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Fluorogenic CRISPR for genomic DNA imaging

Author

Listed:
  • Zhongxuan Zhang

    (Chinese Academy of Science
    The Affiliated Hospital of Southwest Medical University
    University of Chinese Academy of Sciences
    Institute of Zoology, Chinese Academy of Sciences)

  • Xiaoxiao Rong

    (Chinese Academy of Science
    Hebei University, Baoding)

  • Tianjin Xie

    (Chinese Academy of Science
    Southwest University)

  • Zehao Li

    (Chinese Academy of Science
    Hebei University, Baoding)

  • Haozhi Song

    (Chinese Academy of Science)

  • Shujun Zhen

    (Southwest University)

  • Haifeng Wang

    (Tsinghua University)

  • Jiahui Wu

    (University of Massachusetts)

  • Samie R. Jaffrey

    (Cornell University)

  • Xing Li

    (Chinese Academy of Science
    The Affiliated Hospital of Southwest Medical University
    University of Chinese Academy of Sciences
    Institute of Zoology, Chinese Academy of Sciences)

Abstract

Genomic DNA exhibits high heterogeneity in terms of its dynamic within the nucleus, its structure and functional roles. CRISPR-based imaging approaches can image genomic loci in living cells. However, conventional CRISPR-based tools involve expressing constitutively fluorescent proteins, resulting in high background and nonspecific nucleolar signal. Here, we construct fluorogenic CRISPR (fCRISPR) to overcome these issues. fCRISPR is designed with dCas9, an engineered sgRNA, and a fluorogenic protein. Fluorogenic proteins are degraded unless they are bound to specific RNA hairpins. These hairpins are inserted into sgRNA, resulting in dCas9: sgRNA: fluorogenic protein ternary complexes that enable fluorogenic DNA imaging. With fCRISPR, we image various genomic DNA in different human cells with high signal-to-noise ratio and sensitivity. Furthermore, fCRISPR tracks chromosomes dynamics and length. fCRISPR also allows DNA double-strand breaks (DSBs) and repair to be tracked in real time. Taken together, fCRISPR offers a high-contrast and sensitive platform for imaging genomic loci.

Suggested Citation

  • Zhongxuan Zhang & Xiaoxiao Rong & Tianjin Xie & Zehao Li & Haozhi Song & Shujun Zhen & Haifeng Wang & Jiahui Wu & Samie R. Jaffrey & Xing Li, 2024. "Fluorogenic CRISPR for genomic DNA imaging," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-45163-9
    DOI: 10.1038/s41467-024-45163-9
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