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A study of transcriptional activation using CRISPRa and synthetic crRNA:tracrRNA
Poster Title: A study of transcriptional activation using CRISPRa and synthetic crRNA:tracrRNA
Submitted on 07 Feb 2018
Author(s): Maren M. Gross1, Zaklina Stresozka, Elena Maksimova1, Eldon T. Chou1, and Anja van Brabant Smith
Affiliations: Dharmacon part of Horizon Discovery Group
This poster was presented at ADCB/EMBO Conference
Poster Views: 686
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Poster Information
Abstract: The CRISPR-Cas9 system derived from Streptococcus pyogenes has been adapted to upregulate any gene in its endogenous context, enabling gain-of-function or gene activation experiments while avoiding the use of exogenous over-expression plasmids. 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 three transcriptional activators [(VP64, p65 and Rta19) comprising the VPR system]. The guide RNAs target upstream of the transcription start site enabling the dCas9-VPR to up-regulate the expression of the 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. Summary: Here we demonstrate strategies 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 dCas9-VPR stable cell populations. Additionally, we show how the transcriptional activation leads to significant increase in target protein level, resulting in detectable phenotypic effects by inhibiting or activating downstream genes. References: Report abuse »
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