmids expressing the sgRNAs were constructed using p X330 as the matrix plasmid. In order to knockout H2-K1,the constructed plasmids and p SUPER-puro were cotransfected into the m ES cells through electroporation. After screening by puromycin,the result of gene targeting was determined by PCR, and H2-K1 knockout mouse ES cells were further confirmed through sequencing and flow cytometry. We found that H2-K1 in the mouse ES cell was knocked out using CRISPR/Cas9 technology. Through PCR,4clones were determined as one allele knockout（ 19. 0%）,2 clones were determined as two allele knockout（ 9. 5%）. 2 clones were further confirmed as two allele knockout clones by sequencing and flow cytometry. The generated H2-K1 knockout mouse ES cells would provide a reference for the knockout and replacement of MHC class Ⅰ gene.