Fu Yujuan, Yang Xiaoxuan, Li Sihui, Ma Chenyang, An Yao, Cheng Tao, Liang Ying, Sun Shengbai, Cheng Tianyi, Zhao Yongyang, Wang Jianghu, Wang Xiaoyue, Xu Pengfei, Yin Yafei, Liang Hongqing, Liu Nan, Zou Wei, Chen Baohui
Bone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China.
Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou, China.
Nat Commun. 2025 Feb 14;16(1):1640. doi: 10.1038/s41467-025-56735-8.
CRISPR activation (CRISPRa) is a powerful tool for endogenous gene activation, yet the mechanisms underlying its optimal transcriptional activation remain unclear. By monitoring real-time transcriptional bursts, we find that CRISPRa modulates both burst duration and amplitude. Our quantitative imaging reveals that CRISPR-SunTag activators, with three tandem VP64-p65-Rta (VPR), form liquid-like transcriptional condensates and exhibit high activation potency. Although visible CRISPRa condensates are associated with some RNA bursts, the overall levels of phase separation do not correlate with transcriptional bursting or activation strength in individual cells. When the number of SunTag scaffolds is increased to 10 or more, solid-like condensates form, sequestering co-activators such as p300 and MED1. These condensates display low dynamicity and liquidity, resulting in ineffective gene activation. Overall, our studies characterize various phase-separated CRISPRa systems for gene activation, highlighting the foundational principles for engineering CRISPR-based programmable synthetic condensates with appropriate properties to effectively modulate gene expression.
CRISPR激活（CRISPRa）是一种用于内源性基因激活的强大工具，但其最佳转录激活背后的机制仍不清楚。通过监测实时转录爆发，我们发现CRISPRa可调节爆发持续时间和幅度。我们的定量成像显示，具有三个串联VP64-p65-Rta（VPR）的CRISPR-SunTag激活剂形成类液体转录凝聚体，并表现出高激活效力。尽管可见的CRISPRa凝聚体与一些RNA爆发相关，但相分离的总体水平与单个细胞中的转录爆发或激活强度无关。当SunTag支架的数量增加到10个或更多时，会形成类固体凝聚体，隔离诸如p300和MED1等共激活因子。这些凝聚体表现出低动态性和流动性，导致基因激活无效。总体而言，我们的研究表征了用于基因激活的各种相分离CRISPRa系统，突出了设计具有适当特性以有效调节基因表达的基于CRISPR的可编程合成凝聚体的基本原理。
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