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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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    1. Santosh R. Rananaware & Emma K. Vesco & Grace M. Shoemaker & Swapnil S. Anekar & Luke Samuel W. Sandoval & Katelyn S. Meister & Nicolas C. Macaluso & Long T. Nguyen & Piyush K. Jain, 2023. "Programmable RNA detection with CRISPR-Cas12a," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    2. Sagar Sridhara & Hemant N. Goswami & Charlisa Whyms & Jonathan H. Dennis & Hong Li, 2021. "Virus detection via programmable Type III-A CRISPR-Cas systems," Nature Communications, Nature, vol. 12(1), pages 1-10, December.
    3. Anna Nemudraia & Artem Nemudryi & Murat Buyukyoruk & Andrew M. Scherffius & Trevor Zahl & Tanner Wiegand & Shishir Pandey & Joseph E. Nichols & Laina N. Hall & Aidan McVey & Helen H. Lee & Royce A. Wi, 2022. "Sequence-specific capture and concentration of viral RNA by type III CRISPR system enhances diagnostic," Nature Communications, Nature, vol. 13(1), pages 1-12, December.
    4. Cheri M. Ackerman & Cameron Myhrvold & Sri Gowtham Thakku & Catherine A. Freije & Hayden C. Metsky & David K. Yang & Simon H. Ye & Chloe K. Boehm & Tinna-Sólveig F. Kosoko-Thoroddsen & Jared Kehe & Ti, 2020. "Massively multiplexed nucleic acid detection with Cas13," Nature, Nature, vol. 582(7811), pages 277-282, June.
    5. Kean Hean Ooi & Mengying Mandy Liu & Jie Wen Douglas Tay & Seok Yee Teo & Pornchai Kaewsapsak & Shengyang Jin & Chun Kiat Lee & Jingwen Hou & Sebastian Maurer-Stroh & Weisi Lin & Benedict Yan & Gabrie, 2021. "An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing," Nature Communications, Nature, vol. 12(1), pages 1-23, December.
    6. Arturo Carabias & Anders Fuglsang & Piero Temperini & Tillmann Pape & Nicholas Sofos & Stefano Stella & Simon Erlendsson & Guillermo Montoya, 2021. "Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3," Nature Communications, Nature, vol. 12(1), pages 1-12, December.
    7. Shy-Shin Chang & Wen-Han Hsieh & Ting-Shou Liu & Si-Huei Lee & Chih-Hung Wang & Hao-Chang Chou & Yee Hui Yeo & Ching-Ping Tseng & Chien-Chang Lee, 2013. "Multiplex PCR System for Rapid Detection of Pathogens in Patients with Presumed Sepsis – A Systemic Review and Meta-Analysis," PLOS ONE, Public Library of Science, vol. 8(5), pages 1-10, May.
    8. Jeong Moon & Changchun Liu, 2023. "Asymmetric CRISPR enabling cascade signal amplification for nucleic acid detection by competitive crRNA," Nature Communications, Nature, vol. 14(1), pages 1-11, December.
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