Detection of tomato brown rugose fruit virus through CRISPR-Cas12a and CRISPR-Cas9 systems

Tomato brown rugose fruit virus (ToBRFV) is a single-stranded positive-sense RNA virus that targets tomato and pepper plants and is causing significant damage to crops in some regions of the world. ToBRFV is a highly contagious virus that is stable and rapidly spreads by mechanical methods and seeds. As a result, it may spread both locally and over long distances, and it is now recognized as a pandemic in plants. This study investigates the effectiveness of the systems CRISPR-Cas12a and CRISPR-Cas9, in conjugation with recombinase polymerase amplification (RPA), to detect ToBRFV in tomato plant samples collected from the field. The trans-cleavage activity of both nucleases, Cas12a and Cas9, was exploited to process a probe labelled with fluorescein and biotin to be resolved on a lateral flow device, thereby enabling a visual readout. We were able to detect the RNA genome of the virus in about 1 h at a low constant temperature. These results could pave way to offer a rapid, sensitive, and specific method for on-site detection of ToBRFV.