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