Ultrasensitive detection of clinical pathogens through a target-amplification-free collateral-cleavage-enhancing CRISPR-CasΦ tool.
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| Autori principali: | , , , , , , , , , , , , , , , , , , , , |
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| Natura: | Artículo científico |
| Lingua: | en |
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Nature communications
2025
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| author | Chen, Huiyou Song, Fengge Wang, Buhua Huang, Hui Luo, Yanchi Han, Xiaosheng He, Hewen Lin, Shaolu Wan, Liudang Huang, Zhengliang Fu, Zhaoyong Ledesma-Amaro, Rodrigo Yin, Dapeng Mao, Haimei He, Linwen Yang, Tao Chen, Zijing Ma, Yubin Xue, Evelyn Y Wan, Yi Mao, Chuanbin |
| author_facet | Chen, Huiyou Song, Fengge Wang, Buhua Huang, Hui Luo, Yanchi Han, Xiaosheng He, Hewen Lin, Shaolu Wan, Liudang Huang, Zhengliang Fu, Zhaoyong Ledesma-Amaro, Rodrigo Yin, Dapeng Mao, Haimei He, Linwen Yang, Tao Chen, Zijing Ma, Yubin Xue, Evelyn Y Wan, Yi Mao, Chuanbin Chen, Huiyou Song, Fengge Wang, Buhua Huang, Hui Luo, Yanchi Han, Xiaosheng He, Hewen Lin, Shaolu Wan, Liudang Huang, Zhengliang Fu, Zhaoyong Ledesma-Amaro, Rodrigo Yin, Dapeng Mao, Haimei He, Linwen Yang, Tao Chen, Zijing Ma, Yubin Xue, Evelyn Y Wan, Yi Mao, Chuanbin |
| collection | PubMed - marine biology |
| contents | Ultrasensitive detection of clinical pathogens through a target-amplification-free collateral-cleavage-enhancing CRISPR-CasΦ tool. Chen, Huiyou Song, Fengge Wang, Buhua Huang, Hui Luo, Yanchi Han, Xiaosheng He, Hewen Lin, Shaolu Wan, Liudang Huang, Zhengliang Fu, Zhaoyong Ledesma-Amaro, Rodrigo Yin, Dapeng Mao, Haimei He, Linwen Yang, Tao Chen, Zijing Ma, Yubin Xue, Evelyn Y Wan, Yi Mao, Chuanbin CRISPR-Cas Systems Humans Limit of Detection RNA, Guide, CRISPR-Cas Systems Bacteria DNA, Bacterial 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. |
| format | Artículo científico |
| id | pubmed_40280947 |
| institution | PubMed |
| language | en |
| publishDate | 2025 |
| publisher | Nature communications |
| record_format | pubmed |
| spellingShingle | Ultrasensitive detection of clinical pathogens through a target-amplification-free collateral-cleavage-enhancing CRISPR-CasΦ tool. Chen, Huiyou Song, Fengge Wang, Buhua Huang, Hui Luo, Yanchi Han, Xiaosheng He, Hewen Lin, Shaolu Wan, Liudang Huang, Zhengliang Fu, Zhaoyong Ledesma-Amaro, Rodrigo Yin, Dapeng Mao, Haimei He, Linwen Yang, Tao Chen, Zijing Ma, Yubin Xue, Evelyn Y Wan, Yi Mao, Chuanbin CRISPR-Cas Systems Humans Limit of Detection RNA, Guide, CRISPR-Cas Systems Bacteria DNA, Bacterial Ultrasensitive detection of clinical pathogens through a target-amplification-free collateral-cleavage-enhancing CRISPR-CasΦ tool. Chen, Huiyou Song, Fengge Wang, Buhua Huang, Hui Luo, Yanchi Han, Xiaosheng He, Hewen Lin, Shaolu Wan, Liudang Huang, Zhengliang Fu, Zhaoyong Ledesma-Amaro, Rodrigo Yin, Dapeng Mao, Haimei He, Linwen Yang, Tao Chen, Zijing Ma, Yubin Xue, Evelyn Y Wan, Yi Mao, Chuanbin CRISPR-Cas Systems Humans Limit of Detection RNA, Guide, CRISPR-Cas Systems Bacteria DNA, Bacterial 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. |
| title | Ultrasensitive detection of clinical pathogens through a target-amplification-free collateral-cleavage-enhancing CRISPR-CasΦ tool. |
| topic | CRISPR-Cas Systems Humans Limit of Detection RNA, Guide, CRISPR-Cas Systems Bacteria DNA, Bacterial |
| url | https://pubmed.ncbi.nlm.nih.gov/40280947/ |