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

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, Linwen He, Tao Yang, Zijing Chen, Yubin Ma, Evelyn Y. Xue, Yi Wan () and Chuanbin Mao ()
Additional contact information
Huiyou Chen: Hainan University
Fengge Song: Hainan University
Buhua Wang: Hainan University
Hui Huang: People’s Hospital of Haikou
Yanchi Luo: 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
Chuanbin Mao: The Chinese University of Hong Kong

Nature Communications, 2025, vol. 16, issue 1, 1-14

Abstract: 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.

Date: 2025
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DOI: 10.1038/s41467-025-59219-x

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