Identification of SfABCC2 as the critical receptor for Cry1Fa and Cry1Ab in Spodoptera frugiperda via CRISPR-mediated gene knockouts.

Insecticidal proteins from Bacillus thuringiensis (Bt) have been widely used to control major agricultural pests through genetically modified (GM) Bt crops. However, the development of resistance in target pests could undermine the effectiveness of Bt crops. Understanding the mechanisms of action of Bt insecticidal proteins and the resistance mechanisms in pests is crucial for developing effective resistance management strategies to sustain the use of Bt crops. In this study, CRISPR/Cas9 gene editing was used to investigate the functional roles of four genes (SfABCC2, SfABCC3, SfCad1, and SfAPN1) that encode putative receptors for Cry1 proteins in the fall armyworm, Spodoptera frugiperda, a globally significant pest. We created five homozygous knockouts, each with a substantial fragment deletion: SfCad1-KO, SfAPN1-KO, SfABCC2-KO, SfABCC3-KO, and SfC2/C3-KO (a double knockout of SfABCC2 and SfABCC3). Bioassay results revealed that SfCad1-KO, SfAPN1-KO, and SfABCC3-KO strains exhibited no resistance to Cry1Fa or Cry1Ab. In contrast, SfABCC2-KO and SfC2/C3-KO strains demonstrated high levels of resistance to Cry1Fa (>3300-fold) and Cry1Ab (>450-fold), demonstrating that SfABCC2 is pivotal to the insecticidal action of these two Bt proteins. As anticipated, all five knockouts generated in this study did not significantly impact susceptibility to Vip3Aa compared with the control strain. Our findings underscore the critical role of SfABCC2 in mediating Cry1Ab and Cry1Fa toxicity in S. frugiperda. Therefore, resistance monitoring program and resistance management tactics should focus on SfABCC2 mutations in field populations of S. frugiperda.