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

• Main Authors: Konermann, Silvana (Contributor), Brigham, Mark D. (Contributor), Trevino, Alexandro E. (Contributor), Joung, Julia (Contributor), Barcena, Clea (Contributor), Hsu, Patrick D. (Contributor), Habib, Naomi (Author), Gootenberg, Jonathan S. (Contributor), Nishimasu, Hiroshi (Author), Nureki, Osamu (Author), Zhang, Feng (Contributor), Abudayyeh, Omar Osama (Contributor)
• Other Authors: Harvard University- (Contributor), Massachusetts Institute of Technology. Department of Biological Engineering (Contributor), Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences (Contributor), McGovern Institute for Brain Research at MIT (Contributor)
• Format: Article
• Language: English
• Published: 2016-05-23T00:51:14Z.
• Subjects: Article
• Online Access: Get fulltext

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.
National Science Foundation (U.S.) (Graduate Research Fellowship)
United States. Dept. of Energy (Computational Science Graduate Fellowship)
National Institute of Mental Health (U.S.) (DP1-MH100706)
National Institute of Neurological Disorders and Stroke (U.S.) (R01-NS07312401)
National Science Foundation (U.S.)
W. M. Keck Foundation
Kinship Foundation. Searle Scholars Program
Klingenstein Foundation
Vallee Foundation
Simons Foundation