Brizzee, CoreyMysore, KeshavaNjoroge, Teresia M.McConnell, SethCrawford, JackDuman-Scheel, Molly2025-01-152025-01-152023-11Brizzee, C., Mysore, K., Njoroge, T. M., McConnell, S., Crawford, J., & Duman-Scheel, M. (2023). Generation of a Culex Male Mosquito Sex-Separation RNAi Yeast Strain Using Cas-CLOVER and Super PiggyBac Engineering in Saccharomyces cerevisiae. Fermentation, 9(12), Article 12. https://doi.org/10.3390/fermentation9120999https://hdl.handle.net/1805/45272Several emerging mosquito control technologies require mass releases of adult male mosquitoes. Previous studies resulted in the generation of a laboratory female-specific larvicidal yeast strain targeting the GGT gene, which facilitated the laboratory sex separation of male Culex quinquefasciatus mosquitoes. Global deployment of this yeast-based sex-separation technology requires engineering second generation yeast strains which can be used in industrial-scale fermentations to support global mosquito control programs. In this study, the RNA-guided Cas-CLOVER system was used in combination with piggyBac transposase to generate robust Saccharomyces cerevisiae strains with multiple integrated copies of the insecticidal GGT shRNA expression cassette. Top expressing Cas-CLOVER strains killed Culex quinquefasciatus female larvae which consumed the yeast, facilitating male sex separation. Scaled fermentation resulted in kilogram-scale production of the yeast, which can be heat-killed and dried for global deployment to mosquito mass-rearing facilities.enAttribution 4.0 InternationalCulex quinquefasciatusfemale lethalgenetic engineeringArticle