title: Repurposing CRISPR/Cas9 for in situ functional assays.
abstract: RNAi combined with next-generation sequencing has proven to be a powerful and cost-effective genetic screening platform in mammalian cells. Still, this technology has its limitations and is incompatible with in situ mutagenesis screens on a genome-wide scale. Using p53 as a proof-of-principle target, we readapted the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated 9) genome-editing system to demonstrate the feasibility of this methodology for targeted gene disruption positive selection assays. By using novel "all-in-one" lentiviral and retroviral delivery vectors heterologously expressing both a codon-optimized Cas9 and its synthetic guide RNA (sgRNA), we show robust selection for the CRISPR-modified Trp53 locus following drug treatment. Furthermore, by linking Cas9 expression to GFP fluorescence, we use an "all-in-one" system to track disrupted Trp53 in chemoresistant lymphomas in the Eμ-myc mouse model. Deep sequencing analysis of the tumor-derived endogenous Cas9-modified Trp53 locus revealed a wide spectrum of mutants that were enriched with seemingly limited off-target effects. Taken together, these results establish Cas9 genome editing as a powerful and practical approach for positive in situ genetic screens.
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6 Canada,;
authors.forename: Abba
authors.initials: A
authors.lastname: Malina
authors.forename: John R
authors.initials: JR
authors.lastname: Mills
authors.forename: Regina
authors.initials: R
authors.lastname: Cencic
authors.forename: Yifei
authors.initials: Y
authors.lastname: Yan
authors.forename: James
authors.initials: J
authors.lastname: Fraser
authors.forename: Laura M
authors.initials: LM
authors.lastname: Schippers
authors.forename: Marilène
authors.initials: M
authors.lastname: Paquet
authors.forename: Josée
authors.initials: J
authors.lastname: Dostie
authors.forename: Jerry
authors.initials: J
authors.lastname: Pelletier
mesh_terms: D000818:Animals; D064130:CRISPR-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D005260:Female; D018390:Gene Targeting; D016158:Genes, p53 / Q000235:genetics; D005821:Genetic Techniques; D016678:Genome / Q000235:genetics; D054643:INDEL Mutation / Q000235:genetics; D053208:Kaplan-Meier Estimate; D016086:Lentivirus / Q000235:genetics; D008223:Lymphoma / Q000235:genetics / Q000401:mortality / Q000628:therapy; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D015203:Reproducibility of Results
chemical_list: D064130:CRISPR-Associated Proteins
keywords: CRISPR; Cas9; functional screening; genome editing; p53