Repurposing CRISPR/Cas9 for in situ functional assays

A Malina, JR Mills, R Cencic, Y Yan… - Genes & …, 2013 - genesdev.cshlp.org
A Malina, JR Mills, R Cencic, Y Yan, J Fraser, LM Schippers, M Paquet, J Dostie, J Pelletier
Genes & development, 2013genesdev.cshlp.org
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 …
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.
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