Dynamic properties of transcriptional condensates modulate CRISPRa-mediated gene activation
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 quantitat...
Saved in:
| Main Authors: | , , , , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2025-02-01
|
| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-56735-8 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850067478841917440 |
|---|---|
| author | 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 Liang Nan Liu Wei Zou Baohui Chen |
| author_facet | 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 Liang Nan Liu Wei Zou Baohui Chen |
| author_sort | Yujuan Fu |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-92e5b3cac0c8472a8402575d31dd478e |
| institution | DOAJ |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-92e5b3cac0c8472a8402575d31dd478e2025-08-20T02:48:18ZengNature PortfolioNature Communications2041-17232025-02-0116111910.1038/s41467-025-56735-8Dynamic properties of transcriptional condensates modulate CRISPRa-mediated gene activationYujuan Fu0Xiaoxuan Yang1Sihui Li2Chenyang Ma3Yao An4Tao Cheng5Ying Liang6Shengbai Sun7Tianyi Cheng8Yongyang Zhao9Jianghu Wang10Xiaoyue Wang11Pengfei Xu12Yafei Yin13Hongqing Liang14Nan Liu15Wei Zou16Baohui Chen17Bone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of MedicineBone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of MedicineBone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of MedicineWomen’s Hospital, Zhejiang University School of MedicineCenter of Stem Cell and Regenerative Medicine, Zhejiang University School of MedicineWomen’s Hospital, Zhejiang University School of MedicineBone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of MedicineCenter of Stem Cell and Regenerative Medicine, Zhejiang University School of MedicineBone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of MedicineBone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of MedicineBone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of MedicineThe State Key Laboratory of Southwest Karst Mountain Biodiversity Conservation of Forestry Administration, School of Life Science, Guizhou Normal UniversityWomen’s Hospital, Zhejiang University School of MedicineCenter of Stem Cell and Regenerative Medicine, Zhejiang University School of MedicineWomen’s Hospital, Zhejiang University School of MedicineLiangzhu Laboratory, Zhejiang University Medical CenterThe Fourth Affiliated Hospital, Zhejiang University School of MedicineBone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of MedicineAbstract 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.https://doi.org/10.1038/s41467-025-56735-8 |
| spellingShingle | 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 Liang Nan Liu Wei Zou Baohui Chen Dynamic properties of transcriptional condensates modulate CRISPRa-mediated gene activation Nature Communications |
| title | Dynamic properties of transcriptional condensates modulate CRISPRa-mediated gene activation |
| title_full | Dynamic properties of transcriptional condensates modulate CRISPRa-mediated gene activation |
| title_fullStr | Dynamic properties of transcriptional condensates modulate CRISPRa-mediated gene activation |
| title_full_unstemmed | Dynamic properties of transcriptional condensates modulate CRISPRa-mediated gene activation |
| title_short | Dynamic properties of transcriptional condensates modulate CRISPRa-mediated gene activation |
| title_sort | dynamic properties of transcriptional condensates modulate crispra mediated gene activation |
| url | https://doi.org/10.1038/s41467-025-56735-8 |
| work_keys_str_mv | AT yujuanfu dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT xiaoxuanyang dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT sihuili dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT chenyangma dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT yaoan dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT taocheng dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT yingliang dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT shengbaisun dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT tianyicheng dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT yongyangzhao dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT jianghuwang dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT xiaoyuewang dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT pengfeixu dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT yafeiyin dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT hongqingliang dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT nanliu dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT weizou dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation AT baohuichen dynamicpropertiesoftranscriptionalcondensatesmodulatecrispramediatedgeneactivation |