利用CRISPR/Cas9全基因组文库筛选HCT 116细胞增殖相关基因

Genome-scale CRISPR/Cas9 screening for proliferation genes in HCT 116 cells

目的利用CRISPR/Cas9全基因组文库大规模筛选与细胞增殖相关的基因。方法将HCT 116细胞大规模培养至1×10~8个,将CRISPR/Cas9全基因组慢病毒文库大规模感染,加入嘌呤霉素筛选6 d后收集部分细胞,剩余细胞继续培养10 d,收取全部细胞,提取基因组DNA,扩增单链向导RNA(sgRNA)序列、建库并进行深度测序,比较前后两组sgRNA序列数据,根据sgRNA序列数量筛选出与细胞增殖相关的基因。结果与结论筛选出了一批与HCT 116细胞增殖相关的蛋白编码基因及miRNA基因,获得了增殖相关miRNA及其靶基因的调控网络,为进一步验证分析这些基因在HCT 116细胞中的功能奠定了基础。

Objective To screen for genes involved in cell proliferation using the genome-scale CRISPR/Cas9 screen library.Methods HCT 116 cells were cultured to 1×10~8,and transduced with the genome-scale CRISPR/Cas9 screen library lentivirus.Puromycin was added for 6 days.Cells were collected and cultured for 10 days.The cells were harvested and genomic DNA extracted.Then,NGS sequencing was performed.The two sets of data were compared to screen for genes associated with cell proliferation.Results and Conclusion A number of protein coding genes and miRNA genes related to the proliferation of HCT 116 cells were screened.We obtained the regulatory networks of proliferation-related miRNAs and their target genes.This research important for further validation the analysis of the function of these genes in HCT 116 cells.