Specific multivalent molecules boost CRISPR-mediated transcriptional activation
Rui Chen, Xinyao Shi, Xiangrui Yao, Tong Gao, Guangyu Huang, Duo Ning, Zemin Cao, Youxin Xu, Weizheng Liang, Simon Zhongyuan Tian, Qionghua Zhu, Liang Fang, Meizhen Zheng, Yuhui Hu, Huanhuan Cui, Wei Chen
Abstract
CRISPR/Cas-based transcriptional activators can be enhanced by intrinsically disordered regions (IDRs). However, the underlying mechanisms are still debatable. Here, we examine 12 well-known IDRs by fusing them to the dCas9-VP64 activator, of which only seven can augment activation, albeit independently of their phase separation capabilities. Moreover, modular domains (MDs), another class of multivalent molecules, though ineffective in enhancing dCas9-VP64 activity on their own, show substantial enhancement in transcriptional activation when combined with dCas9-VP64-IDR. By varying the number of gRNA binding sites and fusing dCas9-VP64 with different IDRs/MDs, we uncover that optimal, rather than maximal, cis-trans cooperativity enables the most robust activation. Finally, targeting promoter-enhancer pairs yields synergistic effects, which can be further amplified via enhancing chromatin interactions. Overall, our study develops a versatile platform for efficient gene activation and sheds important insights into CRIPSR-based transcriptional activators enhanced with multivalent molecules. Multivalent interactions are crucial in transcriptional regulation. Here, by integrating specific multivalent molecules into dCas9-based activators, the authors provide valuable strategies to refine CRISPRa applications and achieve highly efficient gene transcription.