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

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...

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Veröffentlicht in:	Nature (London) Jg. 517; H. 7536; S. 583 - 588
Hauptverfasser:	Konermann, Silvana, Brigham, Mark D., Trevino, Alexandro E., Joung, Julia, Abudayyeh, Omar O., Barcena, Clea, Hsu, Patrick D., Habib, Naomi, Gootenberg, Jonathan S., Nishimasu, Hiroshi, Nureki, Osamu, Zhang, Feng
Format:	Journal Article
Sprache:	Englisch
Veröffentlicht:	London Nature Publishing Group UK 29.01.2015 Nature Publishing Group
Schlagworte:	45/23 631/337/572/2102 Cell Line, Tumor Clustered Regularly Interspaced Short Palindromic Repeats - genetics CRISPR-Associated Proteins - genetics CRISPR-Associated Proteins - metabolism CRISPR-Cas Systems - genetics Crystal structure DNA, Complementary - biosynthesis DNA, Complementary - genetics Drug resistance Drug Resistance, Neoplasm - drug effects Drug Resistance, Neoplasm - genetics Enzymes Epigenetics Gene expression Gene Expression Regulation, Neoplastic - genetics Gene Library Genetic aspects Genetic engineering Genetic Engineering - methods Genetic Loci - genetics Genetic regulation Genetic research Genetic Testing Genetic transcription Genome, Human - genetics Genomes Genomics Health aspects Humanities and Social Sciences Humans Indoles - pharmacology Melanoma Melanoma - drug therapy Melanoma - genetics multidisciplinary Nucleotide sequence Proteins Proto-Oncogene Proteins B-raf - antagonists & inhibitors Reproducibility of Results RNA, Untranslated - biosynthesis RNA, Untranslated - genetics RNA, Untranslated - metabolism Science Sulfonamides - pharmacology Transcription factors Transcriptional Activation - genetics Up-Regulation - genetics United States
ISSN:	0028-0836, 1476-4687, 1476-4687
Online-Zugang:	Volltext
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Zusammenfassung:	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. The CRISPR-Cas9 system, a powerful tool for genome editing, has been engineered to activate endogenous gene transcription specifically and potently on a genome-wide scale and applied to a large-scale gain-of-function screen for studying melanoma drug resistance. CRISPR-Cas9 used for gene-expression regulation The CRISPR-Cas9 system has emerged as a powerful tool for genome editing and transcriptional regulation of specific genes. Feng Zhang and colleagues have successfully modified the system to specifically and potently activate endogenous gene transcription on a genome-wide scale, such that it can be used for large-scale functional genomics screens. Application to a genome-wide screen of melanoma cells for genes which when overexpressed can confer resistance to a BRAF inhibitor demonstrates the feasibility of such screens, and also led to the discovery of potential new resistance mechanisms.
Bibliographie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 These authors contributed equally to this work.
ISSN:	0028-0836 1476-4687 1476-4687
DOI:	10.1038/nature14136