Pick a Side: Untethered Gel Crawlers That Break Symmetry

Advances in plant synthetic biology are becoming increasingly more important, in line with the need to feed a rapidly expanding global population in the face of climate change and to provide a sustainable platform for the synthesis of clinically-important drugs at large scales. The powerful CRISPR-Cas gene editing system has transformed our synthetic biology capabilities, holding the potential to make highly-specific and efficient genetic modifications in plants, but the efficient delivery of important biomolecules across the plant cell wall to enable CRISPR gene editing remains a challenge. The use of traditional biomolecule delivery strategies in plants (i.e. Agrobacterium-mediated transformation and biolistics) have facilitated translation of this system to enable engineering of more sustainable crops1. However, limitations of these systems hinder their ubiquitous application in situations requiring the delivery of diverse cargo types to a wide range of plant substrates. One such situation is the direct delivery of functional proteins like
CRISPR/Cas ribonucleoprotein complexes to plant somatic cells, which would enable DNA-free plant gene editing. Despite recent advances in nanotechnology-based platforms to aid in the efficient delivery of biomolecules like small interfering RNA2,3 and plasmid DNA4,5, far less progress has been made in enabling efficient protein delivery using these platforms.

Given the lack of a nanoparticle delivery vehicle capable of efficient protein delivery to plants, thus limiting the potential impact of CRISPR gene editing technologies on food security and sustainability, we developed a Genetically-encoded Delivery Vehicle (GDV) capable of delivering diverse biomolecule cargoes to plant somatic cells, offering a promising method for the delivery of functional proteins into plants to address this critical bottleneck to plant genetic engineering. Using a high-translation expression system in tobacco plants6, GDVs were synthesized from the coat proteins (CPs) of Tobacco Mosaic Virus (TMV) for downstream modification and application in biomolecule delivery to plant leaf tissue (Figure 1). We modified GDVs by leveraging a combination of protein engineering and bioorthogonal click chemistry, successfully loading the platform with various cargoes of interest, including fluorescent labels, functional peptides, and active Cas9 RNP complexes, to determine the delivery capabilities of the GDV platform.

This talk will first summarize the development, characterization, and modification of the GDV platform for plant delivery applications. Next, I will present recent results of functional peptide and protein delivery using the GDV platform. This discussion will focus on functional protein cargoes with the potential to enable (i) fluorescent complementation for validation and quantification of GDV delivery efficiency, (ii) electrostatic grafting of plasmid DNA for transient expression assays and (iii) direct RNP delivery for CRISPR/Cas gene editing in plant leaves. We believe that GDVs offer a biodegradable, genetically-encoded and scalable alternative to more traditional nanocarriers used in plant synthetic biology. Boasting a two-pronged modulation strategy that exploits both protein engineering and chemical surface modification, the GDV platform demonstrates the potential to enable DNA-free gene editing in plant somatic tissues via the direct delivery of functional proteins like Cas9 RNPs, realizing a new and powerful tool in the plant synthetic biology toolkit.

1. Zaidi, S. et al. Engineering crops of the future: CRISPR approaches to develop climateresilient and disease-resistant plants. Genome Biol. 21, 289 (2020).

2. Zhang, H. et al. DNA nanostructures coordinate gene silencing in mature plants. Proc. Natl. Acad. Sci. 116, 7543–7548 (2019).

3. Demirer, G. S. et al. Carbon nanocarriers deliver siRNA to intact plant cells for efficient gene knockdown. Sci. Adv. 6, eaaz0495 (2020).

4. Demirer, G. S. et al. High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. Nat. Nanotechnol. 14, 456–464 (2019).

5. Cho, J.-Y. et al. Cellular Delivery of Plasmid DNA into Wheat Microspores Using Rosette Nanotubes. ACS Omega 5, 24422–24433 (2020).

6. Peyret, H. et al. Improving plant transient expression through the rational design of synthetic 5′ and 3′ untranslated regions. Plant Methods 15, 108 (2019).