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Potent transcriptional activation using CRISPRa with synthetic crRNA
EP26744
Poster Title: Potent transcriptional activation using CRISPRa with synthetic crRNA
Submitted on 07 Dec 2017
Author(s): Eldon T. Chou, Žaklina Strezoska, Elena Maksimova, Maren M. Gross, Melissa L. Kelley, and Anja van Brabant Smith
Affiliations: Dharmacon part of Horizon Discovery Group
This poster was presented at ASCB/EMBO Conference
Poster Views: 813
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Poster Information
Abstract: For CRISPR activation (CRISPRa), the guide RNA forms a complex with a nuclease-deactivated Cas9 (dCas9, D10A and H840A), which is in turn fused to transcriptional activators. The machinery then acts upstream of the transcription start site to up-regulate expression of a target gene. CRISPRa provides new tools to identify gene functions that might otherwise go undetected using loss-of-function studies through down-regulation of gene expression or gene knockout. Here we demonstrate a strategy to conduct CRISPRa experiments using chemically synthesized crRNA and tracrRNA molecules. We examine the functionality and advantages of using the synthetic approach for CRISPRa in multiple cell lines. Additionally, we show how the transcriptional activation leads to a significant increase in the target protein level, which in turn causes phenotypic effects by inhibiting or activating downstream genes. The methods presented are broadly applicable as a strategy to up-regulate any gene including systematic functional gene analysis in arrayed screening format.Summary: The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9 proteins) system derived from Streptococcus pyogenes has been adapted to up-regulate any gene in its endogenous context, enabling gain-of-function or gene activation experiments while avoiding the use of exogenous over-expression plasmids. References: Report abuse »
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