Programmable RNA detection with CRISPR-Cas12a

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 and Piyush K. Jain ()
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Santosh R. Rananaware: University of Florida
Emma K. Vesco: University of Florida
Grace M. Shoemaker: University of Florida
Swapnil S. Anekar: University of Florida
Luke Samuel W. Sandoval: University of Florida
Katelyn S. Meister: University of Florida
Nicolas C. Macaluso: University of Florida
Long T. Nguyen: University of Florida
Piyush K. Jain: University of Florida

Nature Communications, 2023, vol. 14, issue 1, 1-14

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-41006-1

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