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bioRxiv · 10.1101/2024.02.22.581525

Rapid, direct, and sequence-specific identification of RNA viruses in various crop plants using CRISPR/Cas13a

Hak, H.·Ostendorp, S.·Reza, A.·Ishgur Greenberg, S.·Pines, G.·Kehr, J.·Spiegelman, Z.
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Abstract

Plant viruses are destructive pathogens causing significant damage to various crop species. Rapid, sensitive, and specific detection is crucial for the effective containment of emerging and resistance-breaking viruses. CRISPR/Cas has been established as a useful tool for plant virus identification. However, its application for on-site, direct detection of viruses from plant tissues is still limited. In this study, we present a rapid method for detecting viruses directly from RNA of different crop species using CRISPR/Cas13a. We successfully applied this method to identify tomato brown rugose fruit virus (ToBRFV) in infected tomato plants and differentiate it from closely related tobamoviruses. ToBRFV could be identified in a 100-fold dilution and early during infection, prior to the onset of viral symptoms. Moreover, CRISPR/Cas13a was used to directly identify cucumber green mottle mosaic virus (CGMMV) in cucumber plants and turnip mosaic virus (TuMV) in Brassica napus plants. Finally, we developed a user-friendly, extraction-free, 15-minute protocol for on-site ToBRFV identification using a portable fluorescent viewer and a mobile phone camera. This protocol was successfully applied for ToBRFV detection in a commercial greenhouse. These results demonstrate that CRISPR/Cas13a is a robust technology for direct, rapid, sensitive, and specific identification of multiple viruses in different crop plants that can be easily implemented for on-site detection.

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Hak, H., Ostendorp, S., Reza, A., Ishgur Greenberg, S., Pines, G., Kehr, J., Spiegelman, Z.. 2024-02-22. Rapid, direct, and sequence-specific identification of RNA viruses in various crop plants using CRISPR/Cas13a. https://doi.org/10.1101/2024.02.22.581525

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