期刊
NATURE
卷 517, 期 7536, 页码 583-U332出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nature14136
关键词
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资金
- NSF Graduate Research Fellowship
- D.O.E. Computational Science Graduate Fellowship
- PRESTO from JST
- JSPS
- CREST program
- JST
- NIMH [DP1-MH100706]
- NINDS [R01-N507312401]
- NSF
- Keck, Searle Scholars
- Klingenstein Foundation
- Vallee Foundation
- Simons Foundation
- Bob Metcalfe
- Grants-in-Aid for Scientific Research [26291010] Funding Source: KAKEN
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.
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