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<p>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 <i>PCDHα</i>−enhancer interactions may persist despite spatial mobility and that BRD4 maintains super-enhancer contacts regulating <i>MYC</i> oncogene expression in cancer cells. CRISPR PRO-LiveFISH can be applied to diverse studies of chromatin dynamics and genome organization in living cells.</p>

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

  • 期刊论文

  • 发表日期：

    2025年11月6日

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  • 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

摘要

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.