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, Feng Zhang*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1813 Scopus citations

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.

Original languageAmerican English
Pages (from-to)583-588
Number of pages6
JournalNature
Volume517
Issue number7536
DOIs
StatePublished - 29 Jan 2015
Externally publishedYes

Bibliographical note

Funding Information:
Acknowledgements We would like to thank S. Shehata, K. Zheng, C. Johannessen, L. Garraway, O. Shalem and members of the Zhang laboratory for assistance and helpful discussions. O.O.A. is supported by a NSF Graduate Research Fellowship, J.S.G. is supported by a D.O.E. Computational Science Graduate Fellowship, H.N. is supported by PRESTO from JST and Grant-in-Aid for Scientific Research (B) from JSPS, O.N. is supported by the CREST program and JST, and F.Z. is supported by the NIMH (DP1-MH100706), the NINDS (R01-NS07312401), NSF, the Keck, Searle Scholars, Klingenstein, Vallee, and Simons Foundations, and Bob Metcalfe. CRISPR reagents are available to the academic community through Addgene, and associated protocols, support forum and computational tools are available via the Zhang laboratory website (http://www.genome-engineering.org).

Publisher Copyright:
© 2015 Macmillan Publishers Limited. All rights reserved.

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