中国动物传染病学报2024，Vol.32Issue(4)：17-24,8.
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
摘要
Abstract
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Key words
CRISPR/Cas9/MDCK cell/genome-wide/gene editing分类
生物科学引用本文复制引用
朱媛媛,许榜丰,闫鸣昊,刘芹防,滕巧泱,苑纯秀,李雪松,李泽君..基于CRISPR/Cas9技术的全基因组基因编辑MDCK细胞文库的构建[J].中国动物传染病学报,2024,32(4):17-24,8.基金项目
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) （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) （JB03）
