Assistant Professor California Institute of Technology Pasadena, California
The powerful CRISPR-Cas gene editing system has transformed our synthetic biology capabilities, making possible highly-specific and efficient genetic modifications. However, the benefits of CRISPR have failed to permeate the plant synthetic biology community as effectively as they have its bacterial and mammalian counterparts. This is largely imparted by the unique physical barriers to CRISPR reagent delivery encountered in plant systems, including a rigid cell wall that hinders intracellular transport of extracellular biomacromolecules. Nanotechnology has emerged as a promising solution to this inherent barrier, enabling the delivery of silencing RNAs and expression plasmids via nanocarriers such as carbon nanotubes and gold nanorods, but these delivery platforms are limited in cargo diversity, scalability of synthesis, and translatability to agriculture.
To overcome these limitations and enable facile cargo delivery to plants for various food security applications, a Genetically-encoded Delivery Vehicle (GDV) was adapted from the Tobacco Mosaic Virus (TMV) viral capsid. Using a highly efficient translational system, TMV-GDVs were synthesized in planta and isolated for downstream modification and application in delivery. Bioorthogonal click chemistry was leveraged for peptide functionalization of the TMV-GDV surface, thus enabling various functionalities. Most notably, in planta fluorescence characterization demonstrates successful particle entry into the epithelial cells of tobacco leaves and offers a method for quantifying modulations in particle delivery efficiency. Additionally, a cell-penetrating peptide (CPP) surface treatment was explored as a method for improving delivery efficiency and for electrostatically grafting negatively-charged plasmid DNA to the GDV surface. GDVs offer a biodegradable, genetically-encoded, and scalable alternative to more traditional nanocarriers used in plant synthetic biology, boasting a two-pronged modulation strategy at the material level that exploits both protein engineering and chemical surface modification to realize a new and powerful tool in the plant synthetic biology toolkit.