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

Engineered 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 poorly tolerated elements 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. We also showed that MSN and NMS were portable across Type I, II, and V CRISPR systems, TALEs, and ZF proteins. Further, as proofs of concepts, we used dCas9-NMS to efficiently reprogram human fibroblasts into iPSCs and demonstrated that MTF TADs are efficacious and well tolerated in therapeutically important primary human cell types. Finally, we leveraged the compact and potent features of these engineered TADs to build new dual and all-in-one CRISPRa AAV systems. Altogether, these compact human TADs, fusion modules, and new delivery architectures should be valuable for synthetic transcriptional control in biomedical applications. Overall design: mRNA sequencing for accessing CRISPR-DREAM specificity in HEK293T cells