title: Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.
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.
authors.affiliation: 1] Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK. [2].
authors.forename: Hiroko
authors.initials: H
authors.lastname: Koike-Yusa
authors.affiliation: 1] Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK. [2].
authors.forename: Yilong
authors.initials: Y
authors.lastname: Li
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
authors.forename: E-Pien
authors.initials: EP
authors.lastname: Tan
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
authors.forename: Martin Del Castillo
authors.initials: Mdel C
authors.lastname: Velasco-Herrera
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Yusa
mesh_terms: D000818:Animals; D001709:Biotechnology; D064113:CRISPR-Cas Systems / Q000235:genetics; D002478:Cells, Cultured; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D053595:Embryonic Stem Cells; D015723:Gene Library; D016678:Genome / Q000235:genetics; D023281:Genomics / Q000379:methods*; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D034622:RNA Interference; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems