Difference between revisions of "Part:BBa K5186013"

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shRNA3(5-HTR1)

To combat mosquito populations, shRNAs (5-HTR1), variants of short hairpin RNAs, are engineered to target mosquitoes' 5-HTR1 gene, which encodes serotonin(5-HT) receptors crucial for immune and nervous systems, utilizing RNAi technique. When shRNA3(5-HTR1) 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(5-HTR1) starting at 1408 nt to achieve degradation. This targeted interference results in neural misfunctions in mosquitoes, eventually causing their deaths without any harm to other non-target organisms.

These shRNAs(5-HTR1) (BBa_K5186011, BBa_K5186012, BBa_K51860013) and other shRNAs targeting mosquitoes' vital survival genes (BBa_K5186014, BBa_K5186015, BBa_K5186016, BBa_K5186017) 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 5-HTR1 (AAEL000528) encodes serotonin(5-HT) receptors, which are located on the cell membrane of neurons and other cell types. In insects, 5-HT receptors play a pivotal role in regulating sleep, feeding, the circadian rhythm, and cognitive functions such as learning and memory, thereby influencing a range of insect behaviors and physiological processes. It has been proved that the silence of the 5-HTR1 gene can lead to significant neural defects in the brains of mosquitoes.

We utilized a professional shRNA designed website(https://rnaidesigner.thermofisher.com/rnaiexpress/) to craft shRNAs targeting the 5-HTR1 gene, which is composed of sense strand, loop and antisense strand.

To address biosafety concerns, aftering gaining the shRNAs candidates, we conducted BLAST searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) of the sense strand sequences against the total RNA database, ensuring their specificity to mosquitoes and no target to other organisms.

Finally, we arranged for the synthesis of DNA oligos encoding the shRNAs(5-HTR1), which were then assembled into the multiple cloning site of the pRS426 yeast shuttle vector, located downstream of pTDH3 and upstream of tTDH1.

Note: Higher scores indicate a greater probability of successful interference.

The shRNAs(5-HTR1) 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