Programmable Multiplexed Nucleic Acid Detection by Harnessing Specificity Defect of CRISPR‐Cas12a
Abstract CRISPR‐Cas12a works like a sophisticated algorithm in nucleic acid detection, yet its challenge lies in sometimes failing to distinguish targets with mismatches due to its specificity limitations. Here, the mismatch profiles, including the quantity, location, and type of mismatches in the C...
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Wiley
2025-01-01
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| Series: | Advanced Science |
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| Online Access: | https://doi.org/10.1002/advs.202411021 |
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| author | Xin Guan Rui Yang Jiongyu Zhang Jeong Moon Chengyu Hou Chong Guo Lori Avery Danielle Scarola Daniel S. Roberts Rocco LaSala Changchun Liu |
| author_facet | Xin Guan Rui Yang Jiongyu Zhang Jeong Moon Chengyu Hou Chong Guo Lori Avery Danielle Scarola Daniel S. Roberts Rocco LaSala Changchun Liu |
| author_sort | Xin Guan |
| collection | DOAJ |
| description | Abstract CRISPR‐Cas12a works like a sophisticated algorithm in nucleic acid detection, yet its challenge lies in sometimes failing to distinguish targets with mismatches due to its specificity limitations. Here, the mismatch profiles, including the quantity, location, and type of mismatches in the CRISPR‐Cas12a reaction, are investigated and its various tolerances to mismatches are discovered. By harnessing the specificity defect of the CRISPR‐Cas12a enzyme, a dual‐mode detection strategy is designed, which includes approximate matching and precise querying of target sequences and develop a programmable multiplexed nucleic acid assay. With the assay, 14 high‐risk human papillomavirus (HPV) subtypes are simultaneously detected, collectively responsible for 99% of cervical cancer cases, with attomolar sensitivity. Specifically, the assay not only distinguishes HPV16 and HPV18, the two most common subtypes but also detects 12 other high‐risk pooled HPV subtypes. To enable low‐cost point‐of‐care testing, the assay is incorporated into a paper‐based microfluidic chip. Furthermore, the clinical performance of the paper‐based microfluidic chip is validated by testing 75 clinical swab samples, achieving performance comparable to that of PCR. This programmable multiplexed nucleic acid assay has the potential to be widely applied for sensitive, specific, and simultaneous detection of different pathogens. |
| format | Article |
| id | doaj-art-0d910158cafe48459ac03971755195ea |
| institution | Kabale University |
| issn | 2198-3844 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Wiley |
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| series | Advanced Science |
| spelling | doaj-art-0d910158cafe48459ac03971755195ea2025-01-29T09:50:19ZengWileyAdvanced Science2198-38442025-01-01124n/an/a10.1002/advs.202411021Programmable Multiplexed Nucleic Acid Detection by Harnessing Specificity Defect of CRISPR‐Cas12aXin Guan0Rui Yang1Jiongyu Zhang2Jeong Moon3Chengyu Hou4Chong Guo5Lori Avery6Danielle Scarola7Daniel S. Roberts8Rocco LaSala9Changchun Liu10Department of Biomedical Engineering University of Connecticut Health Center Farmington Connecticut 06030 USADepartment of Biomedical Engineering University of Connecticut Health Center Farmington Connecticut 06030 USADepartment of Biomedical Engineering University of Connecticut Health Center Farmington Connecticut 06030 USADepartment of Biomedical Engineering University of Connecticut Health Center Farmington Connecticut 06030 USADepartment of Biomedical Engineering University of Connecticut Health Center Farmington Connecticut 06030 USADepartment of Biomedical Engineering University of Connecticut Health Center Farmington Connecticut 06030 USADepartment of Pathology and Laboratory Medicine University of Connecticut Health Center Farmington Connecticut 06030 USADivision of Otolaryngology‐Head & Neck Surgery University of Connecticut Health Center Farmington Connecticut 06030 USADivision of Otolaryngology‐Head & Neck Surgery University of Connecticut Health Center Farmington Connecticut 06030 USADepartment of Pathology and Laboratory Medicine University of Connecticut Health Center Farmington Connecticut 06030 USADepartment of Biomedical Engineering University of Connecticut Health Center Farmington Connecticut 06030 USAAbstract CRISPR‐Cas12a works like a sophisticated algorithm in nucleic acid detection, yet its challenge lies in sometimes failing to distinguish targets with mismatches due to its specificity limitations. Here, the mismatch profiles, including the quantity, location, and type of mismatches in the CRISPR‐Cas12a reaction, are investigated and its various tolerances to mismatches are discovered. By harnessing the specificity defect of the CRISPR‐Cas12a enzyme, a dual‐mode detection strategy is designed, which includes approximate matching and precise querying of target sequences and develop a programmable multiplexed nucleic acid assay. With the assay, 14 high‐risk human papillomavirus (HPV) subtypes are simultaneously detected, collectively responsible for 99% of cervical cancer cases, with attomolar sensitivity. Specifically, the assay not only distinguishes HPV16 and HPV18, the two most common subtypes but also detects 12 other high‐risk pooled HPV subtypes. To enable low‐cost point‐of‐care testing, the assay is incorporated into a paper‐based microfluidic chip. Furthermore, the clinical performance of the paper‐based microfluidic chip is validated by testing 75 clinical swab samples, achieving performance comparable to that of PCR. This programmable multiplexed nucleic acid assay has the potential to be widely applied for sensitive, specific, and simultaneous detection of different pathogens.https://doi.org/10.1002/advs.202411021approximate pattern matchingCRISPR‐Cas12aHR‐HPV detectionpaper‐based microfluidic chipprecise querying |
| spellingShingle | Xin Guan Rui Yang Jiongyu Zhang Jeong Moon Chengyu Hou Chong Guo Lori Avery Danielle Scarola Daniel S. Roberts Rocco LaSala Changchun Liu Programmable Multiplexed Nucleic Acid Detection by Harnessing Specificity Defect of CRISPR‐Cas12a Advanced Science approximate pattern matching CRISPR‐Cas12a HR‐HPV detection paper‐based microfluidic chip precise querying |
| title | Programmable Multiplexed Nucleic Acid Detection by Harnessing Specificity Defect of CRISPR‐Cas12a |
| title_full | Programmable Multiplexed Nucleic Acid Detection by Harnessing Specificity Defect of CRISPR‐Cas12a |
| title_fullStr | Programmable Multiplexed Nucleic Acid Detection by Harnessing Specificity Defect of CRISPR‐Cas12a |
| title_full_unstemmed | Programmable Multiplexed Nucleic Acid Detection by Harnessing Specificity Defect of CRISPR‐Cas12a |
| title_short | Programmable Multiplexed Nucleic Acid Detection by Harnessing Specificity Defect of CRISPR‐Cas12a |
| title_sort | programmable multiplexed nucleic acid detection by harnessing specificity defect of crispr cas12a |
| topic | approximate pattern matching CRISPR‐Cas12a HR‐HPV detection paper‐based microfluidic chip precise querying |
| url | https://doi.org/10.1002/advs.202411021 |
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