Enhanced Diagnosis of Tomato Brown Rugose Fruit Virus (TBRFV) Infections Through CRISPR-Cas12 and CRISPR-Cas9 Technologies

Tomato Brown Rugose Fruit Virus (ToBRFV), a newly identified Tobamovirus that exclusively targets tomato plants, is causing significant damage to agricultural crops worldwide. It was originally discovered in greenhouse tomatoes in Jordan in 2015, and it is now a global danger to tomato and pepper harvests. 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 large distances, and it is now recognized as a pandemic in plants and corpse life. This study investigates the effectiveness of a single-step CRISPR-Cas12a in conjugation with reverse transcription-recombinase polymerase amplification (RT-RPA) for detection of ToBRFV in samples collected from the field. In this study, we employed the CRISPR-Cas12a system for the detection of viral DNA amplicons generated through RT-RPA. Moreover, we show that different ToBRFV signals sensed by the CRISPR complexes may be processed by designed DNA logic circuits. All things considered, the use of CRISPR-Cas9 R-loop for the molecular beacon opening (COLUMBO) platform for multiplexed detection in a single tube, enhances the capabilities of already available CRISPR-based techniques, and shows promise for use in biocomputing and diagnosis. Our innovative approach successfully identified the presence of ToBRFV, in tomato plants. Furthermore, the detection methodology refined by integrating lateral flow strips with a fluorimetry assay, enabling a visual readout. This modification circumvents the costly and labor-intensive RNA purification phase, streamlining the diagnostic process. These methods have the potential to be useful for field-deployable applications since they enable viral diagnoses to be completed within 30 minutes of leaf harvesting. The study proved that the combination of RT-RPA amplification and single-step CRISPR-Cas12a and CRISPR-Cas9 can offer a precise, sensitive, and targeted method for on-site detection of ToBRFV.