Nature Biotechnology, 6 November, 2025, DOI：https://doi.org/10.1038/s41587-025-02887-3

CRISPR live-cell imaging reveals chromatin dynamics and enhancer interactions at multiple non-repetitive loci

Meishuo Liu, Keyun Huang, Jie Zhang, Qingyang Li, Xinming Wang, Buming Gu, Hao Tang, Zhenhai Du, Liangjun Hu, Shutao Qi, Yu Ma, Hongtao Yu, Wei Xie, Xianyang Fang & Haifeng Wang

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.

Article link：https://www.nature.com/articles/s41587-025-02887-3