CRISPR/Cas9技术在HepG2基因组中进行定点基因突变的探索

Exploration of site-directed mutagenesis mediated by CRISPR/Cas9 system in HepG2

目的探索利用CRISPR/Cas9技术在细胞株Hep G2基因组中进行定点突变。方法设计并构建靶向核受体LRH-1和ERRα基因组序列的g RNA质粒。将构建好的g RNA质粒和h Cas9质粒转染Hep G2细胞后,PCR扩增Hep G2基因组中LRH-1和ERRα基因的突变位点,并用SURVEYOR法检测突变情况。结果靶向核受体LRH-1和ERRα基因组序列的g RNA质粒构建成功。Hep G2细胞经g RNA-LRH-1/g RNA-ERRα和h Cas9转染后,针对LRH-1和ERRα的突变位点基因组序列的PCR产物经SURVEYOR法检测结果显示出现两条与预计大小相符的电泳条带。结论在Hep G2细胞中成功利用CRISPR/Cas9技术介导的基因组编辑对LRH-1和ERRα特定基因组序列产生突变,为构建其细胞株敲除模型奠定了基础。

Objective To explore whether CRISPR/Cas9 system could be effective in hepatocarcinoma cell line Hep G2. Methods g RNA plasmids targeting nuclear receptor LRH-1 and ERR α were designed and constructed. After verification by sequencing, g RNA-LRH-1/g RNA-ERR α and h Cas9 plasmids were transfected in Hep G2 cells respectively. Then mutation sites were amplified by PCR and mutation rates were evaluated by SURVEYOR assay. Results g RNA-LRH-1 and g RNA-ERRα recombination plasmids targeting nuclear receptor LRH-1and ERRα were successfully established. After transfecting Hep G2 with g RNA-LRH-1/g RNA-ERRα and h Cas9, the mutation sites were amplified by PCR and subjected to SURVEYOR assay. The electrophoresis results showed that two additional bands with expected size were found in cells transfected with g RNA-LRH-1/g RNA-ERRα and h CAS9.Conclusion The site-directed mutagenesis of LRH-1 and ERRα gene locus were successfully created by genome editing mediated by CRISPR/Cas9 in Hep G2, which provides the basis for the generation of Hep G2-LRH-1/ERR αknockout cell model.