Introduction
Our RNAi-based herbicide, Knaptime is Over, will be sprayed onto patches of invasive plant species, stopping their growth while keeping surrounding organisms safe at the same time. The herbicide will primarily enter the plant through absorption by the stomata [4]. This herbicide involves the termination of a protein essential for the survival and reproduction of the invasive plant. Double-stranded RNA (dsRNA), the key In vitro transcription, an RNA synthesis method that utilizes a purified linear DNA template and T7 phage RNA polymerase [3], will be the primary method used to produce our custom dsRNA. This method can generate up to 200 µg of RNA [3]. Finally, we will apply our dsRNA as a foliar spray [2].
Then, the absorbed dsRNA is cleaved into small interfering RNA (siRNA) by the RNase-III enzyme termed DICER [5]. Measuring approximately 21 nucleotides in length [6], the cleaved siRNA will separate into the sense and antisense strands by the RNA-induced silencing complex (RISC). Sense strands are then discarded and disintegrate in the cell’s cytoplasm, while the antisense strand of the dsRNA remains bound to the RISC complex. In turn, this produces the antisense strand to bind to the sense strand of the mature mRNA that we aim to silence. The specific base pairing allows for both precise and accurate cleavage of the target mRNA, allowing silencing of the gene [7].
DICER cleaves dsRNA into siRNAs. RISC further cleaves the siRNAs sense and antisense strand. Following this procedure, RISC binds the sense and antisense strands together to conduct cleaving of the mRNA resulting in translation inhibition [8]. Subsequently, the plant will no longer be capable of producing the essential protein encoded by the silenced gene [6].
Through laboratory testing, we will determine the amount of herbicide required to efficiently and reliably kill the target plant, and the time it takes for the RNAi to take full effect. We will test the effectiveness of our herbicide on the seeds of knapweed, which have the ability to stay dormant in the soil for 8 to 10 years [9]. If our custom dsRNA cannot penetrate the seeds, we will either target a gene essential to seed production or extend the duration of treatment to be performed annually for at least 8 years. The viscosity of the herbicide itself will resemble that of water, making it suitable for spraying through the most commonly available spray bottles. Using a mobile herbicide sprayer that can identify and spray plants autonomously is a possibility for larger applications.
Our RNAi-based herbicide will only be effective on the plant species we desire to terminate. This is due to the specificity of our siRNA sequence. By choosing the dsRNA antisense strand that is completely unique to the sense strand of the mature mRNA we aim to silence, we can limit the concern that our herbicide will terminate surrounding organisms. RISC would not be able to bind two sense strands together in a different organism than our target species because the dsRNA we produce will only be specific to the plant species we want to terminate. Therefore, no gene silencing would occur in non-targeted species. This process is advantageous as it eliminates the concern of disrupting the ecosystem our herbicide is in. Furthermore, this process allows for easier spraying and removes the potential human and wildlife health risks [10].
