PiggyBac-mediated transgenesis and CRISPR–Cas9 knockout in the greater wax moth, Galleria mellonella

The larvae of the greater wax moth, Galleria mellonella, are gaining prominence as a versatile nonmammalian in vivo model to study host-pathogen interactions. Their ability to be maintained at 37 °C, coupled with a broad susceptibility to human pathogens and a distinct melanization response that serves as a visual indicator for larval health, positions G. mellonella as a powerful resource for infection research. Despite these advantages, the lack of genetic tools, such as those available for zebrafish and Drosophila melanogaster, has hindered development of the full potential of G. mellonella as a model organism. Here we describe a robust methodology for generating transgenic G. mellonella using the PiggyBac transposon system and for precise gene knockouts via CRISPR-Cas9 technology. These advances significantly enhance the utility of G. mellonella in molecular research, paving the way for its widespread use as an inexpensive and ethically compatible animal model in infection biology and beyond.