A nuclear targeting approach enables efficient transgenesis in the milkweed bug Oncopeltus fasciatus

Insects show extreme diversity and have long intrigued biologists. Although gene editing and transgenesis should enable the use of almost any insect species for biological research, species-specific challenges remain and conditions must be optimized carefully. Here, using the milkweed bug Oncopeltus fasciatus, we first generate a useful eye- and body-color mutant strain by CRISPR/Cas9-mediated genome editing. Using this strain, we develop an efficient piggyBac-mediated transgenesis system with a nuclear targeting approach. Incorporating 1x and 3x nuclear localization signals into piggyBac mRNA substantially enhances overall transformation efficiency in O. fasciatus. Taking advantage of the mutant strain and the efficient transgenesis system, we integrated multiple expression cassettes ranging from 1.8 to 7.2 kb, including attP strains for phiC31-mediated integration and histone-labelled strains for live imaging. We further show that the Q system, a binary expression system, is functional in this species, paving the way for sophisticated genetic manipulations. Together, our results expand the genetic toolkit of O. fasciatus as a comparative model insect and provide a practical framework for developing efficient transgenesis in other non-traditional model organisms.