Rapid detection of ACCase W2027C mutation in Leptochloa chinensis (L.) Nees using RAA‐CRISPR/Cas12a: a sensitive and visual tool for herbicide resistance diagnosis

Abstract BACKGROUND Prolonged application of ACCase‐inhibiting herbicides, such as cyhalofop‐butyl, has resulted in the widespread emergence of herbicide‐resistant Leptochloa chinensis populations in paddy fields. Among the numerous target‐site resistance (TSR) mutations in ACCase, the Trp‐2027‐Cys (W2027C) mutation is one of the most prevalent in L. chinensis . The increasing prevalence of herbicide‐resistant L. chinensis highlights the critical need for rapid and precise diagnostic tools to detect resistance mutations. RESULTS In this study, we present a novel detection system that integrates Recombinase‐Aided Amplification (RAA) with CRISPR/Cas12a technology to specifically target the W2027C mutation in the ACCase gene of L. chinensis . The system exhibits high sensitivity, capable of detecting DNA concentrations as low as 2–200 fg/μL, and high specificity, facilitating accurate and visual differentiation of resistant from susceptible plants, thereby offering significant potential for rapid field applications. CONCLUSION This report has described the application of the RAA‐CRISPR system for mutation detection in herbicide resistant weeds, presenting a promising tool for integrated weed management and enabling more timely decision‐making regarding herbicide application and resistance management. © 2025 Society of Chemical Industry.