Citation

  • Authors: Li, Y., Mendiratta, S., Ehrhardt, K., Kashyap, N., White, M. A., Bleris, L.
  • Year: 2016
  • Journal: PLoS One 11 e0144970
  • Applications: in vitro / DNA / jetPRIME
  • Cell types:
    1. Name: HEK-293T
      Description: Human embryonic kidney Fibroblast
      Known as: HEK293T, 293T
    2. Name: SW48
      Description: Human colon adenocarcinoma cells

Abstract

CRISPR/Cas9 is an enabling RNA-guided technology for genome targeting and engineering. An acute DNA binding constraint of the Cas9 protein is the Protospacer Adjacent Motif (PAM). Here we demonstrate that the PAM requirement can be exploited to specifically target single-nucleotide heterozygous mutations while exerting no aberrant effects on the wild-type alleles. Specifically, we target the heterozygous G13A activating mutation of KRAS in colorectal cancer cells and we show reversal of drug resistance to a MEK small-molecule inhibitor. Our study introduces a new paradigm in genome editing and therapeutic targeting via the use of gRNA to guide Cas9 to a desired protospacer adjacent motif.

Pubmed