Objective: To facilitate using the CRISPR/Cas9 gene editing system in human liver and gallbladder cancer cells, we established Cas9 stably expressed human liver and gallbladder cancer cell lines, and validated the gene editing activity of Cas9. Methods: Human liver cancer cell lines (Huh7, PLC/PRF/5, HepG2, Hep3b, SK-HEP-1 and Li-7), human cholangiocarcinoma cells (RBE) and human gallbladder cancer cells (GBC-SD) were infected with 3 Cas9-expressing lentivirus vectors (pLv-EF1α-Cas9-Flag-Neo, pLv-EF1α-Cas9-Flag-Puro, Cas9m1.1), respectively, and Cas9 stably expressed colonies were screened and selected. We extracted the genomic DNA and protein, validated the stable expression of Cas9 by using genomic polymerase chain reaction (PCR) and western blot. Three of cell lines were further infected with Lv-EF1α-mCherry. Then mCherry positive cells were sorted by flow cytometry and infected with designed guide RNA (gRNA) vectors which targeted mCherry gene. Subsequently the gene editing activity of Cas9 was detected by genomic PCR, fluorescence microscopic observation and flow cytometry analysis. Results: One hundred Cas9-expressing human liver and gallbladder cancer cell lines were selected. Among them, 35 cell lines expressed Cas9-Neo, 25 expressed Cas9-puro, and 40 expressed mutant Cas9 (mCas9). We also established 3 cell lines with stable expression of mCherry (Huh7-mCas9-M, PLC/PRF/5-Cas9-M and SK-HEP-1-Cas9-M). The results of genomic PCR and sequencing showed that by lentiviral infection with 2 types of designed gRNA, the long fragment deletion of mCherry gene was found in these 3 cell lines. Moreover, mCherry(-)EGFP(+) cells infected with 2 types of gRNA were observed by fluorescence microscope. The results of flow cytometry showed that mCherry(-)EGFP(+) cells accounted from 0.3% to 93.6%. Conclusion: We successfully establish 100 human liver and gallbladder cancer cell lines with stable expression of Cas9 protein and validate their activities of gene editing.
目的： 建立Cas9蛋白稳定表达的人肝胆癌细胞株，并验证Cas9的基因编辑活性，以便利用CRISPR/Cas9系统开展肝胆细胞基因编辑的研究。 方法： 在人肝癌细胞(Huh7、PLC/PRF/5、HepG2、Hep3b、SK-HEP-1和Li-7)、人胆管癌细胞(RBE)和人胆囊癌细胞(GBC-SD)中，分别感染Cas9表达质粒pLv-EF1α-Cas9-Flag-Neo、pLv-EF1α-Cas9-Flag-Puro和Cas9m1.1，挑选单细胞克隆培养扩增。提取基因组DNA和总蛋白后，通过基因组聚合酶链反应(PCR)和Western blot验证Cas9的稳定表达。选取其中3个细胞株并感染Lv-EF1α-mCherry，流式细胞术分选出mCherry阳性的细胞，再通过慢病毒感染靶向mCherry基因的向导RNA，细胞扩增后，通过基因组PCR、荧光显微镜下观察和流式细胞术检测mCherry被敲除的情况。 结果： 筛选到100个Cas9稳定表达的人肝胆癌细胞株，其中表达Cas9-Neo的细胞35株，表达Cas9-Puro的细胞25株，表达突变型Cas9的细胞40株。建立了3株mCherry稳定表达的细胞系Huh7-mCas9-M、PLC/PRF/5-Cas9-M和SK-HEP-1-Cas9-M。基因组PCR和测序显示，同时感染2种gRNA的细胞基因组内存在mCherry片段缺失。荧光显微镜下，同时感染2种gRNA的细胞可以观察到mCherry(－)EGFP(＋)细胞。流式细胞仪检测结果显示，mCherry(－)EGFP(＋)细胞占0.3%～93.6%。 结论： 成功建立了100个Cas9稳定表达的人肝胆癌细胞株，其中的Cas9蛋白具有基因编辑活性。.
Keywords: CRISPR/Cas9; Cell line; Cholangiocarcinoma; Gallbladder neoplasms; Gene editing; Hepatocellular carcinoma.