Bacillus thuringiensis (Bt) insecticidal toxins have been globally utilized for control of agricultural insects through spraying or transgenic crops.Binding of Bt toxins to special receptors on midgut epithelial cells of target insects is a key step in the mode of action.Previous studies suggested aminopeptidase N1 (APN1) as a receptor or putative receptor in several lepidopteran insects including Helicoverpa armigera through evidence from RNA interefence-based gene silencing approaches.In the current study we tested the role of APNs in the mode of action of Bt toxins using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated gene knockout.Three APN genes (HaAPN1,HaAPN2 and HaAPN5) were individually knocked out in a susceptible strain (SCD) of H.armigera to establish three homozygous knockout strains.Qualitative in vitro binding studies indicated binding of Cry1Ac or Cry2Ab to midgut brush border membrane vesicles was not obviously affected by APN knockout.Bioassay results showed that none of the three knockouts had significant changes in susceptibility to Cry1A or Cry2A toxins when compared with the SCD strain.This suggests that the three HaAPN genes we tested may not be critical in the mode of action of Cry 1A or Cry2A toxins in H.armigera.