CRISPR/Cas9-Mediated Knockout of White Gene Produces Eye Color Mutants in the Southern Green Stinkbug, Nezara viridula.

CRISPR/Cas9 gene editing is a transformative tool for genetic studies in non-model organisms like the southern green stinkbug, Nezara viridula. However, current protocols depend on embryonic microinjection of CRISPR/Cas9, which remains technically difficult. An alternative method of delivering Cas9 ribonucleoprotein directly into female ovaries has been tested in only a few insect species, such as mosquitoes and whiteflies. Here, we developed a simple technique for gene editing by injecting Cas9 ribonucleoprotein into adult Nezara viridula females using ReMOT control or BAPC delivery methods previously described. We observed gene editing of the eye color marker white using the ReMOT method and HhKV ligand. These results demonstrate proof of concept for creating germline mutations in N. viridula. The protocol presented in this study could help advance genetic research in hemipteran pest species.