An optimized genome-wide, virus-free CRISPR screen for mammalian cells

Pooled CRISPR screens have been widely applied to mammalian and other organisms to elucidate the interplay between genes and phenotypes of interest. The most popular method for delivering the CRISPR components into mammalian cells is lentivirus based. However, because lentivirus is not always an option, virus-free protocols are starting to emerge. Here, we demonstrate an improved virus-free, genome-wide CRISPR screening platform for Chinese hamster ovary cells with 75,488 gRNAs targeting 15,028 genes. Each gRNA expression cassette in the library is precisely integrated into a genomic landing pad, resulting in a very high percentage of single gRNA insertions and minimal clonal variation. Using this platform, we perform a negative selection screen on cell proliferation that identifies 1,980 genes that affect proliferation and a positive selection screen on the toxic endoplasmic reticulum stress inducer, tunicamycin, that identifies 77 gene knockouts that improve survivability. [Display omitted] •A recombinase-based method for virus-free, genome-wide CRISPR knockout screens•Utilizes the same libraries and analysis tools as traditional virus-based screens•Avoids noise associated with virus-based random insertions into the genome Although lentivirus-based delivery of genome-wide CRISPR screen components has proven successful, there are situations in, e.g., industry and hospitals where working with live viruses is difficult or simply not an option. For those situations we have developed an alternative to virus-based, genome-wide CRISPR screens that retains compatibility with the software tools developed for analyzing the results, takes a similar amount of time, and offers improved signal-to-noise ratio. CRISPR knockout screening has mostly been performed by using viruses to deliver the required components into cells. In this paper, Xiong et al. demonstrate a virus-free approach that reduces noise and broadens access to CRISPR-based screens.