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Nature. 2015 Jan 29;517(7536):583-8. doi: 10.1038/nature14136. Epub 2014 Dec 10.

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

Author information

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.

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.

PMID:
25494202
PMCID:
PMC4420636
DOI:
10.1038/nature14136
[Indexed for MEDLINE]
Free PMC Article

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