Using targeted genome integration for virus-free genome-wide mammalian CRISPR screen

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

doi:10.1016/j.crmeth.2021.100062

GenomicsCell Reports MethodsAug 1, 2021

Peer-reviewed journal article

Using targeted genome integration for virus-free genome-wide mammalian CRISPR screen

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

Abstract

Pooled CRISPR screens have been widely applied in mammalian cells to discover genes regulating various phenotypes of interest. In such screens, CRISPR components are generally delivered with a lentivirus. However, lentiviral CRISPR screens are limited by unpredictable genome insertion, the requirement of biosafety level II lab facilities and personnel trained to work with viruses. Here we established a virus-free (VF) genome-wide CRISPR screening platform for Chinese hamster ovary (CHO) cells with 74,617 gRNAs targeting 18,353 genes. Each gRNA expression cassette in the library is precisely integrated into a genomic landing pad thus reducing the clonal variation. Using this VF CRISPR screening platform, 338 genes are identified as essential for CHO cell growth and 76 genes were found to be involved in the unfolded protein response (UPR) induced endoplasmic reticulum (ER) stress. Extensive validation of the candidate genes further demonstrated the robustness of this novel non-viral CRISPR screen method.

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