CRISPR-based Transcriptional Activators for Dissecting Human Gene-Regulatory Mechanisms

Résumé fourni par la source

Nuclease-inactivated CRISPR/Cas-based (dCas-based) systems have emerged as powerful technologies to reshape the human epigenome and gene expression synthetically. Despite the increasing adoption of these platforms, their relative potencies lack optimization, and their mechanistic differences are incompletely characterized. Here we expanded the dCas9-based activator toolbox to incorporate human CBP and GCN5 and tested their efficacy as transcriptional activators. For the optimization of the dCas9-toolbox, we showed that affinity-purification-mass spectrometry could be applied to identify the antagonistic gene for these dCas9-based tools in the cellular context, which enables us to improve dCas9-based transcriptional activation by targeted antagonist neutralization through either small-molecule inhibition or RNA interference. Eventually, we systematically compared the most widely adopted dCas9-based transcriptional activators to investigate the interconnections between different aspects of transcription regulation, including eRNA transcription, mRNA transcription, histone acetylation and chromatin spatial rearrangement. We used dCas9-based activators to demonstrate that an intrinsic transcriptional and epigenetic reciprocity can exist between human enhancers and promoters and that enhancer-mediated tracking and engagement of a downstream promoter can be synthetically driven by targeting dCas9-based transcriptional activators to an enhancer. Collectively, our study increases the expanse of the dCas9-activator toolbox, creates new systematic approaches for their functional improvement, and unravels the potential of the dCas9-based activator toolbox to provide new insights into the enhancer-mediated control of human gene expression.

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