Dual direction CRISPR transcriptional regulation screening uncovers gene networks driving drug resistance

Sci Rep. 2017 Dec 18;7(1):17693. doi: 10.1038/s41598-017-18172-6.

Abstract

Pooled CRISPR-Cas9 knock out screens provide a valuable addition to the methods available for novel drug target identification and validation. However, where gene editing is targeted to amplified loci, the resulting multiple DNA cleavage events can be a cause of false positive hit identification. The generation of nuclease deficient versions of Cas9 has enabled the development of two additional techniques - CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) - that enable the repression or overexpression, respectively, of target genes. Here we report the first direct combination of all three approaches (CRISPRko, CRISPRi and CRISPRa) in the context of genome-wide screens to identify components that influence resistance and sensitivity to the BRAF inhibitor, vemurafenib. The pairing of both loss- and gain-of-function datasets reveals complex gene networks which control drug response and illustrates how such data can add substantial confidence to target identification and validation analyses.

MeSH terms

  • CRISPR-Cas Systems / genetics
  • Cell Line, Tumor
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • Clustered Regularly Interspaced Short Palindromic Repeats / physiology
  • DNA Cleavage
  • Drug Evaluation, Preclinical / methods
  • Drug Resistance / genetics*
  • Endonucleases / genetics
  • Gene Editing / methods
  • Gene Expression Regulation / genetics
  • Gene Knockout Techniques / methods*
  • Gene Regulatory Networks / genetics*
  • Genome / genetics
  • Humans
  • Proto-Oncogene Proteins B-raf / antagonists & inhibitors
  • Vemurafenib / pharmacology

Substances

  • Vemurafenib
  • Proto-Oncogene Proteins B-raf
  • Endonucleases