基于CRISPR/Cas9技术的全基因组基因编辑MDCK细胞文库的构建  

Construction of Genome-Wide Gene Editing MDCK Cell Library Based on CRISPR/Cas9 Technology

作　　者：朱媛媛 许榜丰 闫鸣昊 刘芹防[1] 滕巧泱[1] 苑纯秀[1] 李雪松[1] 李泽君[1] ZHU Yuanyuan;XU Bangfeng;YAN Minghao;LIU Qinfang;TENG Qiaoyang;YUAN Chunxiu;LI Xuesong;LI Zejun(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2024年第4期17-24,共8页Chinese Journal of Animal Infectious Diseases

基　　金：中国农业科学院创新工程经费;上海市科技兴农项目(2021-02-08-00-12-F00746);中央级公益性科研院所基本科研业务费专项(JB03)。

摘　　要：本研究利用CRISPR/Cas9基因编辑技术,首先靶向犬全基因组设计合成转录sgRNA的DNA文库,并将DNA文库全部克隆于lentiCRISPR v2慢病毒转移载体上,高通量测序结果显示文库覆盖度高、均一性良好。将质粒文库和慢病毒包装系统质粒共转染293T细胞,收取含慢病毒样病毒的上清液感染MDCK细胞,在最适浓度嘌呤霉素筛选下,收集嘌呤霉素抗性细胞,冻存于-80℃,提取一部分细胞的基因组,通过PCR扩增转录sgRNA的DNA,将PCR产物插入到T载体后,挑取单克隆菌落,测序表明细胞文库中转录的sgRNA具有较好的覆盖度。本实验成功构建了犬全基因组范围转录sgRNA的质粒文库,并成功构建了MDCK细胞全基因组范围的基因编辑细胞库,该文库可作为后续筛选病毒复制关键宿主因子的细胞平台。In this study,the whole genome of canine was first targeted using CRISPR/Cas9 gene editing technology to synthesize DNA library for the transcription of sgRNA.The DNAs were inserted into the lentiCRISPR v2 lentivirus transfer vector to build the plasmid library,which was proved to be high coverage and good uniformity by the high-throughput sequencing.The plasmid library was transfected on 293T cells together with the lentiviral packaging systems to rescue the lentivirus-like viruses.The supernatant containing lentivirus-like viruses was collected and used to infect MDCK cells.The MDCK cells were cultured in the medium containing proper concentration of puromycin and the resistant cells were collected and stored at-80℃.The genome of a part of the harvested cells was extracted and the DNA transcribing sgRNA was amplified by PCR.After the PCR product was inserted into the T vector,the colony was selected and sequenced.The results showed that the sgRNA in the cell library had good coverage.In this study,a genome-wide sgRNA transcription plasmid library and a genome-wide gene editing cell library of MDCK cells were constructed,which could be used as a cell platform for screening key host factors for virus replication.

关 键 词：CRISPR/Cas9 MDCK细胞系 全基因组 基因编辑 

分 类 号：Q78[生物学—分子生物学]