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.
We thank Nachon Charanyanonda Petersen and Saranya Nallapareddy for helping with the FACS and molecular cloning. This work was supported by generous funding from the Novo Nordisk Foundation provided to the Center for Biosustainability at the Technical University of Denmark (NNF10CC1016517 and NNF16OC0021638) and from National Insitute of General Medical Sciences (NIGMS) (R35 GM119850 to N.E.L.). Conceptualization, K.X. and L.E.P.; methodology, K.X. L.E.P. J.S.L. and P.S.; validation, I.V.; investigation, K.X.; resources, K.X. K.J.l.C.K. H.H. S.L. L.M.G. S.L. and P.S.; data curation, L.E.P.; writing – original draft, K.X.; writing – review & editing, L.E.P. and N.E.L.; visualization, K.X. and L.E.P.; supervision, L.E.P. and N.E.L.; project administration, L.E.P. and G.M.L.; funding acquisition, N.E.L. The authors declare no competing interests.We thank Nachon Charanyanonda Petersen and Saranya Nallapareddy for helping with the FACS and molecular cloning. This work was supported by generous funding from the Novo Nordisk Foundation provided to the Center for Biosustainability at the Technical University of Denmark ( NNF10CC1016517 and NNF16OC0021638 ) and from National Insitute of General Medical Sciences (NIGMS) ( R35 GM119850 to N.E.L.).
