CRISPR/Cas12a mediated rapid and efficient detection of Tomato Leaf Curl Karnataka Virus without amplification

Abstract

Transboundary movement of agricultural produce increases the risk of introduction of plant pathogens into newer areas which pose a serious economic threat. This requires a quick, accurate detection method that will help in diagnosing the plant pathogens and make appropriate containment. The diagnostic methods thus developed will also be handy in the early screening for plant pathogens in the asymptomatic stage. Removing the infected plants in the early stage will help in maintaining the field stand to realize the full yield potential. Since vector-mediated transmission occurs at the seedling stage, random screening of the seedlings at the nursery stage will help in the clean planting programme. In this communication, we have compared the differential sensitivities of two methods viz. polymerase chain reaction and also CRISPR/Cas12a. In this regard, we have carried out whole genome sequencing for the ToLCKV isolate (PP763439.1) collected from tomatoes. We have expressed and purified Cas12a protein (187 kDa) and designed two guide RNAs (gRNAs) each for two genes viz. CP and Rep of ToLCKV and formed ribonucleoprotein (RNP) complex (sgRNA + Cas12a). Among the four RNP complexes tested, CPgRNA-1, CPgRNA-2 and REP gRNA1 enabled the detection of ToLCKV virus titre 10-fold lower (0.1 ng) than that of PCR assay (1.0 ng). Additionally, we also used CPgRNA-1 and CPgRNA-2 RNP to detect ToLCKV in field samples with high accuracy.