Publication Information
CRISPR live-cell imaging reveals chromatin dynamics and enhancer interactions at multiple non-repetitive loci
Abstract
Existing methods to visualize dynamic changes in the three-dimensional genome, promoter−enhancer interactions and the influence of epigenetic modifications in non-repetitive loci are limited. Here we introduce CRISPR PRO-LiveFISH (Pooled gRNAs with Orthogonal bases LiveFISH), which combines orthogonal bases from expanded genetic alphabet technology and rational single guide RNA (sgRNA) design to efficiently label multiple non-repetitive loci in living cells. The optimized method allows simultaneous imaging of up to six genomic loci and uses as few as 10 sgRNAs for non-repetitive loci imaging without signal amplification. We demonstrate the method in diverse cell types, including primary cells, and apply it to reveal enhancer−promoter dynamics and a correlation between genomic dynamics and epigenetic states. We also show that PCDHα−enhancer interactions may persist despite spatial mobility and that BRD4 maintains super-enhancer contacts regulating MYC oncogene expression in cancer cells. CRISPR PRO-LiveFISH can be applied to diverse studies of chromatin dynamics and genome organization in living cells.
Preferred citation
Liu, M., Huang, K., Zhang, J. et al. CRISPR live-cell imaging reveals chromatin dynamics and enhancer interactions at multiple non-repetitive loci. Nat Biotechnol (2025). https://doi.org/10.1038/s41587-025-02887-3
Data Availability
The data supporting this study are provided in this article and its supplementary information and Source Data files. Additional data are available below. Source data are provided with this paper.
Additional Data
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Acknowledgments
We are grateful to members of the Wang laboratory for their discussions and comments during the preparation of the manuscript. We thank the Imaging Core Facility of the Technology Center for Protein Sciences at Tsinghua University for microscopy imaging and analysis support. We thank R. Xu and D. S. Liu for their support in exploring fluorescent gRNA labeling methods during the initial stages, which, although not included here, has provided valuable insights. This work was funded by the following grants: the Agriculture Science and Technology Major Project to H.W., the National Key R&D Program of China (2022YFC3400204 to H.W., 2021YFA1301500 to X.F., 2021YFA1100102 to W.X.); the National Natural Science Foundation of China (32270573 to H.W., U1832215 to X.F.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB0570300 to X.F.), the Beijing Natural Science Foundation (F251024 to X.F.) and the Tsinghua-Peking Joint Center for Life Sciences (H.W. and W.X.). H.W. is supported by the Independent Research Fund of the State Key Laboratory of Complex, Severe, and Rare Diseases (2025-I-ZD-005), the Tsinghua University Initiative Scientific Research Program and the Benyuan Charity Fund. W.X. is a recipient of the New Cornerstone Investigator award.