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Potent transcriptional activation with CRISPRa using synthetic crRNA
Poster Title: Potent transcriptional activation with CRISPRa using synthetic crRNA
Submitted on 22 Jun 2018
Author(s): Žaklina Strezoska, Elena Maksimova, Eldon T. Chou, Maren Mayer Gross, Shawn McClelland, Melissa L. Kelley, and Anja van Brabant Smith
Affiliations: Dharmacon, part of Horizon Discovery Group
This poster was presented at Keystone Symposia, Precision Genome Editing with Programmable Nucleases
Poster Views: 567
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Poster Information
Abstract: These data show that using a dual synthetic gRNA approach provides benefits that are applicable from simply upregulating a gene to systematic functional gene analysis in an arrayed screening format.

The CRISPR-Cas system derived from Streptococcus pyogenes has been adapted to upregulate any gene in its endogenous context, enabling overexpression experiments without a need for exogenous overexpression 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 transcriptional activators. The machinery then acts upstream of the transcription start sites to upregulate expression of a target gene. The ease of programming the CRISPRa system with small RNA guides is transformative for performing gain-of function studies as an alternative method for identification of gene functions that might be undiscovered with loss-of function methods alone.

Here we demonstrate a strategy to conduct CRISPRa experiments using synthetic crRNA reagents. We examine the functionality and advantages of using the synthetic guide RNA approach for CRISPRa in multiple cell lines including pooling of crRNAs to enhance target gene activation, as well as activating multiple genes simultaneously. Additionally, we demonstrate transcriptional activation leading to a significant increase in the target’s protein level, which in turn causes phenotypic effects by inhibiting or activating downstream genes. This demonstrates that CRISPRa using synthetic crRNAs could be used for low- or high-throughput studies of downstream signaling and pathway analysis. The methods presented are broadly applicable as a strategy to upregulate any gene including systematic functional gene analysis in an arrayed screening format.
Summary: Here we look at the advantages of a two-part synthetic guide RNA approach for CRISPRa. We tested our methods in multiple cell lines, observed pooling of crRNAs to enhance target activation, and multiplexed gRNAs for simultaneous activation of different targets. We also show CRISPRa leading to an increase in the target’s protein level, which in turn, causes downstream phenotypic effects. References: Report abuse »
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