利用CRISPR/Cas9系统构建SIRT1基因敲除的K562细胞株

Construction of a SIRT1 knockout K562 cell line by using the CRISPR/Cas9

目的构建去乙酰化酶SIRT1基因敲除的K562单克隆细胞株,研究SIRT1基因对细胞增殖的影响。方法设计靶向SIRT1酶活性中的sgRNA编码序列,构建CRISPR/Cas9载体并转染K562细胞,嘌呤霉素筛选后单克隆化并扩增;采用基因组测序、PCR和免疫印迹的方法进行基因敲除效果鉴定;对选定的SIRT1基因敲除的单克隆做生长曲线检测。结果目标质粒测序符合,质粒转染细胞后检测到基因组靶序列位置杂合峰,获得SIRT1基因敲除的单克隆3个,SIRT1基因敲除导致细胞增殖明显减缓。结论成功构建了SIRT1基因敲除的细胞株,该细胞可作为血液系统疾病研究的有力工具。

Objective To construct a monoclonal K562 cell line with SIRT1 gene knockout and investigate the effect of SIRT1 gene on cell proliferation.Methods The sgRNA encoding sequence targeting SIRT1 enzyme activity was designed and the CRISPR/Cas9 vector was constructed and transfected to K562 cells,followed by puromycin selection,being single-cloned and amplified.Genomic sequencing,PCR,and immunoblotting were performed to verify the gene knockout effect,and the growth curves of selected monoclonal cells with SIRT1 gene knockout were measured.Results The sequencing of the target plasmid was consistent with the design,and the hybrid peaks at the target sequence location were observed after plasmid was transfected into cells.Three single clones with SIRT1 gene knockout were obtained,and the SIRT1 gene knockout resulted in a significant slowdown in cell proliferation.Conclusion A cell line with SIRT1 gene knockout was successfully constructed.It can serve as a powerful tool for the study on hematological diseases.