Pollen Transfection via Tuneable Polymeric Silica Nanocapsules : A Non-Transgenic Platform for Plant Genetic Manipulation

Objective

The escalating climate crisis, population growth cause serious challenges to system reliant to plants, posing a critical threat to agricultural productivity and global food security. In the urgent quest to develop resilient crops, CRISPR-Cas9 ribonucleoprotein (RNP) complexes present a powerful and precise genome-editing tool. Their primary advantage lies in enabling non-transgenic improvement by acting transiently without the integration of foreign DNA, thereby potentially streamlining regulatory approval and public acceptance. However, the transformative potential of this technology is severely hampered by a fundamental bottleneck: the efficient delivery of these macromolecules into plant cells. To address the delivery challenge, we propose the development of a novel, sustainable nanocarrier platform centred on a biocompatible silicon core, which serves as a scaffold for a precise layer-by-layer (LbL) assembly technique. This enables the meticulous encapsulation of functional CRISPR-Cas9 RNP complexes, thereby shielding them from enzymatic degradation in the extracellular environment. Cell-penetrating peptides (CPPs) will be incorporated to promote enhanced cellular uptake and internalisation by overcoming the cell wall barrier, while choline-based deep eutectic solvents (DES) will be employed to stabilise the RNP payload and ensure its release upon intracellular entry. By introducing the cargoes into pollen, we aim to achieve heritable edits that are directly transmitted to the progeny seeds following pollination. This method leverages the natural process of fertilisation, offering a transformative, genotype-independent solution for rapid crop improvement. The successful execution of this research will provide a robust and versatile platform technology to accelerate the development of more stress-resilient crops, ultimately contributing to sustainable agriculture and ensuring food security in the face of a rapidly changing climate.