Difference between revisions of "Part:BBa K5186015"

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shRNA(Shaker)

To combat mosquito populations, shRNA (Shaker), a short hairpin RNA, is engineered to target mosquitoes' Shaker gene, which encodes an evolutionarily conserved subunit of voltage-gated potassium channels crucial for neural, immune and muscular development at multiple stages of the mosquito life cycle, utilizing RNAi technique. When shRNA(Shaker) in the freeze dried inactivated yeast cells are digested by mosquitoes, they will be processed into small interfering RNAs (siRNAs), and further specifically target mRNA(Shaker) to achieve degradation. This targeted interference results in neural misfunctions in mosquitoes, eventually causing their deaths without any harm to other non-target organisms.

shRNA(Shaker) and its expression cassette (BBa_K5186017), shRNAs(5-HTR1) (BBa_K5186011, BBa_K5186012, BBa_K51860013) and other shRNAs targeting mosquitoes' vital survival genes (BBa_K5186014, BBa_K5186016) contribute to mosquitoes control solutions and thus make up a part collection. This collection serves as a valuable resource for the iGEM community and researchers, offering a safe, efficient, and environmentally friendly approach to mosquito control.

The gene Shaker(AAEL000242) encodes an evolutionarily conserved subunit of voltage-gated potassium channels, which function as critical regulators of neural and muscular development in divergent species. Interference of this gene leads to impaired nerve function, resulting in paralysis and deaths of the mosquito. (Keshava M. et al, 2020)

This part was engineered to target the Shaker gene, referring to Sh.463 target site (Corey B. et al, 2023). It is designed to assemble into the multiple cloning site of the pRS426 yeast shuttle vector, located downstream of pTDH3 and upstream of tTDH1. It can be utilized for mosquito control by expressing them in S. cerevisiae CEN. PK2-1C, which is subsequently inactivated and freeze-dried to create RNAi-based mosquitocides.

By integrating these yeast RNAi mosquitocides with attractive targeted sugar baits (ATSBs) and the blood-feeding mosquito attractant HMBPP, an emerging non-transgenic mosquito control solution called Moskilla is formed. It capitalizes on the mosquitoes' natural sugar-feeding behavior and the characteristic of Plasmodium's metabolite HMBPP, which stimulates mosquitoes' blood-seeking behavior. And the digestion of these freeze-dried inactivated yeast mosquitocide by mosquitoes will result in their death. This method offers a safe, eco-friendly, and effective strategy for mosquito population management without the need for spraying chemical insecticides or employing transgenic approaches.

Figure 1. Developing process of RNAi yeast mosquitocides.

In the end, 6 shRNAs were engineered to target mosquitoes' vital survival genes (Figure 2a, 2b). To validate the successful production of our shRNAs in yeast, we extracted RNA from S. cerevisiae and conducted electrophoresis. And the designed shRNAs are 55 nts with poly T ending sequence, which corresponds to the electrophoresis result, indicating the successful generation of shRNAs (Figure 2c).

In our experimental validation, mosquitoes were divided into a control group and an experimental group. The experimental group was provided with freeze-dried, inactivated yeast cells engineered to express a variety of shRNAs, including shRNA1 (5-HTR1), shRNA2 (5-HTR1), shRNA3 (5-HTR1), shRNA (Rbfox1), shRNA (Shaker), and shRNA (Irx). Over a five-day observation period, we monitored the impact of these shRNAs on mosquito survival. (Figure 2b)

Our findings revealed that while the control group exhibited a natural mortality rate of 5%, all experimental groups experienced a complete 100% mortality rate by the 3rd day after feeding. This outcome not only confirms the potency of our RNAi-based mosquitocides but also underscores their rapid effect, holding significant promise for the swift control of mosquito populations.

Figure 2. 6 variants of shRNAs targeting mosquitoes' vital survival genes are expressed in S. cerevisiae CEN. PK2-1C. (a) Mosquitoes' vital survival genes 5-HTR1, Rbfox1, Shaker, Irx are chosen to silence, encoding for serotonin receptor, RNA binding proteins, voltage-gated potassium channels and Iroquois-class homeodomain-containing proteins respectively. They involve critical functions including neural, immune, reproductive and muscular development. (b) Genetic circuit and nucleotide sequences of shRNAs expression. (c) Gel electrophoresis analysis of RNA extracted from yeast cells expressing various shRNAs. (d) Mortality of mosquitoes consuming freeze dried inactivated yeast cells expressing various shRNAs.

Mysore, K., Njoroge, T.M., Stewart, A.T.M. et al. (2023). Characterization of a novel RNAi yeast insecticide that silences mosquito 5-HT1 receptor genes. Sci Rep 13, 22511 . https://doi.org/10.1038/s41598-023-49799-3

Mysore, K., Longhua, S., Limb, K.H. et al. (2022). A broad-based mosquito yeast interfering RNA pesticide targeting Rbfox1 represses Notch signaling and kills both larvae and adult mosquitoes. Pathogens, 11(9), 956. https://doi.org/10.3390/pathogens11090956

Corey B., Keshava M., Teresia M. N., Seth McConnell, et al. Targeting Mosquitoes through Generation of an Insecticidal RNAi Yeast Strain Using Cas-CLOVER and Super PiggyBac Engineering in Saccharomyces cerevisiae. J. Fungi. 2023, 9, 1056. https://doi.org/10.3390/jof9111056

Keshava M., Longhua S., Limb K. H., et al. A Yeast RNA-Interference Pesticide Targeting the Irx Gene Functions as a Broad-Based Mosquito Larvicide and Adulticide. Insects. 2021, 12: 986. https://doi.org/10.3390/insects12110986