Abstract
CRISPR/Cas9-mediated transcriptional activation (CRISPRa) is a powerful tool for investigating complex biological phenomena. Although CRISPRa approaches based on the VP64 transcriptional activator have been widely studied in both cultured cells and in animal models and exhibit great versatility for various cell types and developmental stages in vivo, different dCas9-VP64 versions have not been rigorously compared. Here, we compared different dCas9-VP64 constructs in identical contexts, including the cell lines used and the transfection conditions, for their ability to activate endogenous and exogenous genes. Moreover, we investigated the optimal approach for VP64 addition to VP64- and p300-based constructs. We found that MS2-MCP-scaffolded VP64 enhanced basal dCas9-VP64 and dCas9-p300 activity better than did direct VP64 fusion to the N-terminus of dCas9. dCas9-VP64+MCP-VP64 and dCas9-p300+MCP-VP64 were superior to VP64-dCas9-VP64 for all target genes tested. Furthermore, multiplexing gRNA expression with dCas9-VP64+MCP-VP64 or dCas9-p300+MCP-VP64 significantly enhanced endogenous gene activation to a level comparable to CRISPRa-SAM with a single gRNA. Our findings demonstrate improvement of the dCas9-VP64 CRISPRa system and contribute to development of a versatile, efficient CRISPRa platform.
Funder
National Institute of Diabetes and Digestive and Kidney Diseases
Children's Discovery Institute of Washington University and St. Louis Children's Hospital
Japan Society for the Promotion of Science
Cell Science Research Foundation
Publisher
Public Library of Science (PLoS)
Cited by
3 articles.
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