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Dynamic properties of transcriptional condensates modulate CRISPRa-mediated gene activation

Author

Listed:
  • Yujuan Fu

    (Zhejiang University School of Medicine
    Zhejiang University Medical Center
    Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy)

  • Xiaoxuan Yang

    (Zhejiang University School of Medicine
    Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy)

  • Sihui Li

    (Zhejiang University School of Medicine
    Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy)

  • Chenyang Ma

    (Zhejiang University School of Medicine)

  • Yao An

    (Zhejiang University School of Medicine)

  • Tao Cheng

    (Zhejiang University School of Medicine)

  • Ying Liang

    (Zhejiang University School of Medicine
    Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy)

  • Shengbai Sun

    (Zhejiang University School of Medicine)

  • Tianyi Cheng

    (Zhejiang University School of Medicine
    Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy)

  • Yongyang Zhao

    (Zhejiang University School of Medicine
    Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy)

  • Jianghu Wang

    (Zhejiang University School of Medicine
    Guizhou Normal University)

  • Xiaoyue Wang

    (Guizhou Normal University)

  • Pengfei Xu

    (Zhejiang University School of Medicine)

  • Yafei Yin

    (Zhejiang University School of Medicine)

  • Hongqing Liang

    (Zhejiang University School of Medicine)

  • Nan Liu

    (Zhejiang University Medical Center
    Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy)

  • Wei Zou

    (Zhejiang University School of Medicine
    Zhejiang University)

  • Baohui Chen

    (Zhejiang University School of Medicine
    Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy
    Zhejiang Provincial Key Laboratory of Genetic & Developmental Disorders)

Abstract

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.

Suggested Citation

  • Yujuan Fu & Xiaoxuan Yang & Sihui Li & Chenyang Ma & Yao An & Tao Cheng & Ying Liang & Shengbai Sun & Tianyi Cheng & Yongyang Zhao & Jianghu Wang & Xiaoyue Wang & Pengfei Xu & Yafei Yin & Hongqing Lia, 2025. "Dynamic properties of transcriptional condensates modulate CRISPRa-mediated gene activation," Nature Communications, Nature, vol. 16(1), pages 1-19, December.
  • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-56735-8
    DOI: 10.1038/s41467-025-56735-8
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    References listed on IDEAS

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