Room temperature CRISPR diagnostics for low-resource settings
Abstract Maintaining elevated reaction temperatures and multi-step sample preparations increases the costs and complexity of diagnostics, impeding their deployment in low-resource settings. Here, we develop a one-pot, room temperature recombinase polymerase amplification (RPA)-CRISPR reaction that r...
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| Format: | Article |
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Nature Portfolio
2025-01-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-025-86373-5 |
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| author | Eric A. Nalefski Selma Sinan Jason L. Cantera Anastasia G. Kim Remy M. Kooistra Rafael E. Rivera Jordyn P. Janshen Sanchita Bhadra Joshua D. Bishop Andrew D. Ellington Ilya J. Finklestein Damian Madan |
| author_facet | Eric A. Nalefski Selma Sinan Jason L. Cantera Anastasia G. Kim Remy M. Kooistra Rafael E. Rivera Jordyn P. Janshen Sanchita Bhadra Joshua D. Bishop Andrew D. Ellington Ilya J. Finklestein Damian Madan |
| author_sort | Eric A. Nalefski |
| collection | DOAJ |
| description | Abstract Maintaining elevated reaction temperatures and multi-step sample preparations increases the costs and complexity of diagnostics, impeding their deployment in low-resource settings. Here, we develop a one-pot, room temperature recombinase polymerase amplification (RPA)-CRISPR reaction that removes these critical challenges. We show that RPA amplification is reduced by several orders of magnitude at 25 °C as compared to 37 °C. Similarly, when coupled to RPA, the performance of multiple Cas12a orthologs, including the widely used LbCas12a, is severely compromised at temperatures below 37 °C. To mitigate these limitations, we identify the ortholog TsCas12a as a highly active nuclease at 25 °C and develop a single-protocol RPA-Cas12a detection reaction with this enzyme. A quantitative kinetic analysis reveals that fast nuclease activation is more critical than higher steady-state trans-cleavage activity for room temperature diagnostic applications. RPA-TsCas12a reactions performed at 25 °C effectively detected HPV-16 in crudely prepared cervical swab samples with high sensitivity and specificity using both optical and lateral flow readouts. The reactions developed herein reduce the complexity and equipment requirements for affordable diagnostics in low- and middle-income countries. |
| format | Article |
| id | doaj-art-c5f73570047a4c16b6d2f32730a0fbf1 |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-c5f73570047a4c16b6d2f32730a0fbf12025-02-02T12:24:42ZengNature PortfolioScientific Reports2045-23222025-01-0115111310.1038/s41598-025-86373-5Room temperature CRISPR diagnostics for low-resource settingsEric A. Nalefski0Selma Sinan1Jason L. Cantera2Anastasia G. Kim3Remy M. Kooistra4Rafael E. Rivera5Jordyn P. Janshen6Sanchita Bhadra7Joshua D. Bishop8Andrew D. Ellington9Ilya J. Finklestein10Damian Madan11Global Health Labs, IncDepartment of Molecular Biosciences, University of Texas at AustinGlobal Health Labs, IncGlobal Health Labs, IncGlobal Health Labs, IncGlobal Health Labs, IncGlobal Health Labs, IncDepartment of Molecular Biosciences, University of Texas at AustinGlobal Health Labs, IncDepartment of Molecular Biosciences, University of Texas at AustinDepartment of Molecular Biosciences, University of Texas at AustinGlobal Health Labs, IncAbstract Maintaining elevated reaction temperatures and multi-step sample preparations increases the costs and complexity of diagnostics, impeding their deployment in low-resource settings. Here, we develop a one-pot, room temperature recombinase polymerase amplification (RPA)-CRISPR reaction that removes these critical challenges. We show that RPA amplification is reduced by several orders of magnitude at 25 °C as compared to 37 °C. Similarly, when coupled to RPA, the performance of multiple Cas12a orthologs, including the widely used LbCas12a, is severely compromised at temperatures below 37 °C. To mitigate these limitations, we identify the ortholog TsCas12a as a highly active nuclease at 25 °C and develop a single-protocol RPA-Cas12a detection reaction with this enzyme. A quantitative kinetic analysis reveals that fast nuclease activation is more critical than higher steady-state trans-cleavage activity for room temperature diagnostic applications. RPA-TsCas12a reactions performed at 25 °C effectively detected HPV-16 in crudely prepared cervical swab samples with high sensitivity and specificity using both optical and lateral flow readouts. The reactions developed herein reduce the complexity and equipment requirements for affordable diagnostics in low- and middle-income countries.https://doi.org/10.1038/s41598-025-86373-5 |
| spellingShingle | Eric A. Nalefski Selma Sinan Jason L. Cantera Anastasia G. Kim Remy M. Kooistra Rafael E. Rivera Jordyn P. Janshen Sanchita Bhadra Joshua D. Bishop Andrew D. Ellington Ilya J. Finklestein Damian Madan Room temperature CRISPR diagnostics for low-resource settings Scientific Reports |
| title | Room temperature CRISPR diagnostics for low-resource settings |
| title_full | Room temperature CRISPR diagnostics for low-resource settings |
| title_fullStr | Room temperature CRISPR diagnostics for low-resource settings |
| title_full_unstemmed | Room temperature CRISPR diagnostics for low-resource settings |
| title_short | Room temperature CRISPR diagnostics for low-resource settings |
| title_sort | room temperature crispr diagnostics for low resource settings |
| url | https://doi.org/10.1038/s41598-025-86373-5 |
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