(53f) Engineered Nanomaterials Enable Transgene-Free Plant Genetic Transformations

Demirer, G. S., University of California
Zhang, H., University of California
Matos, J., University of California
Goh, N., University of California
Cunningham, F., UC Berkeley
Sung, Y., UC Berkeley
Staskawicz, B., University of California
Landry, M., Chan Zuckerberg Biohub

115%;background:white"> letter-spacing:.15pt">Engineered nanomaterials enable transgene-free plant
genetic transformations

.5in;line-height:115%"> font-family:Helvetica">Plant biotechnology is an essential component of
agricultural engineering, small molecule synthesis, and bioenergy efforts1.
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 DNA delivery tools
such as Agrobacterium either limit the range of plant species that can
be transformed, or in the case of biolistic delivery, exhibit low
transformation efficiencies and tissue damage due to the use of high biolistic force2.
Furthermore, both methods yield uncontrolled transgene integration into the plant
genome, and even if DNA is delivered with engineered non-integrating Agrobacterium,
this pathogenÕs use elicits genetically modified organism (GMO) regulatory
purview. Helvetica;color:black">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.

.5in;line-height:115%"> font-family:Helvetica;color:black">I 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 nm3,4,5.
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.
Here, I describe a CNT-based gene delivery platform which can efficiently
deliver plasmid or linear DNA into line-height:115%;font-family:Helvetica;color:black">both model and
agriculturally-relevant crop plants, without mechanical aid, in a non-toxic and
non-integrating manner3. Notably, this combination of features is
not attainable with existing plant transformation approaches. I show 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), and Gossypium hirsutum
(cotton, non-model, hard to transform), and in monocot species Triticum
(wheat, non-model)3,6. 11.0pt;line-height:115%;font-family:Helvetica;color:black">I next discuss CNT
delivery of CRISPR gene vectors 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 plant leaves and seeds.

.5in;line-height:115%"> font-family:Helvetica;color:black">Moreover, I validate that CNTs with
different surface chemistries can be used to deliver other important biomolecules,
such as small interfering RNAs, for efficient DNA-free gene knock-down applications
in intact plants5. In a separate study, I systematically investigate
the effect of certain biomaterial parameters (shape, size, aspect ratio,
stiffness, and cargo attachment loci) on plant cell internalization, and gene
silencing pathways and efficiencies4. Additionally, I explain how these
nanomaterials not only facilitate biomolecule transport into plant cells but
also protect polynucleotides from nuclease degradation5 font-family:Helvetica">. Lastly, transcriptomic
analyses of the impact of nanomaterials versus Agrobacterium on plants
demonstrates the utility of nanomaterials for plant biotechnology.

background:white"> Helvetica;color:black">CNT-based plant transformation is a breakthrough for
biotechnology applications where transient protein expression without gene
integration is desired, such as the delivery of CRISPR 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.

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text-indent:-.25in;line-height:115%"> 115%;font-family:Helvetica">1.    
Helvetica">Demirer, G.S. 115%;font-family:Helvetica">, Landry, M.P. Delivering Genes to Plants. AIChE

text-indent:-.25in;line-height:115%"> 115%;font-family:Helvetica">2.    
F.J., Goh, N., Demirer, G.S. et al. Nanoparticle-Mediated Delivery
Towards Advancing Plant Genetic Engineering. Cell Press Trends in

text-indent:-.25in;line-height:115%"> 115%;font-family:Helvetica">3.    
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">4.    
H.*, Demirer, G.S.* et al. DNA Nanostructures Coordinate Gene Silencing
in Mature Plants. PNAS (2019).

text-indent:-.25in;line-height:115%"> 115%;font-family:Helvetica">5.    
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">6.    
M. P., Demirer, G.S. Mature plant transformation with
nanoparticle-grafted gene vectors. U.S. Provisional patent filed via UC
Berkeley, March 2017.  

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