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Ultrasensitive detection of clinical pathogens through a target-amplification-free collateral-cleavage-enhancing CRISPR-CasΦ tool

Author

Listed:
  • Huiyou Chen

    (Hainan University
    Hainan University
    Hainan University)

  • Fengge Song

    (Hainan University
    Hainan University
    Hainan University)

  • Buhua Wang

    (Hainan University
    Hainan University)

  • Hui Huang

    (People’s Hospital of Haikou)

  • Yanchi Luo

    (Hainan University
    Hainan University)

  • Xiaosheng Han

    (People’s Hospital of Haikou)

  • Hewen He

    (Ltd)

  • Shaolu Lin

    (Ltd)

  • Liudang Wan

    (Ltd)

  • Zhengliang Huang

    (Ltd)

  • Zhaoyong Fu

    (Ltd)

  • Rodrigo Ledesma-Amaro

    (Imperial College London)

  • Dapeng Yin

    (Hainan Center for Disease Control and Prevention)

  • Haimei Mao

    (Products Quality Supervision and Testing Institute of Hainan Province)

  • Linwen He

    (Hainan University)

  • Tao Yang

    (The Chinese University of Hong Kong)

  • Zijing Chen

    (The Chinese University of Hong Kong)

  • Yubin Ma

    (The Chinese University of Hong Kong)

  • Evelyn Y. Xue

    (The Chinese University of Hong Kong)

  • Yi Wan

    (Hainan University
    Hainan University
    Hainan University)

  • Chuanbin Mao

    (The Chinese University of Hong Kong)

Abstract

Clinical pathogen diagnostics detect targets by qPCR (but with low sensitivity) or blood culturing (but time-consuming). Here we leverage a dual-stem-loop DNA amplifier to enhance non-specific collateral enzymatic cleavage of an oligonucleotide linker between a fluophore and its quencher by CRISPR-CasΦ, achieving ultrasensitive target detection. Specifically, the target pathogens are lysed to release DNA, which binds its complementary gRNA in CRISPR-CasΦ to activate the collateral DNA-cleavage capability of CasΦ, enabling CasΦ to cleave the stem-loops in the amplifier. The cleavage product binds its complementary gRNA in another CRISPR-CasΦ to activate more CasΦ. The activated CasΦ collaterally cleaves the linker, releasing the fluophore to recover its fluorescent signal. The cycle of stem-loop-cleavage/CasΦ-activation/fluorescence-recovery amplifies the detection signal. Our target amplification-free collateral-cleavage-enhancing CRISPR-CasΦ method (TCC), with a detection limit of 0.11 copies/μL, demonstrates enhanced sensitivity compared to qPCR. It can detect pathogenic bacteria as low as 1.2 CFU/mL in serum within 40 min.

Suggested Citation

  • Huiyou Chen & Fengge Song & Buhua Wang & Hui Huang & Yanchi Luo & Xiaosheng Han & Hewen He & Shaolu Lin & Liudang Wan & Zhengliang Huang & Zhaoyong Fu & Rodrigo Ledesma-Amaro & Dapeng Yin & Haimei Mao, 2025. "Ultrasensitive detection of clinical pathogens through a target-amplification-free collateral-cleavage-enhancing CRISPR-CasΦ tool," Nature Communications, Nature, vol. 16(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-59219-x
    DOI: 10.1038/s41467-025-59219-x
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    References listed on IDEAS

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