Delivery of siRNA-containing tetrahedral RNA nanostructures into bioenergy crops using LDH stabilized sprays

Genetic engineering plays a pivotal role in accelerating breeding efforts and enhancing traits in ways that are unattainable through classical breeding techniques. This is critical for alleviating the global pressures on food and renewable energy resources driven by rapid economic growth, population expansion, and climate change. However, conventional genetically modified organism (GMO)-based approaches are not only time-consuming and technically challenging but also dependent on specific germplasm. Additionally, the regulatory and public acceptance challenges surrounding GMOs remain significant, impeding the development of bioenergy crops with improved traits. Recent advances in small interfering RNA (siRNA) delivery into plants present a promising, non-GMO strategy for transiently modulating gene expression and altering specific traits in a very targeted and environmentally benign manner. Current siRNA delivery methods primarily rely on viral vectors or foliar applications of long double-stranded RNA (dsRNA), which are randomly processed into siRNAs. While viral vectors can offer a one-time solution for long-lived perennials, their development and testing remain labor-intensive. Furthermore, the production of long dsRNA is costly, and their cellular uptake, modulation efficiency, and persistence are often suboptimal. Silvec proposes an innovative two-component siRNA delivery system consisting of RNA nanostructures with a novel nanoring architecture capable of delivering 6 to 12 highly specific siRNAs, and engineered nanosheets as nanocarriers to enhance delivery efficiency and cellular uptake. This system aims to effectively deliver high concentrations of siRNAs into plants, transiently modulating key traits such as biomass yield, crop quality, and resistance to biotic and abiotic stresses. Designed for precision and efficiency, this system specifically targets monocotyledonous species, including switchgrass and other prairie grasses which are often resistant to genetic modification approaches, with the goal of enhancing key agronomic traits without altering the plant’s DNA through precise gene silencing mechanisms. This process can also provide broad anti-pest plant protection as well by utilizing highly targeted siRNAs to target insects and other pests of interest. This Phase I project will explore the ability of RNA Nanorings to load, protect, deliver, and release multiple siRNAs, thereby inducing targeted gene silencing in plants. Concurrently, the project will assess the effectiveness of the nanosheets in stabilizing the Nanoring structures and enhancing gene silencing efficiency. Additionally, the use of predicted siRNAs encapsulated within the Nanorings is expected to reduce the production of suboptimal siRNAs during dsRNA processing in plant cells, thereby improving RNA interference (RNAi) efficiency, minimizing off-target effects, and significantly reducing RNA production costs. This initiative marks the first attempt to utilize supramolecular RNA nanostructures in combination with LDH nanosheets to modulate gene expression in crops, aiming to facilitate rapid trait improvements in field conditions. Upon successful completion of Phase I, Silvec will be well-positioned during Phases II and III to develop cost-effective, non-GMO commercial solutions for improving traits in food and bioenergy crops using customized Nanorings and engaging in field trials to demonstrate efficacy under real-life conditions. Moreover, this technology will provide academic researchers with a powerful tool to explore multiple gene functions through an innovative, non-transgenic approach, leveraging the extensive siRNA loading capacity of Nanoring RNAs. This advancement is expected to significantly enhance functional genome analysis and accelerate the application of gene function knowledge in the post-genomic era.