(Invited) Carbon Nanotubes for Plant Genetic Engineering

Abstract

Plants will benefit from genetic engineering to feed our growing global population in the midst of climate change, to provide our most clinically-relevant drugs, and for our environmental remediation efforts. New generation genome engineering technologies, such as CRISPR/Cas, hold the potential to alter plant genes in a precise and targeted manner. The first step of any plant genetic engineering application is to deliver important biomolecular cargoes into the desired subcellular locations of plant cells. However, the limitations of traditional plant gene delivery methods, i.e. Agrobacterium and gene gun, prevent plant genetic engineering from reaching its full potential.Given the lack of efficient biomolecule delivery vehicles to plants and potential impact of plant biotechnology on food security and sustainability, we developed nanoparticle-based platforms to efficiently deliver small interfering RNA1,2 and plasmid DNA3,4 cargoes into plant cells in a species-independent manner, which eliminates the use of gene gun and addresses the main delivery bottlenecks of plant genetic engineering. We show that pristine single walled carbon nanotubes (SWCNTs) can adsorb small interfering RNA molecules through pi-pi stacking for plant delivery, whereas a covalently modified and positively charged SWCNT platform is more efficient for large double-stranded DNA delivery to mature plant leaf cells.This talk will first briefly summarize the development, characterization and biological verification of each nanoparticle platform for plant delivery applications. Next, I will present our recent results of CRISPR/Cas gene editing in plant leaves using the SWCNT DNA delivery platform5. The talk will be concluded by discussing the several assays performed to demonstrate the effect of nanoparticles on plant health, including an RNA-sequencing analysis of nanoparticle-treated plant leaves6. We believe the SWCNT delivery technologies that are discussed in this talk will have an immense impact on food security and sustainability by enabling the generation of improved plants that can rapidly reach the market in a cost-effective manner. Demirer G.S.*, Zhang H.* et al., DNA Nanostructures Coordinate Gene Silencing in Mature Plants. PNAS (2019). Demirer G.S. et al., Carbon Nanocarriers Deliver siRNA to Intact Plant Cells for Efficient Gene Knockdown. Science Advances (2020). Demirer G.S. et al., High Aspect Ratio Nanomaterials Enable Delivery of Functional Genetic Material Without Transgenic DNA Integration in Mature Plants. Nature Nanotechnology (2019). Demirer G.S. et al., Carbon nanotube–mediated DNA delivery without transgene integration in intact plants. Nature Protocols (2019). Demirer G.S. et al., Nanotechnology to advance CRISPR–Cas genetic engineering of plants. Nature Nanotechnology (2021).González-Grandío E., Demirer, G.S. et al. Carbon nanotube biocompatibility in plants is determined by their surface chemistry. bioRxiv (2021).