The global deployment of RNAi yeast insecticides involves transitioning from the use of laboratory yeast strains to more robust strains that are suitable for scaled fermentation. In this investigation, the RNA-guided Cas-CLOVER system was used in combination with Piggybac transposase to produce robust Saccharomyces cerevisiae strains with multiple integrated copies of the Sh.463 short hairpin RNA (shRNA) insecticide expression cassette. This enabled the constitutive high-level expression of an insecticidal shRNA corresponding to a target sequence that is conserved in mosquito Shaker genes, but which is not found in non-target organisms. Top-expressing Cas-CLOVER strains performed well in insecticide trials conducted on Aedes, Culex, and Anopheles larvae and adult mosquitoes, which died following consumption of the yeast. Scaled fermentation facilitated the kilogram-scale production of the yeast, which was subsequently heat-killed and dried. These studies indicate that RNAi yeast insecticide production can be scaled, an advancement that may one day facilitate the global distribution of this new mosquito control intervention.

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 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.

Cas-CLOVER is an emerging high-fidelity genome editing system that enables precise and efficient cell engineering. In this study, we applied Cas-CLOVER to establish a robust, gene-edited platform in suspension-adapted CHO-K1 cells supporting cell line development (CLD) for biopharmaceutical production. An attractive strategy for high yield clone selection is the use of glutamine synthetase (GS) knockout CHO cells. The primary GS gene resides on chromosome 5 (GS5), while a recently identified GS pseudogene is located on chromosome 1 (GS1). To compare editing efficiency, we evaluated Cas-CLOVER and Cas9 at both GS loci using the Neon™ Transfection System. Cas-CLOVER achieved 84% editing at GS5 and 74% at GS1, markedly higher than Cas 9. Leveraging Cas-CLOVER’s dual-guide RNA design, we generated a GS5 single knockout (GS5-SKO) and subsequently a double knockout (GS-DKO) line at both the GS5 and GS1 loci, both with none detected off-target mutations analyzed in 40 predictably off-target sites. For functional validation, these cell lines were engineered with the proprietary Harbor-IN transposase system to stably express trastuzumab. Using an optimized protocol, the resulting GS-DKO platform, termed CleanCut GS CHO, enabled stringent selection and yielded high-producing clones with cell-specific productivity exceeding 100 pg/cell/day and antibody titers greater than 5 g/L in 24 deep well-plate fed-batch cultures after 14 days. The antibody titer stability analysis showed consistency over 60 generations. Collectively, these findings establish Cas-CLOVER as a versatile genome editing tool for developing high-yield CHO host platforms in CLD.