Developing Nanomaterial Platforms for Gene Delivery and Transgene-Free Plant Genetic Engineering

Plant biotechnology is an essential component of agricultural engineering, small molecule synthesis, and bioenergy efforts. Maximizing the throughput of producing and testing genetically engineered plants is important for both academic research and the agro-industry, and requires a toolset that is (i) plant species-independent, and (ii) capable of high performance despite the physical barriers presented in intact plant tissues, such as the plant cell wall. Currently used plant biotic delivery tools limit the range of plant species that can be transformed, and in the case of biolistic delivery, exhibit low transformation efficiencies and tissue damage due to the use of high force. Furthermore, both methods yield uncontrolled and random transgene integration into the plant nuclear genome, which then elicits strict genetically modified organism (GMO) regulatory purview and public concern for consumption. To-date, there has yet to be a plant transformation method that enables high-efficiency gene delivery, without transgene integration, in a plant species-independent manner./nI have recently shown it is possible to introduce DNA and RNA into intact plant cells without external force with engineered nanomaterials that are below the plant cell wall size-exclusion limit of ~20 nm. Among these nanomaterials, carbon nanotubes (CNTs) possess several optimal criteria for gene delivery into intact plants: high aspect ratio, exceptional tensile strength, biocompatibility, and biomolecular cargo protection from cellular degradation. In this dissertation, I describe a CNT-based gene delivery platform which can efficiently deliver plasmid or linear DNA into both model and agriculturally-relevant crop plants, without mechanical aid, in a non-toxic and non-integrating manner. Notably, this combination of features is not attainable with existing plant transformation approaches. CNT gene delivery enables strong transient expression of reporter and functional proteins without DNA integration in dicot species Nicotiana benthamiana (model), Eruca sativa (arugula, non-model), Gossypium hirsutum (cotton, non-model, hard to transform), and in monocot species Triticum aestivum (wheat, non-model). This technology can be used to deliver CRISPR/Cas9 gene editing plasmids as a method to achieve stable genome editing in plants while circumventing GMO labeling, through the transient expression of a nuclease protein and guide RNA in model and crop plants./nMoreover, CNTs with different surface chemistries are developed and used to deliver other important biomolecules, such as small interfering RNAs, for efficient DNA-free gene knock-down applications in intact plants. In a separate study, I systematically investigated the effect of certain nanomaterial parameters (shape, size, aspect ratio, stiffness, and cargo attachment loci) on plant cell internalization, and gene silencing pathways and efficiencies using easily programmable DNA nanostructures as nanomaterial scaffolds. Additionally, in this dissertation, I demonstrate how these nanomaterials not only facilitate biomolecule transport into plant cells but also protect polynucleotides from nuclease degradation inside the cells, which increase their efficiencies and permit the usage of a lower cargo dose./nCNT-based plant transformation is a breakthrough for biotechnology applications where transient protein expression without gene integration is desired, such as the delivery of CRISPR/Cas9 cargoes to achieve permanent genome editing. Furthermore, CNT-based gene delivery is rapid, cost-effective, amenable to multiplexing and can aid high-throughput screening in mature plants. This enables (i) rapid identification of genotypes that result in desired phenotypes, (ii) mapping and optimization of plant biosynthetic pathways, and (iii) maximization of plant-mediated therapeutics synthesis. Therefore, nanomaterials promise to overcome the long-standing plant genome engineering limitations in a species-independent and non-integrating manner and can newly enable variety of different life sciences applications.

Advisor: Landry, Markita P