Dual knockout of susceptibility genes AtKRP6 and AtWRKY23 confers nematode resistance without affecting the host fitness

Background The root-knot nematode, Meloidogyne incognita , is responsible for significant yield losses worldwide. Breeding nematode-resistant crops has traditionally provided a sustainable management strategy. However, regulatory challenges associated with genetically modified crops and the emergence of nematode populations capable of overcoming resistance have limited the efficacy of resistance breeding. As an alternative strategy, CRISPR/Cas9 technology has been employed to confer nematode resistance by selectively knocking out host susceptibility ( S ) genes in various crop plants. Nevertheless, targeting a single S gene has often failed to reduce nematode populations below economically acceptable thresholds, likely due to functional redundancy among nematode-responsive S genes. Results Here, we demonstrate a proof-of-concept using multiplex genome editing (polycistronic tRNA–gRNA system) to simultaneously knock out two distinct nematode-responsive S genes, AtWRKY23 and AtKRP6 , which were specifically selected based on their strong and localized induction upon M. incognita infection. This expression pattern, confirmed by promoter: GUS reporter assays and RT-qPCR analyses, minimizes potential fitness costs associated with gene disruption. Compared to the wild-type, nematode multiplication per plant was reduced by 29.22%, 26.75% and 56.45% in AtKRP6 knockout (KO), AtWRKY23 KO and dual KO lines, respectively, highlighting the increased efficacy of multiplex editing in suppressing nematode populations below the threshold level. Conclusion Taken together, this study lays the foundation for developing durable nematode resistance in crops through simultaneous knockout of multiple susceptibility genes with minimal impact on host plant fitness.