Abstract
Insect pest population control via sterile insect technique markedly benefits from separation by sex prior to release. To simplify this process, traditional genetics has been deployed to develop genetic sexing strains (GSSs) for several disease vectors and agricultural pests of vast economic significance, although very few are applied in the field due to associated fitness costs and instability. In this study, we generated a method to engineer cisgenic GSS (CGSS) in insects. We use CRISPR/Cas9-mediated homology-directed repair to seamlessly translocate a sex-specific alternatively spliced intron into a dominant phenotypic gene generating a genetically stable strain that enables sex-sorting by eye. To achieve this feat, we use Ceratitis capitata as our model and relied on the sex-specifically spliced intron of its endogenous transformer gene, which we seamlessly inserted a copy into the pupal colouration white pupae gene. This minimal modification resulted in the generation of a homozygous strain we term IMPERIAL that was genetically and phenotypically stable where all female pupae are brown while male pupae are white with overall good fitness. By minimally editing the genome, our novel CGSS approach can be applied to other pests that may aid more efficient and economically suitable pest control.