Novel approach of genetic perturbation

 

In order to systematically determine gene function, the perturbations on gene expression is required. In contrast to the well-established genetic perturbation, loss-of-function (LOF), the gain-of-function (GOF) is confined to the overexpression of cDNA library, which has a lot limitations. Konermann et al. built a CRISPR-Cas9 complex for the regulation of effective transcriptional activation at target genomic loci. The article was published online in the Nature, recently.

 

Cas9 is a widely used programmable DNA-binding proteins for mediating endogenous gene expression, due to simpler programming compared with other custom DNA-binding domains. dCas9-activatior, an RNA-guided transcription activator concerted from Cas9, gains low or ineffective upregulation with individual single-guide RNAs (sgRNAs). Researchers built a Cas9-sgRNA-targetDNA tertiary complex, which can mediate robust upregulation of endogenous genes. They demonstrated multiplexed upregulation of ten genes, as well as long intergenic non-coding RNA (lincRNA) transcripts. By gene activation, researchers also synthesized a screen library for sorting genes are able to confer resistance to a BRAF inhibitor. Some of the screening  results matched with previously study of drug resistance, and novel candidates emerged. The findings indicates Cas9-based activators has potential as a effective technology for genetic perturbation.

 

Reference:
Nature. 2014 Dec 10. 10.1038/nature14136

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S1152	PLX-4720	PLX4720 is a potent and selective inhibitor of B-RafV600E with IC50 of 13 nM in a cell-free assay, equally potent to c-Raf-1(Y340D and Y341D mutations), 10-fold selectivity for B-RafV600E than wild-type B-Raf.

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