(346a) Nanomaterials Enable DNA-Free siRNA-Guided Gene Silencing in Intact Plants

Demirer, G. S., University of California
Zhang, H., University of California
Goh, N., University of California
Chang, R., University of California
Landry, M., Chan Zuckerberg Biohub

115%;background:white"> letter-spacing:.15pt">Nanomaterials enable DNA-free siRNA-guided gene silencing
in intact plants

.5in;line-height:115%"> font-family:Helvetica">Plants are central in providing over 25% of our most
clinically-relevant drugs, are at the core of our sustainability efforts, and
will benefit from genetic engineering to feed our growing population in the
midst of climate change. Plant biotechnology is currently limited by the cost,
ease, and throughput of methods for probing plant genetics and gene expression
profiles. Consequently, less than a dozen complete biosynthetic pathways are
known for plant natural products that have been reconstituted heterologously,
compared to the ~1000 known biosynthetic pathways in bacteria and fungi. Post-transcriptional
gene silencing (PTGS) is a prominent tool in plants for discovery of new
biosynthetic pathways, increased production of valuable small molecules, and to
confer resistance to plant diseases. While the most expedient method of
utilizing PTGS would be to directly deliver small interfering RNA (siRNA) to
cells, plants have a cell wall which presents a significant barrier to exogenous
biomolecule delivery. Consequently, viral vectors combined with Agrobacterium
delivery is the preferred method of siRNA delivery (coded as DNA) to intact
plant cells. However, most viruses and Agrobacteria are limited in their
host range, often do not result in uniform gene silencing, yield random DNA
integration that can adversely and unpredictably affect the cell operation, and
are subject to genetically modified organism (GMO) regulatory oversight.  

.5in;line-height:115%"> font-family:Helvetica">While nanoparticle-mediated RNA delivery has been
extensively explored in animals, its potential for plants remains
under-studied. To-date, there has yet to be a nanoparticle-based delivery
platform of siRNA into intact plant cells. In our prior work, we have shown single-walled
carbon nanotubes (SWNTs) functionalized with cationic polymers enable plasmid
DNA delivery without the use of external force into model and crop plants for
genetic transformations1. However, the same nanoparticle chemistry is
ineffective for siRNA delivery to plants. In this study, we develop a different
SWNT surface chemistry and RNA loading strategy for siRNA delivery into intact
plants. Pi-pi adsorption of sense and antisense siRNA strands onto pristine
SWNTs followed by equimolar delivery of these suspensions in mature Nicotiana
plant leaves results in up to 95% gene silencing at the mRNA
transcript level2. We further show that nanotubes are non-toxic, and
that SWNT-based delivery provides a significant delay in intracellular siRNA
degradation, suggesting nanoparticles protect the cargo from nuclease

.5in;line-height:115%"> font-family:Helvetica">To elucidate the underlying principles of plant nanoparticle
internalization process, we systematically investigate the effect of certain
nanomaterial parameters (shape, size, aspect ratio, stiffness and cargo
attachment loci) on plant cell uptake, and gene silencing pathways and
efficiencies3. By leveraging the facile programmability of DNA
nanostructures and DNA origami, we designed a set of nanostructures with varying
structural and mechanical properties. Results reveal that nanostructures with
sizes below " ms mincho>_ Helvetica">10 nm and higher stiffness have higher plant cellular
internalization, although size or stiffness alone are not mutually-exclusive
contributors3. When loaded with siRNA, these DNA nanostructures also
able to silence genes in plant leaves with efficiencies that match
nanostructure internalization trends. Interestingly, we show that the
employment of certain plant endogenous gene silencing mechanism can be altered
by the DNA nanostructure shape and the siRNA attachment locus on the

.5in;line-height:115%"> font-family:Helvetica">In summary, we show that nano-scale technologies such as
carbon nanotubes and DNA nanostructures can now be rationally programmed to efficiently
internalize into intact plant cells and significantly knock-down important
endogenous genes for (i) rapid and species-independent identification of
genotypes that result in desired phenotypes, (ii) mapping and optimization of
plant biosynthetic pathways, and (iii) maximization of plant-mediated
therapeutics synthesis, enabling many diverse life sciences applications based
on RNA interference in plants.   


text-indent:-.25in;line-height:115%"> 115%;font-family:Helvetica">1.    
Helvetica">Demirer, G.S. 115%;font-family:Helvetica">, Zhang, H. et al. High Aspect Ratio Nanomaterials
Enable Delivery of Functional Genetic Material Without Transgenic DNA
Integration in Mature Plants. Nature Nanotechnology (2019).

text-indent:-.25in;line-height:115%"> 115%;font-family:Helvetica">2.    
Helvetica">Demirer, G.S. 115%;font-family:Helvetica">, Zhang, H., et al. Nanotubes effectively deliver
siRNA to intact plant cells and protect siRNA against nuclease degradation. bioRxiv

text-indent:-.25in;line-height:115%"> 115%;font-family:Helvetica">3.    
H., Demirer, G.S. et al. DNA Nanostructures Coordinate Gene Silencing in
Mature Plants. PNAS (2019).