Compact engineered human transactivation modules enable potent and versatile synthetic transcriptional control Barun Mahata 1 , Jing Li 1 , Alan Cabrera 1 , Daniel A. Brenner 1 , Rosa Selenia Guerra- Resendez 1,2 , Jacob Goell 1 , Kaiyuan Wang 1 , Mario Escobar 3 , Yannie Guo 1 , Abinand Krishna Parthasarathy 1 , Isaac B. Hilton 1,2,3* 1 Department of Bioengineering, Rice University, Houston, TX, USA 2 Systems, Synthetic, and Physical Biology Graduate Program, Rice University, Houston, TX, USA 3 Department of BioSciences, Rice University, Houston, TX, USA * To whom correspondence should be addressed. Tel: 713-348-8247; Email: isaac.hilton@rice.edu Abstract 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 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 eN3x9) 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. Introduction Nuclease deactivated CRISPR-Cas (dCas) systems can be used as programmable transcriptional modulators in cells and organisms 1-7 . For CRISPR/Cas based transactivation (CRISPRa) approaches, transcriptional activators can be recruited to genomic regulatory elements using direct fusions to dCas proteins 8-10 , antibody-mediated recruitment in tandem with dCas proteins 11 , or using engineered gRNA aptamer architectures 12, 13 . High levels of CRISPRa-driven transactivation have been achieved by shuffling 14 , reengineering 15 , or combining 8, 16 transactivation domains (TADs) and/or chromatin modifiers. However, many of the transactivation components used in CRISPRa systems have coding sizes that are restrictive for applications such as viral vector-based delivery. Moreover, most of the transactivation modules that display high potencies harbor components derived from viral pathogens, which could hamper clinical or in vivo use. Finally, there is an untapped repertoire of thousands of human transcription factors (TFs) and chromatin modifiers 17-19 that has yet to be systematically tested and optimized as . CC-BY-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 21, 2022. ; https://doi.org/10.1101/2022.03.21.485228 doi: bioRxiv preprint