Generation of a Culex Male Mosquito Sex-Separation RNAi Yeast Strain Using Cas-CLOVER and Super PiggyBac Engineering in Saccharomyces cerevisiae
Abstract
:1. Introduction
2. Materials and Methods
2.1. Yeast Strain Construction
2.2. Evaluation of GGT.566 Expression
2.3. Whole Genome Sequencing (WGS) of Yeast Strains
2.4. Pilot Fermentations
2.5. Mosquito Assays
3. Results
3.1. Production and Evaluation of Culex Sex-Separation Yeast Strains
3.1.1. Generation of a Robust Yeast Strain with Multiple Integrations of the GGT.566 shRNA Expression Cassette
3.1.2. WGS Reveals the GGT.566 Expression Cassette Integration Sites
3.2. Assessment of Female-Specific Yeast Larvicide Activity
3.3. Performance of Strains in Pilot Fermentations
3.4. Evaluation of the Larvicidal Activity of Yeast Produced during the Scaled Fermentation
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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shRNA Type | Strain | Genotype | Original Auxotrophy | Restored Genotype |
---|---|---|---|---|
GTT.566 | DMT9-47.9R #2 | MATa, ura3∆0, leu2∆0, Piggybac (leu2d/PTDH3-shRNA_566-TCYC1, PTDH3-shRNA_566-TCYC1, PTDH3-shRNA_566-TCYC1), CEN/ARS (URA3/SPBase_Sc-CO) | Uracil | MATa, Piggybac (leu2d/PTDH3-shRNA_566-TCYC1, PTDH3-shRNA_566-TCYC1, PTDH3-shRNA_566-TCYC1), CEN/ARS (URA3/SPBase_Sc-CO) |
Control | DMT4-347.1R a | MATa, ura3∆0, leu2∆0, his3∆0, trp1∆0, PiggyBac (LEU2/PTDH3-shRNA_Ctrl-TCYC1), 2um (URA3/SPBase_Sc-CO), PiggyBac (HIS3/PTDH3-shRNA_Ctrl-TCYC1), CEN/ARS (URA3/SPBase_Sc-CO) PiggyBac (trp1d/PTDH3-shRNA_Ctrl-TCYC1), CEN/ARS (URA3/SPBase_Sc-CO) | Uracil | MATa, PiggyBac (LEU2/PTDH3-shRNA_Ctrl-TCYC1), 2um (URA3/SPBase_Sc-CO), PiggyBac (HIS3/PTDH3-shRNA_Ctrl-TCYC1), CEN/ARS (URA3/SPBase_Sc-CO) PiggyBac (trp1d/PTDH3-shRNA_Ctrl-TCYC1), CEN/ARS (URA3/SPBase_Sc-CO) |
Strain | Integrations | GGT.566 Copies | Total Copies | Genomic Integration Site | 5′ Flanking Sequence (60 bp) | 3′ Flanking Sequence (60 bp) |
---|---|---|---|---|---|---|
DMT9-47.9R #2 | 3 | 3 | 9 | Chromosome VII (883,906) | AAGTTCTTGGTGTTTTAAAACTAAAAA-AAAGACTAACTATAAAAGTAGAATTTAAGAAGT | GAAATAGATTTACAGAATTACAATCAATACCTACCGTCTTATATACTTATTAGTCAAGT |
Chromosome VIII (245,747) | ACTATATACAACATGAAGGACTTTATAAATTCTTGATCAATCTTTCACACTAATTTTATA | CAGTTCTACACTATTTTTGTTTGCTTGACTGTAAAAACATTCTATTTTATGATTAGTTGC | ||||
Chromosome X (526,821) | AGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTTAATAGTTATGTTAGTA | GAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAAACGCGTGTACGCATGTA |
Treatment | Male:Female a | p-Value |
---|---|---|
Food Control | 1:1 | 1 |
DMT4-347.1R (50% Yeast + 50% Koi Food) | 1:1 | 0.86 |
DMT9-47.9R #2 (50% Yeast + 50% Koi Food) | 5:1 | 2.1 × 10−9 |
Treatment | Male/Female a | p-Value |
---|---|---|
Food Control | 1:1 | 0.72 |
DMT9-47.9R #2 (50% Yeast + 50% Koi Food) | 3.5:1 | 8.4 × 10−10 |
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Brizzee, C.; Mysore, K.; Njoroge, T.M.; McConnell, S.; Crawford, J.; Duman-Scheel, M. Generation of a Culex Male Mosquito Sex-Separation RNAi Yeast Strain Using Cas-CLOVER and Super PiggyBac Engineering in Saccharomyces cerevisiae. Fermentation 2023, 9, 999. https://doi.org/10.3390/fermentation9120999
Brizzee C, Mysore K, Njoroge TM, McConnell S, Crawford J, Duman-Scheel M. Generation of a Culex Male Mosquito Sex-Separation RNAi Yeast Strain Using Cas-CLOVER and Super PiggyBac Engineering in Saccharomyces cerevisiae. Fermentation. 2023; 9(12):999. https://doi.org/10.3390/fermentation9120999
Chicago/Turabian StyleBrizzee, Corey, Keshava Mysore, Teresia M. Njoroge, Seth McConnell, Jack Crawford, and Molly Duman-Scheel. 2023. "Generation of a Culex Male Mosquito Sex-Separation RNAi Yeast Strain Using Cas-CLOVER and Super PiggyBac Engineering in Saccharomyces cerevisiae" Fermentation 9, no. 12: 999. https://doi.org/10.3390/fermentation9120999