Abstract
Gene overexpression through the targeting of transcription activation domains to regulatory DNA via catalytically defective Cas9 (dCas9) represents a powerful approach to investigate gene function as well as the mechanisms of gene control. To date, the most efficient dCas9-based activator is the Synergistic Activation Mediator (SAM) system whereby transcription activation domains are directly fused to dCas9 as well as tethered through MS2 loops engineered into the gRNA. Here, we show that dCas9 fused to the catalytic domain of the histone acetyltransferase CBP is a more potent activator than the SAM system at some loci, but less efficient at other locations in Drosophila cells. Our results suggest that different rate-limiting steps in the transcription cycle are affected by dCas9-CBP and the SAM system, and that comparing these activators may be useful for mechanistic studies of transcription as well as for increasing the number of hits in genome-wide overexpression screens.
Highlights
Gene overexpression through the targeting of transcription activation domains to regulatory DNA via catalytically defective Cas[9] represents a powerful approach to investigate gene function as well as the mechanisms of gene control
We describe a Drosophila defective Cas9 (dCas9)-CBP EpiEffector that proved to be more efficient in activating some genes than the Synergistic Activation Mediator (SAM) system that targets three different transcription activation domains to the genome[9]
This indicates that dCas9-CBP and SAM target different rate-limiting steps in the transcription cycle, and suggests that dCas9-CBP could be useful for overexpressing genes that are refractory to activation by the SAM system
Summary
Gene overexpression through the targeting of transcription activation domains to regulatory DNA via catalytically defective Cas[9] (dCas9) represents a powerful approach to investigate gene function as well as the mechanisms of gene control. DCas[9] can be fused to histone or DNA-modifying protein domains Such EpiEffectors have been used to identify and characterize functional regulatory elements in mammalian cells[1]. We describe a Drosophila dCas9-CBP EpiEffector that proved to be more efficient in activating some genes than the Synergistic Activation Mediator (SAM) system that targets three different transcription activation domains to the genome[9]. We find that both dCas9-CBP and SAM can function from a distance of tens of kb to activate gene expression, but genomic loci respond differently to these two activators This indicates that dCas9-CBP and SAM target different rate-limiting steps in the transcription cycle, and suggests that dCas9-CBP could be useful for overexpressing genes that are refractory to activation by the SAM system
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