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

Insects show extreme diversity and have long intrigued biologists. Recent technological advancements, such as gene editing and transgenesis, should in principle enable the use of almost any insect species for biological research. However, in practice, 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 using CRISPR/Cas9-mediated genome editing. Then we use this strain to develop an efficient piggyBac -mediated transgenesis system using a nuclear targeting approach. We show that incorporating 1× and 3× nuclear localization signals (NLS) into piggyBac mRNA substantially enhances overall transformation efficiency in O. fasciatus . Taking advantage of both the useful mutant strain and the efficient transgenesis system, we successfully integrated multiple expression cassettes ranging from 1.8 to 7.2 kb, including attP strains for phiC31-mediated site-specific integration and histone-labelled strains for live fluorescence imaging. We further provide evidence that the Q system, a binary expression system, is functional in this species, paving the way for future sophisticated genetic manipulations including functional assays for cis -regulatory elements. Together, our results not only expand the genetic toolkit of O. fasciatus as a comparative model insect, but also provide a practical framework for developing efficient transgenesis in other non-traditional model organisms.