(315e) Award Submission: Carbon Nanomaterials Enable Plant Genome Engineering without Transgene Integration

Authors: 
Demirer, G. S., University of California
Zhang, H., University of California
Matos, J., University of California
Aditham, A., University of California
Staskawicz, B., University of California
Landry, M., Chan Zuckerberg Biohub

115%;background:white"> letter-spacing:.15pt">Carbon nanomaterials enable plant genome engineering without
transgene integration

.5in;line-height:115%"> font-family:Helvetica">Food security is threatened by decreasing crop yields
and increased consumption in the light of climate change, population growth,
and a shortage of arable land. To mitigate these factors, genetic engineering
of plants can be employed to create crops that have higher yields and nutritional
value, and are resistant to herbicides, insects, diseases, and abiotic stresses
such as drought. Despite the recent significant advancements in genome editing
(such as CRISPR), most plant species still remain difficult to genetically engineer.
The two bottlenecks of generating transgenic plants are (i) efficient biomolecule
delivery into plant cells through the rigid cell wall and (ii) the regeneration
of transformed tissues1. The workhorse method of plant DNA delivery,
Agrobacterium, limits the range of plant species that can be
transformed and results in uncontrolled transgene integration, hence eliciting
a genetically modified organism (GMO) regulatory purview of edited plants. To-date,
there has yet to be a plant transformation method that enables high-efficiency
plasmid DNA delivery, without transgene integration, in a plant
species-independent manner for intact plants.

.5in;line-height:115%"> font-family:Helvetica;color:black">Here, we describe the generation, validation,
and optimization of a nanomaterial-based gene delivery platform that can
efficiently deliver genes into 115%;font-family:Helvetica;color:black">both model and agriculturally relevant crop
plants, without mechanical aid, in a non-toxic and non-integrating manner; a
combination of features that is not attainable with existing plant
transformation approaches. More specifically, we chemically modified the
surface of single-walled carbon nanotubes (SWNTs) with a cationic polymer
(polyethylenimine, PEI) to adsorb the negatively charged genetic cargoes via
electrostatic attractions. Delivery of plasmid DNA to tobacco, arugula, cotton,
and wheat leaves with these modified SWNTs results in strong transient expression
of reporter and functional proteins2,3. We verified that the transgene
does not integrate into plant nuclear genome with a highly sensitive digital
droplet PCR analysis2. Next, we show chemically modified SWNTs can deliver
CRISPR plasmids encoding the nuclease protein Cas9 and guide RNAs targeting genes
of interest. Through transient expression of Cas9 and guide RNA in plant cells,
we obtain stable font-family:Helvetica;color:black">editing of endogenous plant genes with
efficiencies comparable to 115%;font-family:Helvetica">Agrobacterium 11.0pt;line-height:115%;font-family:Helvetica">-mediated delivery in tobacco
leaves. Gene editing rates are quantified using a combination of techniques,
such as the restriction site loss assay, Sanger sequencing and TIDE/ICE
analysis, and deep amplicon sequencing. We also demonstrate the value of our nano-scale
gene delivery platform by editing genes in plant seeds with CRISPR-SWNT
delivery, which could eliminate the need for laborious tissue culture protocols
to regenerate edited plants. Lastly, we
demonstrate the compatibility of nanomaterials for plant biomolecule delivery
via RNA sequencing analysis.

.5in;line-height:115%"> font-family:Helvetica;color:black">Plant genome editing using carbon nanotubes
loaded with CRISPR vectors is a breakthrough advancement addressing two crucial
bottlenecks in the generation of transgenic plants: (i)
gene delivery to enable transient expression without gene integration and (ii) production
of stable gene editing without tissue regeneration. Additionally,
non-integrating DNA delivery nanotechnologies could enable genetic engineering
of plants in a manner that line-height:115%;font-family:Helvetica;color:black">circumvents GMO labeling in
the U.S. to facilitate introduction of engineered crops to the market in a time-
and cost-effective manner.

text-indent:-.25in;line-height:115%"> 115%;font-family:Helvetica">1.    
Cunningham,
F.J., Goh, N., Demirer, G.S. et al. Nanoparticle-Mediated Delivery
Towards Advancing Plant Genetic Engineering. Cell Press Trends in
Biotechnology 
(2018).

text-indent:-.25in;line-height:115%"> 115%;font-family:Helvetica">2.    
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">3.    
Landry
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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