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Several 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 <i>GGT</i> gene, which facilitated the laboratory sex separation of male <i>Culex quinquefasciatus</i> 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 <i>Saccharomyces cerevisiae</i> strains with multiple integrated copies of the insecticidal GGT shRNA expression cassette. Top expressing Cas-CLOVER strains killed <i>Culex quinquefasciatus</i> 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.