Repurposing CRISPR/Cas9 for in situ functional assays

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...

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Bibliographic Details
Published in:	Genes & development 2013-12, Vol.27 (23), p.2602-2614
Main Authors:	Malina, Abba, Mills, John R, Cencic, Regina, Yan, Yifei, Fraser, James, Schippers, Laura M, Paquet, Marilène, Dostie, Josée, Pelletier, Jerry
Format:	Article
Language:	eng
Subjects:	Animals Clustered Regularly Interspaced Short Palindromic Repeats - genetics CRISPR-Associated Proteins - genetics CRISPR-Associated Proteins - metabolism Female Gene Targeting Genes, p53 - genetics Genetic Techniques Genome - genetics INDEL Mutation - genetics Kaplan-Meier Estimate Lentivirus - genetics Lymphoma - genetics Lymphoma - mortality Lymphoma - therapy Mice Mice, Inbred C57BL Mutation Reproducibility of Results Resource/Methodology
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Summary:	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.
ISSN:	0890-9369 1549-5477