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. 2014 Mar;32(3):267-73.
doi: 10.1038/nbt.2800. Epub 2013 Dec 23.

Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library

Hiroko Koike-Yusa  1 , Yilong Li  1 , E-Pien Tan  2 , Martin Del Castillo Velasco-Herrera  2 , Kosuke Yusa  2
Affiliations

Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library

Hiroko Koike-Yusa et al. Nat Biotechnol. 2014 Mar.

Abstract

Identification of genes influencing a phenotype of interest is frequently achieved through genetic screening by RNA interference (RNAi) or knockouts. However, RNAi may only achieve partial depletion of gene activity, and knockout-based screens are difficult in diploid mammalian cells. Here we took advantage of the efficiency and high throughput of genome editing based on type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems to introduce genome-wide _targeted mutations in mouse embryonic stem cells (ESCs). We designed 87,897 guide RNAs (gRNAs) _targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9. Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4 previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system.

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