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

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.