A CRISPR-based lateral flow assay for plant genotyping and pathogen diagnostics

Abstract

Efficient pathogen diagnostics and genotyping methods enable effective disease management and breeding, improve crop productivity and ensure food security. However, current germplasm selection and pathogen detection techniques are laborious, time-consuming, expensive, and not easy to mass-scale application in the field. Here, we optimized a field-deployable lateral flow assay, Bio-SCAN, as a highly sensitive tool to precisely identify elite germplasm and detect mutations, transgenes, and phytopathogens in less than 1 hour, starting from sample isolation to result output using lateral flow strips. As a proof of concept, we genotyped various wheat germplasms for the Lr34 and Lr67 alleles conferring broad-spectrum resistance to stripe rust, confirmed the presence of synthetically produced herbicide-resistant alleles in the rice genome, and screened for the presence of transgenic elements in the genome of transgenic tobacco and rice plants with 100% specificity. We also successfully applied this new assay to the detection of phytopathogens, including viruses and bacterial pathogens in Nicotiana benthamiana, and two destructive fungal pathogens (Puccinia striiformis f. sp. tritici and Magnaporthe oryzae Triticum) in wheat. Our results illustrate the power of Bio-SCAN in crop breeding, genetic engineering, and pathogen diagnostics to enhance food security. The high sensitivity, simplicity, versatility, and in-field deployability make the Bio-SCAN as an attractive molecular diagnostic tool for diverse applications in agriculture.