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Programmable RNA detection with CRISPR-Cas12a
CRISPR is a prominent bioengineering tool and the type V CRISPR-associated protein complex, Cas12a, is widely used in diagnostic platforms due to its innate ability to cleave DNA substrates. Here we demonstrate that Cas12a can also be programmed to directly detect RNA substrates without the need for...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Journal Experts
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9949221/ https://www.ncbi.nlm.nih.gov/pubmed/36824842 http://dx.doi.org/10.21203/rs.3.rs-2549171/v1 |
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author | Rananaware, Santosh R. Vesco, Emma K. Shoemaker, Grace M. Anekar, Swapnil S. Sandoval, Luke Samuel W. Meister, Katelyn S. Macaluso, Nicolas C. Nguyen, Long T. Jain, Piyush K. |
author_facet | Rananaware, Santosh R. Vesco, Emma K. Shoemaker, Grace M. Anekar, Swapnil S. Sandoval, Luke Samuel W. Meister, Katelyn S. Macaluso, Nicolas C. Nguyen, Long T. Jain, Piyush K. |
author_sort | Rananaware, Santosh R. |
collection | PubMed |
description | CRISPR is a prominent bioengineering tool and the type V CRISPR-associated protein complex, Cas12a, is widely used in diagnostic platforms due to its innate ability to cleave DNA substrates. Here we demonstrate that Cas12a can also be programmed to directly detect RNA substrates without the need for reverse transcription or strand displacement. We discovered that while the PAM-proximal “seed” region of the crRNA exclusively recognizes DNA for initiating trans-cleavage, the PAM-distal region or 3’-end of the crRNA can tolerate both RNA and DNA substrates. Utilizing this property, we developed a method named Split Activators for Highly Accessible RNA Analysis or ‘SAHARA’ to detect RNA sequences at the PAM-distal region of the crRNA by merely supplying a short ssDNA or a PAM containing dsDNA to the seed region. Notably, SAHARA is Mg(2+) concentration- and pH-dependent, and it was observed to work robustly at room temperature with multiple orthologs of Cas12a. SAHARA also displayed a significant improvement in the specificity for target recognition as compared to the wild-type CRISPR-Cas12a, at certain positions along the crRNA. By employing SAHARA we achieved amplification-free detection of picomolar concentrations of miRNA-155 and hepatitis C virus RNA. Finally, SAHARA can use a PAM-proximal DNA as a switch to control the trans-cleavage activity of Cas12a for the detection of both DNA and RNA targets. With this, multicomplex arrays can be made to detect distinct DNA and RNA targets with pooled crRNA/Cas12a complexes. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes. |
format | Online Article Text |
id | pubmed-9949221 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Journal Experts |
record_format | MEDLINE/PubMed |
spelling | pubmed-99492212023-02-24 Programmable RNA detection with CRISPR-Cas12a Rananaware, Santosh R. Vesco, Emma K. Shoemaker, Grace M. Anekar, Swapnil S. Sandoval, Luke Samuel W. Meister, Katelyn S. Macaluso, Nicolas C. Nguyen, Long T. Jain, Piyush K. Res Sq Article CRISPR is a prominent bioengineering tool and the type V CRISPR-associated protein complex, Cas12a, is widely used in diagnostic platforms due to its innate ability to cleave DNA substrates. Here we demonstrate that Cas12a can also be programmed to directly detect RNA substrates without the need for reverse transcription or strand displacement. We discovered that while the PAM-proximal “seed” region of the crRNA exclusively recognizes DNA for initiating trans-cleavage, the PAM-distal region or 3’-end of the crRNA can tolerate both RNA and DNA substrates. Utilizing this property, we developed a method named Split Activators for Highly Accessible RNA Analysis or ‘SAHARA’ to detect RNA sequences at the PAM-distal region of the crRNA by merely supplying a short ssDNA or a PAM containing dsDNA to the seed region. Notably, SAHARA is Mg(2+) concentration- and pH-dependent, and it was observed to work robustly at room temperature with multiple orthologs of Cas12a. SAHARA also displayed a significant improvement in the specificity for target recognition as compared to the wild-type CRISPR-Cas12a, at certain positions along the crRNA. By employing SAHARA we achieved amplification-free detection of picomolar concentrations of miRNA-155 and hepatitis C virus RNA. Finally, SAHARA can use a PAM-proximal DNA as a switch to control the trans-cleavage activity of Cas12a for the detection of both DNA and RNA targets. With this, multicomplex arrays can be made to detect distinct DNA and RNA targets with pooled crRNA/Cas12a complexes. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes. American Journal Experts 2023-02-17 /pmc/articles/PMC9949221/ /pubmed/36824842 http://dx.doi.org/10.21203/rs.3.rs-2549171/v1 Text en https://creativecommons.org/licenses/by/4.0/This work is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) , which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. https://creativecommons.org/licenses/by/4.0/License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License (https://creativecommons.org/licenses/by/4.0/) |
spellingShingle | Article Rananaware, Santosh R. Vesco, Emma K. Shoemaker, Grace M. Anekar, Swapnil S. Sandoval, Luke Samuel W. Meister, Katelyn S. Macaluso, Nicolas C. Nguyen, Long T. Jain, Piyush K. Programmable RNA detection with CRISPR-Cas12a |
title | Programmable RNA detection with CRISPR-Cas12a |
title_full | Programmable RNA detection with CRISPR-Cas12a |
title_fullStr | Programmable RNA detection with CRISPR-Cas12a |
title_full_unstemmed | Programmable RNA detection with CRISPR-Cas12a |
title_short | Programmable RNA detection with CRISPR-Cas12a |
title_sort | programmable rna detection with crispr-cas12a |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9949221/ https://www.ncbi.nlm.nih.gov/pubmed/36824842 http://dx.doi.org/10.21203/rs.3.rs-2549171/v1 |
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