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CRISPR/Cas9-sg RNA(single guide RNA)全基因组文库筛选技术是在哺乳动物中进行系统基因组分析的有力工具.本文通过在人急性单核白血病细胞系THP1上优化CRISPR/Cas9-sg RNA人类全基因组慢病毒文库感染体系,构建裸鼠皮下移植肿瘤模型,并通过优化测序建库方法,对肿瘤组织进行二代测序以及利用生物信息学分析获得筛选结果.裸鼠肿瘤模型表明,感染sg RNA文库后的THP1细胞成瘤能力下降,推测可能与某些促癌基因的敲除有关;通过对二代测序数据进行生物信息学分析得到了一些候选基因.实验结果表明,在人单核细胞白血病细胞系上进行CRISPR/Cas9-sg RNA全基因组文库筛选是可行的,这项工作为在其他白血病细胞系或原代细胞上的筛选应用奠定了一定基础.
CRISPR / Cas9-sg RNA (single guide RNA) genome-wide library screening is a powerful tool for systematic genome analysis in mammals By optimizing CRISPR / Cas9-sg RNA in human acute monocytic leukemia cell line THP1 Genomic lentivirus library infection system was constructed to construct a subcutaneous tumor model in nude mice and the second generation of tumor tissues was sequenced and the screening results were obtained by bioinformatics analysis by optimizing the sequencing library construction.The nude mice tumor model showed that the infection of sg RNA library The decreased THP1 cell tumorigenic ability may be related to the knockdown of some oncogenes.Several candidate genes were obtained by bioinformatics analysis of the second generation sequencing data.The experimental results show that in the human monocytic leukemia cells It is feasible to screen the whole genome library of CRISPR / Cas9-sg RNA, and this work will lay a foundation for the screening of other leukemia cell lines or primary cells.