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

Silvana Konermann, Mark D. Brigham, Alexandro E. Trevino, Julia Joung, Omar O. Abudayyeh, Clea Barcena, Patrick D. Hsu, Naomi Habib, Jonathan S. Gootenberg, Hiroshi Nishimasu, Osamu Nureki and Feng Zhang ()
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Silvana Konermann: Broad Institute of MIT and Harvard, 75 Ames Street
Mark D. Brigham: Broad Institute of MIT and Harvard, 75 Ames Street
Alexandro E. Trevino: Broad Institute of MIT and Harvard, 75 Ames Street
Julia Joung: Broad Institute of MIT and Harvard, 75 Ames Street
Omar O. Abudayyeh: Broad Institute of MIT and Harvard, 75 Ames Street
Clea Barcena: Broad Institute of MIT and Harvard, 75 Ames Street
Patrick D. Hsu: Broad Institute of MIT and Harvard, 75 Ames Street
Naomi Habib: Broad Institute of MIT and Harvard, 75 Ames Street
Jonathan S. Gootenberg: Broad Institute of MIT and Harvard, 75 Ames Street
Hiroshi Nishimasu: Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi Bunkyo, Tokyo 113-0032, Japan
Osamu Nureki: Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi Bunkyo, Tokyo 113-0032, Japan
Feng Zhang: Broad Institute of MIT and Harvard, 75 Ames Street

Nature, 2015, vol. 517, issue 7536, 583-588

Abstract: 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.

Date: 2015
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DOI: 10.1038/nature14136

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