清脆的
Cas9
基因组编辑
生物
基因
亚基因组mRNA
计算生物学
基因表达调控
遗传学
基因组
作者
Silvana Konermann,Mark D. Brigham,Alexandro E. Trevino,Julia Joung,Omar O. Abudayyeh,Clea Bárcena,Patrick Hsu,Naomi Habib,Jonathan S. Gootenberg,Hiroshi Nishimasu,Osamu Nureki,Feng Zhang
出处
期刊:Nature
[Springer Nature]
日期:2014-12-10
卷期号:517 (7536): 583-588
被引量:2260
摘要
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. 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.
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