PiggyBac mediated transgenesis and CRISPR/Cas9 knockout in the greater waxmoth,Galleria mellonella

The larvae of the greater waxmoth, Galleria mellonella , are gaining prominence as a versatile non-mammalian 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 melanisation response that serves as a visual indicator for larval health, positions Galleria as a powerful resource for infection research. Despite these advantages, the lack of genetic tools, such as those available for zebrafish and fruit flies, has hindered development of the full potential of Galleria as a model organism. In this study, we describe a robust methodology for generating transgenic Galleria using the PiggyBac transposon system and for precise gene knockouts via CRISPR/Cas9 technology. These advances significantly enhance the utility of Galleria in molecular research, opening the way to its widespread use as an inexpensive and ethically compatible animal model for infection biology and beyond.