Journal article
An optimized genome-wide, virus-free CRISPR screen for mammalian cells
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark1
University of California at San Diego2
Department of Biotechnology and Biomedicine, Technical University of Denmark3
Section for Protein Science and Biotherapeutics, Department of Biotechnology and Biomedicine, Technical University of Denmark4
Ajou University5
Technical University of Denmark6
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.
Language: | English |
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Publisher: | Elsevier |
Year: | 2021 |
Pages: | 100062 |
ISSN: | 26672375 |
Types: | Journal article |
DOI: | 10.1016/j.crmeth.2021.100062 |
ORCIDs: | 0000-0001-8045-0252 , 0000-0001-7700-3654 , Pedersen, Lasse Ebdrup , Karottki, Karen Julie la Cour , Grav, Lise Marie and Kildegaard, Helene Faustrup |