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

Abstract 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.