Ultrasensitive detection of clinical pathogens through a target-amplification-free collateral-cleavage-enhancing CRISPR-CasΦ tool
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.
Authors
- Liudang Wan
- Chuanbin Mao (ORCID: https://orcid.org/0000-0002-8142-3659)
- Huiyou Chen (ORCID: https://orcid.org/0000-0002-5996-4272)
- Buhua Wang (ORCID: https://orcid.org/0000-0002-7026-6360)
- Shih‐Ching Lin (ORCID: https://orcid.org/0000-0003-2960-5348)
- Xiaosheng Han
- Hui Huang (ORCID: https://orcid.org/0000-0001-5716-4012)
- Evelyn Y. Xue (ORCID: https://orcid.org/0000-0002-4483-1952)
- Rodrigo Ledesma‐Amaro (ORCID: https://orcid.org/0000-0003-2631-5898)
- Zhengliang Huang (ORCID: https://orcid.org/0000-0002-8457-6394)
- Yu‐Bin Ma
- Fengge Song (ORCID: https://orcid.org/0000-0003-2296-4225)
- Zijing Chen (ORCID: https://orcid.org/0000-0002-7996-2004)
- Dapeng Yin
- Yi Wan (ORCID: https://orcid.org/0000-0002-0667-3279)
- Tao Yang (ORCID: https://orcid.org/0000-0002-7698-3440)
- Zhang Fu
- Yanchi Luo (ORCID: https://orcid.org/0009-0007-6267-3912)
- Haimei Mao
- Linwen He
- Hewen He
Institutions
- Chinese University of Hong Kong (HK)
- Hainan University (CN)
- Haikou City People's Hospital (CN)
- Hainan Center for Disease Control & Prevention (CN)
- Guangdong Testing Institute for Product Quality Supervision (CN)
- Imperial College London (GB)
- Hainan Medical University (CN)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-04-26
- DOI
- https://doi.org/10.1038/s41467-025-59219-x
- Citations
- 50
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 39.49
Funders
- National Natural Science Foundation of China
- Hainan University
- National Natural Science Foundation of China-Yunnan Joint Fund