Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex

  • Nature. 2015 Jan 29;517(7536):583-8. doi: 10.1038/nature14136.
Silvana Konermann  1 ,  Mark D Brigham  1 ,  Alexandro E Trevino  1 ,  Julia Joung  2 ,  Omar O Abudayyeh  1 ,  Clea Barcena  1 ,  Patrick D Hsu  1 ,  Naomi Habib  3 ,  Jonathan S Gootenberg  4 ,  Hiroshi Nishimasu  5 ,  Osamu Nureki  6 ,  Feng Zhang  1
Affiliations
  • 1. 1] Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, Massachusetts 02142, USA [2] McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [3] Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [4] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
  • 2. 1] Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, Massachusetts 02142, USA [2] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
  • 3. Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, Massachusetts 02142, USA.
  • 4. 1] Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, Massachusetts 02142, USA [2] McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [3] Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [4] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [5] Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
  • 5. 1] Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi Bunkyo, Tokyo 113-0032, Japan [2] JST, PRESTO 2-11-16 Yayoi Bunkyo, Tokyo 113-0032, Japan.
  • 6. Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi Bunkyo, Tokyo 113-0032, Japan.
PMID: 25494202 DOI: 10.1038/nature14136
Abstract

Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.