A small library crRNA-enhanced CRISPR-Cas12a system for ultrasensitive point-of-care test of hantavirus M gene
The integration of CRISPR-Cas systems with isothermal nucleic acid amplification (INA) holds transformative potential for point-of-care diagnostics, yet technical challenges such as limited sensitivity, cross-contamination risks, and incompatibility between amplification and detection phases hinder...
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Elsevier
2025-06-01
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| Series: | Sensing and Bio-Sensing Research |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2214180425000777 |
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| author | Jian Zhou Xue-mei Ren Xin Wang Pu Xu Zhuo Li |
| author_facet | Jian Zhou Xue-mei Ren Xin Wang Pu Xu Zhuo Li |
| author_sort | Jian Zhou |
| collection | DOAJ |
| description | The integration of CRISPR-Cas systems with isothermal nucleic acid amplification (INA) holds transformative potential for point-of-care diagnostics, yet technical challenges such as limited sensitivity, cross-contamination risks, and incompatibility between amplification and detection phases hinder their clinical adoption. Here, we present a novel small library CRISPR/Cas12a crRNA (SLCC) platform for ultrasensitive detection of the hantavirus M gene, a conserved target critical for diagnosing hemorrhagic fever with renal syndrome (HFRS). The SLCC platform incorporates three key innovations: machine learning-guided crRNA design to target highly conserved viral regions; multi-crRNA collaborative signal amplification to enhance Cas12a's collateral cleavage activity, and a single-tube workflow integrating reverse transcription, recombinase polymerase amplification (RT-RPA), and CRISPR detection. Experimental validation demonstrated that the combinatorial six-crRNA strategy achieved an 85-fold improvement in sensitivity over single-crRNA systems (limit of detection (LoD): 0.086 pM vs. 7.31 pM for DNA of amplification). The optimized one-step RT-RPA/CRISPR-Cas12a workflow reduced assay time, while maintaining high specificity, as evidenced by concordant results with clinical samples and negligible cross-reactivity against SARS-CoV-2, HBV, and mycoplasma pneumoniae. Notably, the platform achieved a 42.29-fold lower LoD for RNA detection compared to single-crRNA CRISPR-Cas system, with fluorescence signal amplification plateauing within 45 min. The SLCCA platform integration of RNA reverse transcription amplification and Cas12a enzymatic cleavage within a single-tube workflow, combined with lateral flow strip-based signal readout, which achieves a sensitivity of 500 pM for RNA detection, demonstrating a 10–20-fold enhancement in the LoD compared to single-crRNA systems CRISPR-Cas diagnostic approaches. The advancements in the small library crRNA strategy address critical barriers in CRISPR-based diagnostics by offering a convenient and field-deployable solution for rapid, highly sensitive pathogen detection in resource-limited settings. This study establishes SLCC as a versatile framework that can adapt to emerging infectious disease surveillance and point-of-care applications. |
| format | Article |
| id | doaj-art-7d81e6d9d7634ceca4d3ecf1c2fa6427 |
| institution | DOAJ |
| issn | 2214-1804 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
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| series | Sensing and Bio-Sensing Research |
| spelling | doaj-art-7d81e6d9d7634ceca4d3ecf1c2fa64272025-08-20T02:39:33ZengElsevierSensing and Bio-Sensing Research2214-18042025-06-014810081110.1016/j.sbsr.2025.100811A small library crRNA-enhanced CRISPR-Cas12a system for ultrasensitive point-of-care test of hantavirus M geneJian Zhou0Xue-mei Ren1Xin Wang2Pu Xu3Zhuo Li4Department of Laboratory Medicine, The First Affiliated Hospital of Xi'an Medical University, Xi'an 710077, ChinaDepartment of Laboratory Medicine, The First Affiliated Hospital of Xi'an Medical University, Xi'an 710077, ChinaDepartment of Laboratory Medicine, The First Affiliated Hospital of Xi'an Medical University, Xi'an 710077, ChinaDepartment of Laboratory Medicine, The First Affiliated Hospital of Xi'an Medical University, Xi'an 710077, ChinaCorresponding author.; Department of Laboratory Medicine, The First Affiliated Hospital of Xi'an Medical University, Xi'an 710077, ChinaThe integration of CRISPR-Cas systems with isothermal nucleic acid amplification (INA) holds transformative potential for point-of-care diagnostics, yet technical challenges such as limited sensitivity, cross-contamination risks, and incompatibility between amplification and detection phases hinder their clinical adoption. Here, we present a novel small library CRISPR/Cas12a crRNA (SLCC) platform for ultrasensitive detection of the hantavirus M gene, a conserved target critical for diagnosing hemorrhagic fever with renal syndrome (HFRS). The SLCC platform incorporates three key innovations: machine learning-guided crRNA design to target highly conserved viral regions; multi-crRNA collaborative signal amplification to enhance Cas12a's collateral cleavage activity, and a single-tube workflow integrating reverse transcription, recombinase polymerase amplification (RT-RPA), and CRISPR detection. Experimental validation demonstrated that the combinatorial six-crRNA strategy achieved an 85-fold improvement in sensitivity over single-crRNA systems (limit of detection (LoD): 0.086 pM vs. 7.31 pM for DNA of amplification). The optimized one-step RT-RPA/CRISPR-Cas12a workflow reduced assay time, while maintaining high specificity, as evidenced by concordant results with clinical samples and negligible cross-reactivity against SARS-CoV-2, HBV, and mycoplasma pneumoniae. Notably, the platform achieved a 42.29-fold lower LoD for RNA detection compared to single-crRNA CRISPR-Cas system, with fluorescence signal amplification plateauing within 45 min. The SLCCA platform integration of RNA reverse transcription amplification and Cas12a enzymatic cleavage within a single-tube workflow, combined with lateral flow strip-based signal readout, which achieves a sensitivity of 500 pM for RNA detection, demonstrating a 10–20-fold enhancement in the LoD compared to single-crRNA systems CRISPR-Cas diagnostic approaches. The advancements in the small library crRNA strategy address critical barriers in CRISPR-based diagnostics by offering a convenient and field-deployable solution for rapid, highly sensitive pathogen detection in resource-limited settings. This study establishes SLCC as a versatile framework that can adapt to emerging infectious disease surveillance and point-of-care applications.http://www.sciencedirect.com/science/article/pii/S2214180425000777CRISPR-Cas12aHantavirus M geneIsothermal amplificationMulti-crRNA strategyPoint-of-care testingUltrasensitive detection |
| spellingShingle | Jian Zhou Xue-mei Ren Xin Wang Pu Xu Zhuo Li A small library crRNA-enhanced CRISPR-Cas12a system for ultrasensitive point-of-care test of hantavirus M gene Sensing and Bio-Sensing Research CRISPR-Cas12a Hantavirus M gene Isothermal amplification Multi-crRNA strategy Point-of-care testing Ultrasensitive detection |
| title | A small library crRNA-enhanced CRISPR-Cas12a system for ultrasensitive point-of-care test of hantavirus M gene |
| title_full | A small library crRNA-enhanced CRISPR-Cas12a system for ultrasensitive point-of-care test of hantavirus M gene |
| title_fullStr | A small library crRNA-enhanced CRISPR-Cas12a system for ultrasensitive point-of-care test of hantavirus M gene |
| title_full_unstemmed | A small library crRNA-enhanced CRISPR-Cas12a system for ultrasensitive point-of-care test of hantavirus M gene |
| title_short | A small library crRNA-enhanced CRISPR-Cas12a system for ultrasensitive point-of-care test of hantavirus M gene |
| title_sort | small library crrna enhanced crispr cas12a system for ultrasensitive point of care test of hantavirus m gene |
| topic | CRISPR-Cas12a Hantavirus M gene Isothermal amplification Multi-crRNA strategy Point-of-care testing Ultrasensitive detection |
| url | http://www.sciencedirect.com/science/article/pii/S2214180425000777 |
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