Engineering enhanced biolistic delivery systems for efficient plant transformation and genome editing
Date
2025-08
Authors
Thorpe, Connor
Major Professor
Advisor
Jiang, Shan
Wang, Kan
Whitham, Steve
Kirillova, Alina
Chang, Boyce
Committee Member
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Biolistic delivery has long served as a foundational method for plant transformation, particularly for species and genotypes recalcitrant to Agrobacterium-mediated transformation. Despite its broad applicability, the gene gun platform has remained largely unchanged for over four decades, resulting in major limitations in delivery efficiency, reproducibility, and compatibility with modern genome editing tools. This dissertation presents a comprehensive suite of innovations designed to modernize and dramatically enhance the performance of biolistic delivery systems for plant biotechnology.
First, I engineered a novel Flow Guiding Barrel (FGB), an aerodynamic attachment for the PDS-1000/He gene gun, which improves the focus, speed, and uniformity of particle dispersion. This modification leads to 4x to 30x improvements in transformation efficiency, depending on the application and tissue type, without increasing cellular damage. The FGB represents the first mechanical innovation to the gene gun in over 40 years and is compatible with standard gene gun infrastructure.
Second, I developed a rapid, room-temperature synthesis platform for producing monodisperse gold microparticles ranging from 400 nm to 1600 nm in diameter. These particles exhibit exceptional uniformity and reduced aggregation compared to commercial polydisperse particles, resulting in significantly enhanced protein binding, delivery precision, and reproducibility. This advancement enables rational optimization of particle size for different biological applications and plant systems.
Third, I designed and screened a library of synthetic, tunable lipid-based delivery agents tailored for complexing and delivering CRISPR-Cas9 ribonucleoproteins (RNPs) into plant cells. By systematically varying tail length and amine-rich headgroup chemistry, I identified lead compounds that exhibit >90% protein binding, >60% release, minimal cytotoxicity, and >15% genome editing efficiency, far outperforming commercial reagents like TransIT-2020 and TransIT-X2. These lipids enable high-efficiency, DNA-free genome editing, offering a path forward for transgene-free trait development and regulatory simplification.
Collectively, these innovations represent a transformative leap forward for biolistic technologies, bridging materials science and plant molecular biology to overcome critical bottlenecks in gene and protein delivery. The modularity and scalability of this platform make it adaptable for a wide range of crops and delivery targets, from functional genomics to commercial trait development. This work not only reinvents the gene gun for the era of precision genome editing but also lays the foundation for the next generation of plant transformation technologies.
Series Number
Journal Issue
Is Version Of
Versions
Series
Academic or Administrative Unit
Type
dissertation