利用CRISPR/Cas方法建立RNaseL基因稳定敲除细胞株

Generation of RNase L knockout cell lines by CRISPR/Cas system

目的利用CRISPR/Cas9技术建立人RNase L基因稳定敲除的细胞株。方法根据Gen Bank中RNase L基因序列,设计RNase L敲除的小指导RNA(sgRNA)序列,将其克隆到p Cas-Guide真核表达载体中,获得p Cas指导RNA(gRNA)载体;设计供体DNA的同源臂序列,通过搭桥PCR将左同源臂、潮霉素B抗性基因和右同源臂扩增成为一条片段作为同源重组的修复模板,并将其克隆到p Back Zero-T表达载体上,获得p Back Zero-T-RNase LK载体。将上述两个载体共转染HEK293细胞,用潮霉素B筛选,通过Western印迹和DNA测序等技术验证RNase L从基因组上敲除。结果与结论成功构建了载体p Cas-gRNA和p Back Zero-T-RNase LK,Western印迹和DNA测序结果表明,成功获得5株RNase L缺失的细胞株,为探讨RNase L的生物学功能和研究其分子机制奠定了基础。

Objective To establish RNase L gene knockout HEK293 cell lines using CRISPR/Cas9 system. Methods Small guide RNA （sgRNA） sequences of human RNase L were designed and sgRNAs were inserted into pCas-Guide and pCas-guide RNA（gRNA） vectors were obtained. The donor DNA sequences of the homologous arm were designed for RNase L knockout. In the presence of the right homologous arm, the resistance gene of hygromycin B and the left homologous arm as templates of homology-directed repair, the donor DNA template was amplified by overlopping PCR and cloned into the pBackZero-T expression vector and pBackZero-T-RNase LK vector was obtained. The pCas-gRNA vector and pBackZero-T-RNase LK vector were co-transfected into HEK293 cells to establish the stable expression cell line of RNase L gene knockout. Cells were cultured with hygromycin B, while Western blotting and DNA sequencing were used to analyze the gene of RNase L knockout from genome. Results and Conclusion The pCas-gRNA vector and pBackZero-T-RNase LK vector were successfully constructed. Five RNase L gene knockout HEK293 cell lines were generated, contributing to the study of the biological function and molecular mechanism of RNase L.