Cas9与耐药基因研究是啥关系nature.pdfVIP

ARTICLE doi:10.1038/nature14136 Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex Silvana Konermann1,2,3,4*, Mark D. Brigham1,2,3,4*, Alexandro E. Trevino1,2,3,4 , Julia Joung1,4, Omar O. Abudayyeh1,2,3,4 , 1,2,3,4 1,2,3,4 1 1,2,3,4,5 6,7 6 Clea Barcena , Patrick D. Hsu , Naomi Habib , Jonathan S. Gootenberg , Hiroshi Nishimasu , Osamu Nureki Feng Zhang1,2,3,4 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 per