Versatile in vivo regulation of tumor phenotypes by dCas9-mediated transcriptional perturbation

Proc Natl Acad Sci U S A. 2016 Jul 5;113(27):E3892-900. doi: 10.1073/pnas.1600582113. Epub 2016 Jun 20.


Targeted transcriptional regulation is a powerful tool to study genetic mediators of cellular behavior. Here, we show that catalytically dead Cas9 (dCas9) targeted to genomic regions upstream or downstream of the transcription start site allows for specific and sustainable gene-expression level alterations in tumor cells in vitro and in syngeneic immune-competent mouse models. We used this approach for a high-coverage pooled gene-activation screen in vivo and discovered previously unidentified modulators of tumor growth and therapeutic response. Moreover, by using dCas9 linked to an activation domain, we can either enhance or suppress target gene expression simply by changing the genetic location of dCas9 binding relative to the transcription start site. We demonstrate that these directed changes in gene-transcription levels occur with minimal off-target effects. Our findings highlight the use of dCas9-mediated transcriptional regulation as a versatile tool to reproducibly interrogate tumor phenotypes in vivo.

Keywords: CRISPR; cancer genetics; cancer models; cancer therapeutic resistance; gene regulation.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins / genetics*
  • CRISPR-Associated Protein 9
  • DNA Damage
  • DNA Modification Methylases / metabolism
  • DNA Repair Enzymes / metabolism
  • Dacarbazine / analogs & derivatives
  • Drug Resistance, Neoplasm
  • Endonucleases / genetics*
  • Gene Expression Regulation, Leukemic*
  • Genetic Techniques*
  • Leukemia, Experimental*
  • Mice
  • Sequence Analysis, RNA
  • Temozolomide
  • Tumor Suppressor Protein p53 / metabolism
  • Tumor Suppressor Proteins / metabolism


  • Bacterial Proteins
  • Tumor Suppressor Protein p53
  • Tumor Suppressor Proteins
  • Dacarbazine
  • DNA Modification Methylases
  • MGMT protein, mouse
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases
  • DNA Repair Enzymes
  • Temozolomide