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

Kai Xiong, Karen Julie la Cour Karottki, Hooman Hefzi, Songyuan Li, Lise Marie Grav, Shangzhong Li, Philipp Spahn, Jae Seong Lee, Ildze Ventina, Gyun Min Lee, Nathan E. Lewis, Helene Faustrup Kildegaard, Lasse Ebdrup Pedersen*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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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. 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.
Original languageEnglish
Article number100062
JournalCell Reports Methods
Issue number4
Number of pages16
Publication statusPublished - 2021


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