Compact engineered human transactivation modules enable potent and versatile synthetic transcriptional control

Abstract

AbstractEngineered transactivation domains (TADs) combined with programmable DNA binding platforms have revolutionized synthetic transcriptional control. Despite recent progress in programmable CRISPR/Cas-based transactivation (CRISPRa) technologies, the TADs used in these systems often contain components from viral pathogens and/or are prohibitively large for many applications. Here we defined and optimized minimal TADs built from human mechanosensitive transcription factors (MTFs). We used these components to construct potent and compact multipartite transactivation modules (MSN, NMS, and eN3×9) and to build the CRISPR-dCas9 recruited enhanced activation module (CRISPR-DREAM) platform. We found that CRISPR-DREAM was specific, robust across mammalian cell types, and efficiently stimulated transcription from diverse regulatory loci within the human genome. We also showed that MSN and NMS were portable across Type I, II, and V CRISPR systems, TALEs, and ZF proteins, and further that these TADs permitted superior multiplexed transactivation. Finally, as a proof of concept, we used dCas9-NMS to efficiently reprogram human fibroblasts into iPSCs. Altogether, the compact human TADs, design rules, and fusion proteins we have developed here could be valuable for applications where sophisticated synthetic transactivation is needed.