Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.
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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. |
Year of Publication | 2015
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Journal | Nature
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Volume | 517
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Issue | 7536
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Pages | 583-8
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Date Published | 2015 Jan 29
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ISSN | 1476-4687
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URL | |
DOI | 10.1038/nature14136
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PubMed ID | 25494202
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PubMed Central ID | PMC4420636
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Grant list | R01 NS062849 / NS / NINDS NIH HHS / United States
R01-NS07312401 / NS / NINDS NIH HHS / United States
DP1-MH100706 / DP / NCCDPHP CDC HHS / United States
DP1 MH100706 / MH / NIMH NIH HHS / United States
R01 NS073124 / NS / NINDS NIH HHS / United States
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