Repurposing CRISPR/Cas9 for in situ functional assays (original) (raw)

  1. John R. Mills1,6,7,
  2. Regina Cencic1,
  3. Yifei Yan2,
  4. James Fraser1,
  5. Laura M. Schippers1,
  6. Marilène Paquet3,
  7. Josée Dostie1 and
  8. Jerry Pelletier1,4,5,8
  9. 1Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6 Canada,;
  10. 2Département de Biochimie et Médecine Moléculaire, Université de Montréal, Quebec H3C 3J7, Canada;
  11. 3Département de Pathologie et de Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec J2S 2M2, Canada;
  12. 4Department of Oncology, McGill University, Montreal, Quebec H3G 1Y6, Canada;
  13. 5The Rosalind and Morris Goodman Cancer Research Center, McGill University, Montreal, Quebec H3G 1Y6, Canada
  14. ↵6 These authors contributed equally to this work.

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.

Footnotes