Oral Presentation 29th Lorne Cancer Conference 2017

CRISPR loss of function screens for understanding and targeting of β-catenin driven cancers (#20)

Joseph Rosenbluh 1 2 , Han Xu 3 , Francisca Vazquez 3 , David Root 3 , Aviad Tsherniak 3 , William Hahn 3 4
  1. Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia
  2. The Hudson Institute, Clayton, VIC, Australia
  3. The Broad Institute of Harvard and MIT, Cambridge, MA, USA
  4. Medical Oncology, Dana Farber Cancer Institute, Boston, MA, USA

Loss of function genetic screens permit systematic study of how genes function in normal homeostasis and how they are deregulated in disease. Using genome scale shRNA pooled screens in 216 cancer cell lines our previous work revealed new insights into the mechanisms associated with deregulation of β-catenin in colon cancer and identified new targets for drug development1. Due to the high prevalence of RNAi off target effects we evaluated CRISPR/Cas9 as an orthogonal approach to modulate gene function2. Although we found CRISPR as a highly specific loss of function methodology, we3 and others4 have recently reported a gene independent off-target effect in CRISPR/Cas9 experiments induced by Cas9 mediated DNA cleavage of amplified genomic regions. To overcome this hurdle, we utilized CRISPRi which uses a catalytically inactive Cas9 (dCas9) fused to a transcription repressor domain (KRAB). Single guide RNAs (sgRNAs) that tether the KRAB-dCas9 fusion protein to transcription start sites (TSS) suppress gene expression by interfering with the transcriptional machinery without induction of DNA damage. We define roles for identifying highly efficient CRISPRi sgRNAs and identify bidirectional promoters as an off target in CRISPRi experiments. We further developed an array of medium throughput functional assays that include genetic interaction profiling and proteomic profiling2. We demonstrate that combining these methodologies is a highly efficient approach for identifying gene that are required for β-catenin driven cancers.

  1. Rosenbluh J et al. β-catenin driven cancers require a YAP1 transcriptional complex for survival and tumorigenesis. Cell, 151(7):1457-1473, 2012.
  2. Rosenbluh J et al. Integrated genetic and proteomic interrogation of WNT/β-catenin cancer dependencies. Cell Systems, 3(3):302-316, 2016.
  3. Aguirre AJ et al. Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting. Cancer Discovery, 6(8):914-929, 2016.
  4. Munoz DM et al. CRISPR Screens Provide a Comprehensive Assessment of Cancer Vulnerabilities but Generate False-Positive Hits for Highly Amplified Genomic Regions. Cancer Discovery, 6(8):900-913, 2016.