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 germplas m 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 infield deployability make the Bio-SCAN as an attractive molecular diagnostic tool for diverse applications in agriculture.

CRISPR/Cas-Based Diagnostics in Agricultural Applications

“…The reagents have a longer shelf life and can be applicable in resource limited field settings. , Bio-SCAN showed success in the detection of diverse phytopathogens, i.e., viruses, bacteria, and fungi (Table ). Moreover, it demonstrated effectiveness in the detection of herbicide resistance alleles in rice plants and fungal resistance alleles in wheat germplasm . Additionally, it was able to identify typical transgenic sequences (such as the CaMV 35S and Ubi promoters) in total genomic DNA isolated from genetically modified crops (such as transgenic rice and tobacco).…”

“…The Bio-SCAN method is a more efficient and versatile tool for plant pathogen detection and GMO identification . In contrast, the Cas9 based visual colorimetric assay requires a more complex process involving several enzymes and cyclic probes, limiting its range of applications.…”

“…Both CASLFA and Bio-SCAN methods have successfully identified a wide range of targets and have proven to be effective at the point-of-care. However, the Bio-SCAN method has several advantages over other techniques, including lower costs, increased stability, adaptability, and the ability to detect a broad range of elite plant alleles and phytopathogens such as viruses, bacteria, and fungi . The use of commercial LFA strips, low-maintenance reagents, and redesigned primers and sgRNA have made the Bio-SCAN platform simple and adaptable for field application .…”

“…Diverse types of pathogens or diseases can be detected by a single CRISPR system by targeting nucleic acids from different samples. By altering the gRNA, multiple targets can be recognized in a single system . No changes to proteins or experimental settings are necessary in the CRISPR system for target detection.…”

Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

“…Cas12f also targets dsDNA and ssDNA and is better than Cas12a at discriminating single-nucleotide polymorphisms in ssDNA ( Harrington et al., 2018 ). The Bio-SCAN toolkit uses RPA and CRISPR to make the Bio-SCAN as an attractive molecular diagnostic tool for diverse populations applications in agriculture ( Sánchez et al., 2022 ). The CRISPR technology is already being used in a variety of different ways.…”