CAJ | 학술논문

目的：探索利用CRISPR/Cas9技术在细胞株HepG2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gRNA质粒。将构建好的gRNA质粒和hCas9质粒转染HepG2细胞后，PCR扩增HepG2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gRNA质粒构建成功。HepG2细胞经gRNA-LRH-1/gRNA-ERRα和hCas9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HepG2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。
목적：탐색이용CRISPR/Cas9기술재세포주HepG2기인조중진행정점돌변。방법설계병구건파향핵수체LRH-1화ERRα기인조서렬적gRNA질립。장구건호적gRNA질립화hCas9질립전염HepG2세포후，PCR확증HepG2기인조중LRH-1화ERRα기인적돌변위점，병용SURVEYOR법검측돌변정황。결과파향핵수체LRH-1화ERRα기인조서렬적gRNA질립구건성공。HepG2세포경gRNA-LRH-1/gRNA-ERRα화hCas9전염후，침대LRH-1화ERRα적돌변위점기인조서렬적PCR산물경SURVEYOR법검측결과현시출현량조여예계대소상부적전영조대。결론재HepG2세포중성공이용CRISPR/Cas9기술개도적기인조편집대LRH-1화ERRα특정기인조서렬산생돌변，위구건기세포주고제모형전정료기출。
Objective To explore whether CRISPR/Cas9 system could be effective in hepatocarcinoma cell line HepG2. Methods gRNA plasmids targeting nuclear receptor LRH-1 and ERRαwere designed and con-structed. After verification by sequencing, gRNA-LRH-1/gRNA-ERRα and hCas9 plasmids were transfected in HepG2 cells respectively. Then mutation sites were amplified by PCR and mutation rates were evaluated by SUR-VEYOR assay. Results gRNA-LRH-1 and gRNA-ERRαrecombination plasmids targeting nuclear receptor LRH-1 and ERRαwere successfully established. After transfecting HepG2 with gRNA-LRH-1/gRNA-ERRαand hCas9, 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 gRNA-LRH-1/gRNA-ERRαand hCAS9. Conclusion The site-directed mutagenesis of LRH-1 and ERRαgene locus were successfully created by genome ed-iting mediated by CRISPR/Cas9 in HepG2, which provides the basis for the generation of HepG2-LRH-1/ERRαknockout cell model.