In‐Field Molecular Diagnostics of Plant Pathogens Using Bioluminescent CRISPR‐Guided Caspase Assay

In-field molecular diagnostics of plant pathogens are critical for crop disease management and precision agriculture, but tools are still lacking. Herein, we present a bioluminescent molecular diagnostic assay capable of detecting viable pathogens directly in minimally processed plant samples, enabling rapid and precise in-field crop disease diagnosis. The assay, called bioluminescent craspase diagnostics (BioCrastics), leverages newly discovered RNA-activated protease of CRISPR (Craspase) with enzymatic luminescence to generate a cascaded amplification, thus bypasses nucleic acid purification and amplification while achieving sub-nanogram sensitivity for fungal pathogens. Using wheat stripe rust as a proof of concept, we demonstrate direct pathogen detection in crude leaf homogenates within 40 min, early identification of infections 6 days prior to symptom emergence. Notably, the assay, via targeting pathogenic RNAs, specifically quantifies viable fungi, overcoming false positives from dead pathogens-a limitation of PCR-based methods that impairs disease risk assessment. Featuring simplified sample processing, portable detection, and species-specific accuracy, BioCrastics establishes a field-deployable tool that bridges the gap between laboratory-level precision and on-farm diagnostic needs for crop disease management.