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...
Uložené v:
| Vydané v: | Nature (London) Ročník 517; číslo 7536; s. 583 - 588 |
|---|---|
| Hlavní autori: | , , , , , , , , , , , |
| Médium: | Journal Article |
| Jazyk: | English |
| Vydavateľské údaje: |
London
Nature Publishing Group UK
29.01.2015
Nature Publishing Group |
| Predmet: | |
| ISSN: | 0028-0836, 1476-4687, 1476-4687 |
| On-line prístup: | Získať plný text |
| Tagy: |
Pridať tag
Žiadne tagy, Buďte prvý, kto otaguje tento záznam!
|
| Shrnutí: | 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. |
|---|---|
| Bibliografia: | 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 |