Specific multivalent molecules boost CRISPR-mediated transcriptional activation via optimal cis-trans interaction and functional chromatin looping [ChIA-PET]

Synthetic transcriptional activators based on CRISPR/Cas technology can be enhanced by intrinsically disordered regions (IDRs). However, the potential of all IDRs in this regard remains uncertain, and the mechanisms underlying their influence are a subject of debate. Here, we examined 12 well-known IDRs by fusing them to the dCas9-VP64 activator. Our findings reveal that seven IDRs could augment activation, albeit independently of their phase separation properties. Moreover, modular domains (MDs), which facilitate multivalent interactions but are ineffective in enhancing dCas9-VP64 activity on their own, showed substantial enhancement in transcriptional activation when combined with dCas9-VP64-IDR. By varying the number of gRNA binding sites and combining dCas9-VP64 with the IDRs and MDs of different multivalent capabilities, we uncovered that optimal, rather than maximal, cis-trans cooperativity enables the most robust activation. Finally, targeting promoter-enhancer pairs with our IDR-enhanced activation system yielded synergistic effects, potentially further amplifiable by induced functional chromatin looping. To verify that the increased transcriptional activation resulted from enhanced long-range chromatin interactions, we examined HS2-associated looping before and after ABA treatment using ChIA-PET