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

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

  • Nat Biotechnol. 2025 Nov 6. doi: 10.1038/s41587-025-02887-3.
Meishuo Liu # 1,  Keyun Huang # 1,  Jie Zhang # 2 3,  Qingyang Li 1,  Xinming Wang 1,  Buming Gu 1,  Hao Tang 1,  Zhenhai Du 4 5 6,  Liangjun Hu 4 5,  Shutao Qi 7 8,  Yu Ma 1,  Hongtao Yu 7 8,  Wei Xie 9 10,  Xianyang Fang 11,  Haifeng Wang 12
Affiliations

Affiliations

  • 1 School of Life Sciences, Tsinghua-Peking Joint Center for Life Sciences, Center for Synthetic and Systems Biology, State Key Laboratory of Complex, Severe, and Rare Diseases, Tsinghua University, Beijing, China.
  • 2 School of Life Sciences, Tsinghua University, Beijing, China.
  • 3 State Key Laboratory of RNA Innovation, Science and Engineering, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
  • 4 Center for Stem Cell Biology and Regenerative Medicine, MOE Key Laboratory of Bioinformatics, New Cornerstone Science Laboratory, School of Life Sciences, Tsinghua University, Beijing, China.
  • 5 Tsinghua-Peking Joint Center for Life Sciences, Beijing, China.
  • 6 State Key Laboratory of RNA Innovation, Science and Engineering, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.
  • 7 New Cornerstone Science Laboratory, School of Life Sciences, Westlake University, Hangzhou, China.
  • 8 Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China.
  • 9 Center for Stem Cell Biology and Regenerative Medicine, MOE Key Laboratory of Bioinformatics, New Cornerstone Science Laboratory, School of Life Sciences, Tsinghua University, Beijing, China. [email protected].
  • 10 Tsinghua-Peking Joint Center for Life Sciences, Beijing, China. [email protected].
  • 11 State Key Laboratory of RNA Innovation, Science and Engineering, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. [email protected].
  • 12 School of Life Sciences, Tsinghua-Peking Joint Center for Life Sciences, Center for Synthetic and Systems Biology, State Key Laboratory of Complex, Severe, and Rare Diseases, Tsinghua University, Beijing, China. [email protected].
  • # Contributed equally.
PMID: 41199023 DOI: 10.1038/s41587-025-02887-3
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.

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