Programmable RNA detection with CRISPR-Cas12a
Abstract Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-di...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2023-09-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-023-41006-1 |
| _version_ | 1797558132865499136 |
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| author | Santosh R. Rananaware Emma K. Vesco Grace M. Shoemaker Swapnil S. Anekar Luke Samuel W. Sandoval Katelyn S. Meister Nicolas C. Macaluso Long T. Nguyen Piyush K. Jain |
| author_facet | Santosh R. Rananaware Emma K. Vesco Grace M. Shoemaker Swapnil S. Anekar Luke Samuel W. Sandoval Katelyn S. Meister Nicolas C. Macaluso Long T. Nguyen Piyush K. Jain |
| author_sort | Santosh R. Rananaware |
| collection | DOAJ |
| description | Abstract Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target. Our method, Split Activator for Highly Accessible RNA Analysis (SAHARA), detects picomolar concentrations of RNA without sample amplification, reverse-transcription, or strand-displacement by simply supplying a short DNA sequence complementary to the seed region. Beyond RNA detection, SAHARA outperforms wild-type CRISPR-Cas12a in specificity towards point-mutations and can detect multiple RNA and DNA targets in pooled crRNA/Cas12a arrays via distinct PAM-proximal seed DNAs. 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. |
| first_indexed | 2024-03-10T17:26:06Z |
| format | Article |
| id | doaj.art-39af6d98bb7648069f26b3fe592cdc59 |
| institution | Directory Open Access Journal |
| issn | 2041-1723 |
| language | English |
| last_indexed | 2024-03-10T17:26:06Z |
| publishDate | 2023-09-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj.art-39af6d98bb7648069f26b3fe592cdc592023-11-20T10:09:33ZengNature PortfolioNature Communications2041-17232023-09-0114111410.1038/s41467-023-41006-1Programmable RNA detection with CRISPR-Cas12aSantosh R. Rananaware0Emma K. Vesco1Grace M. Shoemaker2Swapnil S. Anekar3Luke Samuel W. Sandoval4Katelyn S. Meister5Nicolas C. Macaluso6Long T. Nguyen7Piyush K. Jain8Department of Chemical Engineering, University of FloridaDepartment of Chemical Engineering, University of FloridaDepartment of Chemical Engineering, University of FloridaDepartment of Chemical Engineering, University of FloridaDepartment of Biology, CLAS, University of FloridaDepartment of Chemical Engineering, University of FloridaDepartment of Chemical Engineering, University of FloridaDepartment of Chemical Engineering, University of FloridaDepartment of Chemical Engineering, University of FloridaAbstract Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target. Our method, Split Activator for Highly Accessible RNA Analysis (SAHARA), detects picomolar concentrations of RNA without sample amplification, reverse-transcription, or strand-displacement by simply supplying a short DNA sequence complementary to the seed region. Beyond RNA detection, SAHARA outperforms wild-type CRISPR-Cas12a in specificity towards point-mutations and can detect multiple RNA and DNA targets in pooled crRNA/Cas12a arrays via distinct PAM-proximal seed DNAs. 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.https://doi.org/10.1038/s41467-023-41006-1 |
| spellingShingle | Santosh R. Rananaware Emma K. Vesco Grace M. Shoemaker Swapnil S. Anekar Luke Samuel W. Sandoval Katelyn S. Meister Nicolas C. Macaluso Long T. Nguyen Piyush K. Jain Programmable RNA detection with CRISPR-Cas12a Nature Communications |
| 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 |
| url | https://doi.org/10.1038/s41467-023-41006-1 |
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