Nanoparticle-Mediated Delivery towards Advancing Plant Genetic Engineering

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Opinion

Nanoparticle-Mediated Delivery towards
Advancing Plant Genetic Engineering

Francis J. Cunningham , 1 ' 6 Natalie S. Goh , 1,6 Gozde S. Demirer , 1 Juliana L. Matos , 2,3 and
Markita P. Landry 1,3A5 -*' @

Genetic engineering of plants has enhanced crop productivity in the face of
climate change and a growing global population by conferring desirable genetic
traits to agricultural crops. Efficient genetic transformation in plants remains a
challenge due to the cell wall, a barrier to exogenous biomolecule delivery.
Conventional delivery methods are inefficient, damaging to tissue, or are only
effective in a limited number of plant species. Nanoparticles are promising
materials for biomolecule delivery, owing to their ability to traverse plant cell
walls without external force and highly tunable physicochemical properties for
diverse cargo conjugation and broad host range applicability. With the advent of
engineered nuclease biotechnologies, we discuss the potential of nanoparticles
as an optimal platform to deliver biomolecules to plants for genetic engineering.

Current Biomolecule Delivery Methods for Genetic Engineering in Plants

Food security has been threatened with decreasing crop yields and increasing food consumption
in the wake of population growth, climate change, increasing shortage of arable land, and crop
usage as raw materials [1,2], Classical plant breeding to obtain plants with preferred genotypes
requires crossing and selection of multiple plant generations, which disallows introduction of traits
that do not currently exist in the species. A technique that enables specific horizontal gene transfer
stands to greatly benefit the agricultural industry by conferring desirable traits to plants, such as
increased yield, abiotic stress tolerance, and disease and pest resistance [3].

Genetic engineering has recently seen major advances in animal systems, though progress has
lagged in plants. When compared to the numerous and diverse gene and protein delivery
methods developed for animal systems, significantly fewer methods exist for plants (Figure 1 ,
Key Figure). Broadly, modern genetic transformation of plants entails two major steps: genetic
cargo delivery and regeneration of the transformed plant, the necessity and difficulty of the latter
being highly dependent on what delivery method is used and whether stable transformation is
desired. Regeneration procedures involve three parts: the induction of competent totipotent
tissue, tissue culture to form calli (see Glossary), and selection and progeny segregation.
Regeneration protocols are dominated by complex hormone mixtures, which are heavily
species and tissue dependent, making protocol optimization the key to increasing procedure
efficacy. The challenge of genetic cargo delivery to plants is attributed to the presence of the
multilayered and rigid plant cell wall, otherwise absent in animal cells, which poses an additional
physical barrier for intracellular delivery of biomolecules and is one of the key reasons for the
slower implementation and employment of genetic engineering tools in plants [4j.

Amongst conventional plant biomolecule delivery approaches, Agrobacterium -mediated and
biolistic particle delivery are the two most established and preferred tools for plant genetic
transformations (Box 1). Current biomolecule delivery methods to plants experience challenges

Highlights

Plant biotechnology is key to ensuring
food and energy security; however,
biomolecule delivery and progeny
regeneration continue to be key chal¬
lenges in plant genetic engineering.

Conventional biomolecule delivery
methods in plants have critical draw¬
backs, such as low efficiency, narrow
species range, limited cargo types,
and tissue damage.

Advances in nanotechnology have cre¬
ated opportunities to overcome limita¬
tions in conventional methods:
nanoparticles are promising for spe¬
cies-independent passive delivery of
DNA, RNA, and proteins.

The advent of nuclease-based gen¬
ome editing (eg., CRISPR-Cas9) has
ushered in a new era of precise genetic
engineering that, among other
impacts, has enabled the development
of genetically engineered crops with¬
out harsh regulatory restrictions.

The potential of nanoparticles to over¬
come limitations in conventional deliv¬
ery makes them excellent candidates
for delivery of nuclease-based genome
editing cargo, thus making nanoparti¬
cle delivery a critical technology for the
advancement of plant genetic
engineering.

department of Chemical and
Biomolecular Engineering, University
of California Berkeley, Berkeley, CA
94720, USA

department of Plant and Microbial
Biology, University of California,
Berkeley, CA 94720, USA
innovative Genomics Institute (IGI),
Berkeley, CA 94720, USA

882 Trends in Biotechnology, September 20f 8, Vol. 36, No. 9 https://doi.Org/10.10f6/j.tibtech.2018.03.009

© 2018 Elsevier Ltd. All rights reserved.

Trends in Biotechnology

that hinder their scope of use (Table 1). Methods such as electroporation, biolistics, Agro¬
bacterium -mediated delivery, or cationic delivery typically target immature plant tissue (calli,
meristems, or embryos). These methods require the regeneration of genetically modified
progeny plants, which can be time-consuming and challenging, whereby efficient protocols
have only been developed for a narrow range of plant species. Biolistic particle delivery
circumvents the cell wall via mechanical force, but often damages portions of target tissue
in the process and yields low levels of gene expression that is often sparse and sporadic.
Agrobacterium- mediated delivery is subject to orthogonal challenges, the largest being that
Agrobacterium displays narrow host and tissue specificity, even between specific cultivars of
the same species [5], Agrobacterium generally experiences lower transformation efficiency for
both delivery and regeneration in monocotyledonous plants (monocots) over dicotyledon¬
ous plants (dicots). Additionally, Agrobacterium yields random DNA integration, which can
cause disruption of important genes, or insertion into sections of the genome with poor or
unstable expression [6]. Random DNA integration, however, can be prevented by utilizing
magnifection with nonintegrating viruses [7], or by using a plasmid deficient in transfer DNA(T-
DNA) insertion [8].

In sum, plant genetic engineering has lagged behind progress in animal systems; conventional
methods of biomolecule delivery to plants remain challenged by intracellular transport through
cell walls, and in turn limit plant genetic transformation efficacy. To date, plant biotechnology
lacks a method that allows passive delivery of diverse biomolecules into a broad range of
plant phenotypes and species without the aid of external force and without causing tissue
damage. We posit nanotechnology as a key driver in the creation of a transformational tool to
address delivery challenges and enhance the utility of plant genetic engineering.

Nanoparticle-Mediated Biomolecule Delivery in Animal Systems

Nanoparticles as Molecular Transporters in Living Systems

Nanotechnology has advanced a variety of fields, including manufacturing, energy, and
medicine. Of particular interest is the use of nanoparticles (NPs) (Box 2) as molecular trans¬
porters in cells, an area that has largely focused on molecular delivery in animal systems. NPs
allow manipulation on a subcellular level, giving rise to a previously unattainable degree of
control over exogenous interactions with biological systems. Therefore, the impact of NPs as
drug and gene delivery vehicles in animals has been nothing short of revolutionary.

The small size of NPs and their highly tunable chemical and physical properties have enabled
NP engineering for NPs to bypass biological barriers and even localize NPs in subcellular
domains of CHO and HeLa cells, among others [10-13]. NPs serve as nonviral, biocompatible,
and noncytotoxic vectors that can transport a range of biomolecules [small molecules, DNA,
siRNA, miRNA, proteins, and ribonucleoproteins (RNPs)] [14-19] to biological cells. To this end,
various features of NPs, including size, shape, functionalization, tensile strength, aspect ratio,
and charge, have been tuned for efficient intracellular biomolecule delivery to animal systems.
Furthermore, ‘smart’ NPs have been developed to achieve responsive release of cargo for
increased control of site-specificity [20]. Various NPs have been manufactured and are
responsive to a range of stimuli, including temperature [21], pH [22], redox [23], and the
presence of enzymes [24],

Outlook and Implications for Nanocarriers in Plant Science

In contrast to the proliferate studies demonstrating NP-mediated delivery in animals, analogous
research in plants is relatively sparse (Figure 1 ), owing to the transport challenge imposed by the plant
cell wall, which renders biomolecule delivery more challenging than for most mammalian systems.

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California Institute for Quantitative
Biosciences (QB3), University of
California Berkeley, Berkeley, CA
94720, USA

5 Chan-Zuckerberg Biohub, San
Francisco, CA 94158, USA
6 These authors contributed equally to
this work

@ Twitter: @l_andry_lab

Correspondence:
landry@berkeley.edu (M.P. Landry).

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Nevertheless, knowledge gained from biomolecule delivery to animals provides a blueprint for
translation to plant systems, and could accelerate advancements in NP-mediated plant biomolecule
delivery. NP-mediated delivery may overcome the three foremost limitations of current delivery
techniques in plant systems by controlling NP size to traverse the cell wall, tuning charge and surface
properties to carry diverse cargo, and greater breadth in utility across plant species.

NP-mediated delivery in animals has successfully carried many types of cargo indiscriminately,
whereby certain methods for plants, such as Agrobacterium, can only deliver DNA. For instance,
Wang and colleagues report NP-mediated RNP delivery to mammalian cells via lipid encapsulation
[25]. Additionally, plastid engineering is not achievable with Agrobacterium, which only targets the
plant nuclear genome and cannot target the chloroplast or mitochondrial genomes. Conversely,
targeting moieties can be attached to NPs to obtain subcellular localization and modification of the
desired genome. Hoshino and coworkers demonstrate the delivery of quantum dots to the
nucleus and mitochondria of Vero kidney cells using respective localizing signal peptides [26].
Active targeting and controlled release is not achievable with conventional plant biomolecule
delivery methods, but has been demonstrated in animal systems with NP-based delivery. Davis
and colleagues designed a polymeric NP with a human transferrin protein-targeting ligand and
polyethylene-glycol (PEG) on the NP exterior to deliver siRNA to human melanoma tumor cells,
specifically [15]. Additionally, Lai and coworkers accomplished stimuli-responsive controlled
release of drug molecules and neurotransmitters encapsulated within mesoporous silica NPs
(MSNs) to neuroglial cells [27]. Drawing inspiration from progress in NP-mediated delivery for
animal systems, NP-mediated controlled delivery and release of biomolecules without species
limitations in plants is a forthcoming goal.

NP-Mediated Biomolecule Delivery to Plants

NP-Plant Interactions

To date, most literature on NP-plant systems focuses on plant-based metallic nanomaterial
synthesis [28], agrochemical delivery [29], and NP uptake, showing both valuable and deleterious
effects on plant growth [30,31 ]. Dicot and monocot plants exhibit variable degrees of direct uptake
of many NP types, including MSNs [32], carbon nanotubes (CNTs) [33], quantum dots [34], and
metal/metal oxide NPs [35-37]. Once uptaken, certain types of NPs exhibit phytotoxicity via
vascular blockage, oxidative stress, or DNA structural damage [30]. Conversely, NPs have been
shown to improve root and leaf growth, and chloroplast production [31]. Tradeoffs between
phytotoxicity and growth enhancement as a function of species, growth conditions, NP proper¬
ties, and dosage are not well understood and call for more studies with afocus on NP physical and
chemical properties. Closing the knowledge gap in plant physiological response to NP uptake is
important and should be pursued in parallel with the enhancement of plant science using
engineered nanomaterials, as the ‘nanorevolution’ in targeted delivery to animals suggests
tremendous potential for analogous progress in plants.

Heuristics for Nanocarrier Design

While a complete structure-function landscape of physical and chemical NP properties that drive
cargo loading and cellular internalization remains elusive, a heuristic approach to nanocarrier design
is a useful starting point. NP uptake and transport throughout plant tissue is limited by pore
diameters, setting size exclusion limits (SELs) for various tissues and organs that are discussed
extensively in the literature [30,38-43]. The cell wall is commonly thought to exclude particles >5-
20 nm, although recently NPs up to 50 nm in diameter have been reported as cell wall-permeable
through unclear mechanisms [38,41]. For genetic engineering applications, where cytosolic or
nuclear localization is necessary to affect gene function, the plasma and nuclear membranes pose
additional barriers to delivery. In practice, the cell wall (SEL <50 nm) plays a dominant role in NP size

Glossary

Callus: a mass of undifferentiated
cells that can be used to regenerate
plants.

Cultlvars: short for cultivated
varieties, a group of plants with
desired characteristics that have
been selected from a naturally
occurring species and are passed
through propagation.
Dicotyledonous plants: one of the
two major groups of flowering plants.
The eponymous term originates from
the presence of two embryonic
leaves upon germination.

Additionally, dicots can be
distinguished from monocots by a
number of characteristics that
include leaf veins, vascular bundles,
root development, floral bundles, and
pollen. See monocotyledonous
plants.

Electroporation: a physical
transfection method where an
electric field is applied to create
temporary pores in cell membranes
for the uptake of genetic cargo into a
cell.

Explant: any segment of a plant that
is removed to initiate a culture.

In planta: a transformation paradigm
involving the genetic transformation
of any segment of a plant without
the need for tissue culture and
regeneration.

Magnifection: delivering virus
vectors using Agrobacterium T-DNA
transfer.

Meristems: regions of tissue
containing undifferentiated cells.

Monocotyledonous plants: one of

the two major groups of flowering
plants that have one embryonic leaf
upon germination. Monocots include
crops that make up the majority of a
balanced diet, such as rice, wheat,
and barley. See dicotyledonous
plants.

Passive delivery: transport of cargo
across cell wall and membrane to an
intracellular location without the use
of mechanical force.

Protoplasts: plant cells with their
cell walls removed, typically through
either mechanical or enzymatic
means.

Recalcitrant: a species of plant that
is difficult to genetically transform
and regenerate into mature plants.
Often used in the context of
Agrobacterium-mediated
transformation.

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internalization limitations, as the cell membrane SEL is much larger (>500 nm) [38]. NP charge and
shape greatly influence cell membrane translocation and thus these properties are central to
nanocarrier optimization [44], Plant cellular uptake can occur through energy-dependent (endocy-
tosis) and energy-independent (direct penetration) pathways that are not well understood. It is
commonly reported that internalization is faster and more efficient for cationic NPs versus anionic
NPs, due to cationic NP binding with the negatively charged cell membrane [44], This charge
preference has been demonstrated in protoplasts and walled plant cells [45,46].

Endosomal escape is critical for subcellular delivery, as vesicle-entrapped NPs can be trafficked for
degradation or exocytosis, and remain inaccessible for downstream processing if trapped in the
endosome. Subcellular localization of NPs in plants is not well understood but will depend on the
uptake pathway, as endocytic proteins and vesicle cargo play a role in endosome fate [47], whereby
direct cell penetration bypasses endosomal vesicle formation entirely. Serag and colleagues report
CNT internalization in protoplasts through both direct penetration and endocytosis, supporting prior
demonstrations in mammalian cells that high aspect ratio NPs undergo vesicle-free internalization
[48,49]. However, for Serag and colleagues, direct penetration was only observed for cell wall-
impermeable multiwalled CNTs in protoplasts [48,49], motivating further studies for plant cell wall
internalization by high aspect ratio NPs. Wong and colleagues have demonstrated passive internali¬
zation of single-walled CNTs in extracted chloroplasts [1 29] through a mechanism dependent on NP
size and zeta potential [130]. Cationic, pH-buffering polymers are well-known endosome disruption
agents [50] that can function as ligands to improve endosomal escape. Chang and colleagues report
energy-independent internalization to walled root cells by organically functionalized spherical MSNs
[51 ]. Notably, endocytosed single-walled CNTs in plants are trafficked to vacuoles but localize in the
cytosol when loaded with DNA [33,48].

Most NPs are amenable to surface adsorption (physisorption) of biomolecules as a simple conju¬
gation strategy. However, physisorption may be unstable depending on the specific NP and cargo,
and thus electrostatic interactions are preferablefor noncovalent cargo loading [52], Cationic surface
chemistry not only enhances endocytic uptake and escape, but is also amenable to electrostatic
loading of genetic cargo via attraction with negatively charged DNA and RNA. Covalent NP surface
functionalization is typically achieved by one of many of ‘click’ chemistries [53]. Notably, covalent
attachment of thiolated DNA and proteins to gold NPs has shown recent success [54] but the field
remains open to new strategies for covalent bioconjugation, especially for applications in plants. As
an alternative to surface functionalization, porous NPs such as MSNs can be internally loaded with
macromolecules or small chemicals alike, for controlled intracellular release [55].

NPs with some or all of the properties mentioned above have demonstrated successful biomolecule
delivery in plants and are good starting points for choosing the appropriate NP, ligand, and cargo for a
given application. However, it should be noted that nanocarrier design is a complex, multivariable
optimization process, such that success will likely require tweaking of these heuristics for different
systems until a complete NP structure-function relationship is established for plant systems.

Nanomaterials for Plant Genetic Engineering

NPs are valuable materials for intracellular biomolecule delivery, owing to their ability to cross
biological membranes, protect and release diverse cargoes, and achieve multifaceted targeting
via chemical and physical tunability. Such properties have enabled NPs to revolutionize targeted
delivery and controlled release in mammalian systems. However, nanocarrier delivery in plants
remains largely underexplored due to the cell wall, which is typically overcome by chemical or
mechanical aid (Figure 1). Passive biomolecule delivery to plants is promising for minimally
invasive, species-independent, in vivo genetic engineering of plants, especially for transient

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Transgene: a gene taken from an
organism and transferred into the
genome of another. Consequently,
transgene integration results in
transgenic plants.

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Key Figure

Nanoparticle (NP)-Mediated Genetic Cargo Delivery to Animals and Plants

(a) NPs classes commonly employed in genetic cargo delivery

r

Bio-inspired NPs

• Calcium phosphate

• Chitosan

• Liposomes

Genetic cargo delivered a-d

"A

i

c3

DNA RNA Protein RNP

Carbon-based NPs

• Single-walled carbon
nanotubes
Multiwalled carbon nanotubes
• Fullerenes

Genetic cargo delivered e g

DNA RNA Protein RNP

Genetic cargo has been delivered in:
^ Both animal and plant systems
O Animal systems only

£ Plant systems only

ftl—T \ V'

yj Neither system

Silicon-based NPs

• Silica spheres
Mesoporous silica NPs

(MSNs)

• Silicon carbide
Genetic cargo delivered h-k

DNA

RNA

Protein

C©

RNP

(b) Modes of NP-mediated cargo delivery

Metallic / Magnetic NPs

• Gold
■ Silver
Iron oxide

o

Genetic cargo delivered 1

DNA RNA Protein RNP

Polymeric NPs

> Polyethylene-glycol (PEG)

• Poly(lactic-co-glycolic acid)
(PLGA)

• Polyethylenimine (PEI)

Genetic cargo delivered l-s

DNA

RNA

Protein RNP

Trends in Biotechnology

Figure 1. (A) NPs commonly used for biomolecule delivery in both animal and plant systems cover five major categories: bio-inspired, carbon-based, silicon-based,
polymeric, and metallic/magnetic. We provide a visual comparison of delivery of various genetic cargo [DNA, RNA, proteins (site-specific recombinases or nucleases),
and ribonucleoprotein (RNP)] with each of the five NP types across animal and plant systems. It is evident that NP-mediated delivery has been utilized with a greater
variety of genetic cargo in animals than in plants, (b) NP-mediated cargo delivery is conducted via several means. Physical methods include creating transient pores in
the cell membrane with electric fields, soundwaves, or light, magnetofection, microinjection, and biolistio particle delivery. Nonphysical methods include the use of

cationic carriers, incubation, and infiltration. a [64], b [86], c [87], d [88], e [89], f [68], g [90], h [91], '[45], '[92], k [58], '[93], m [94], n [95], °[96], p [97], q [98], r [99], s [81], ’[1 00], u [63],
v [101], W [1 02], x [54].

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Box 1. Common Gene Delivery Methods in Plants

Agrobacterium-Mediated Transformation

Agrobacterium tumefaciens is a soil bacterium that infects a wide range of dicots, causing crown gall disease. The
formation of a gall on the host plant is achieved via the stable transfer, integration, and expression of bacterial DNA in
infected plants. Engineering of the Agrobacterium plasmid by substitution of the gall-inducing virulence genes with
genes of interest confers the ability of Agrobacterium to transform the host plant. For this reason, Agrobacterium has
been harnessed as a tool for plant genetic transformation since the early 1980s [107],

Genetic transformation occurs through a process involving T-DNA export, targeting, and insertion into the plant nuclear
genome. The export of T-DNA from the bacterium to the plant cell is facilitated by the activity of virulence genes present
in the tumor inducing-plasmid of Agrobacterium, but are not themselves transferred. These virulence genes are
expressed in the presence of phenolic inducers, such as acetosyringone, produced by wounded plant cells. Agro¬
bacterium attaches to plant cells, where border sequences on either side of the T-strand (a single-stranded copy of the
T-DNA sequence) are cleaved. The T-strand is then carried by a transporter with a nuclear localization sequence and
integrated into the plant nuclear genome. Integration occurs at random positions in the genome via nonhomologous
recombination, a repair pathway for double-stranded breaks in DNA.

Gene Gun-Mediated Transformation

A form of biolistic particle delivery (also called particle bombardment), the gene gun, is a physical method that is
commonly utilized for plant genetic transformations. Developed in 1982 by Sanford and colleagues [1 08], the process
involves gold or tungsten microparticles (or microcarriers) coated with genetic cargo that are accelerated by pressurized
helium (He) gas into plant cells, rupturing cell walls and membranes. The gene gun consists of three main parts: a rupture
disk, macrocarrier (holding microcarrier particles), and stopping screen. The rupture disk is a membrane designed to
burst at a critical pressure of He gas. When He gas is accelerated to the desired pressure, the rupture disk bursts,
creating a shock wave that propels the macrocarrier towards the plant cells. The macrocarrier’s momentum is stopped
by the stopping screen, which allows genetic cargo-loaded microcarriers to pass and enter the plant cells.

Unlike Agrobacterium-mediated transformation, biolistic delivery can result in transformation of the nuclear, plastidal, or
mitochondrial genomes due to the nonspecific localization of genetic cargo. Consequently, more DNA needs to be
delivered with biolistic delivery than Agrobacterium-mediatecl delivery when targeting the nuclear genome.

expression in somatic tissue (Table 2). The potential of NP-based plant delivery methods is
underscored by the limitations of in vitro plant studies in general, wherein regeneration capacity
varies widely across species, genotype, and even within a single plant depending on develop¬
mental age of source tissue [56]. Currently, stable transformation requires progeny regeneration
from embryogenic calli regardless of the delivery method (Table 2). Thus, parallel optimization of
delivery and regeneration is necessary to improve efficiency and expand stable transformation
capabilities to all plant species.

In 2007, Torney and colleagues were the first to demonstrate NP co-delivery of DNA and chemicals
to Nicotians tabacunn plants via biolistic delivery of 100-200-nm gold-capped MSNs [45]. In this
study, a chemical expression inducer was loaded into MSN pores (~3 nm) that were subsequently
covalently capped with gold NPs. The capped MSNs were then coated with GFP plasmids and
delivered by gene gun to N. tabacum cotyledons, wherein GFP expression was triggered upon
uncapping and release of the expression inducer [45]. This seminal paper demonstrated proof of
concept that strategies common for NP delivery of DNA to mammalian systems can be adapted to
plants. Notably, gold MSNs were also used for biolistic co-delivery of DNA and proteins, namely
GFP and Cre-recombinase, demonstrating the ability of MSNs to deliver proteins for gene editing
[58]. Many delivery strategies still require a gene gun, electromagnetic field, or protoplast PEG-
transfection [58-63] as NP structure-function parameters have not yet been fully optimized to
passively bypass the cell wall (T able 3). However, for systems where mechanical or chemical aid is
necessary for NP internalization, the small size and high surface area of nanocarriers still offers

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Table 1. Scope of Use Summary for Plant Biomolecule Delivery Methods

Delivery method

Adverse effects of delivery

Target species/tissue

Cargo type and size 3

Limitations

Physical

Biolistic particle-
(gene gun)
mediated delivery

Damage to target tissue & cargo, low
penetration depth, random
integration

Depends on tissue type b /
oalli, embryos, leaves

DNA, siRNA, miRNA,
ribonucleoproteins
(RNPs), large cargo
size

Targeting leaves requires detachment from
plant, which limits time to observe delivery
effects; targeting embryos requires laborious
regeneration protocols, the effectiveness of
which is highly species/cultivar-dependent

Electroporation

Damage to target tissue, nonspecific
transport of material through pores
may lead to improper cell function

Unlimited/protoplasts 0 ,
meristems, pollen grains

Nucleic acids (DNA,

SiRNA, miRNA)

Limited cargo-carrying capacity

Chemical

Polymer-mediated

delivery

High charge densities induce
cytotoxicity

Species amenable to
protoplast regeneration/
protoplasts 0

Nucleic acids (DNA,

SiRNA, miRNA)

Regeneration is highly inefficient for most
species in transient studies and requires
tissue culture

Biological

Agrobacterium-
mediated delivery

Can lead to apoptosis and necrosis,
random integration

Narrow range of plant
species, especially
restricted from monooots 0 /
mature plants, immature
tissue, protoplasts

Limited to DNA, large
cargo size

Leaf-targeted delivery is transient and gene
edits are not transmitted to progeny, but allow
diverse biological studies; requires tissue
culture (except Arabidopsis) to generate
progeny; exhibits high host-speoificity

Viral delivery

Virus integration (can be mitigated by
using nonintegrating viruses)

Host plant species
restriotions/mature plants,
meristems

Nucleic acids (DNA,

SiRNA, miRNA), very
limited cargo size

Highly limited cargo-carrying capacity

“While most biomolecule delivery methods to plants can deliver a variety of gene editing reagents, DNA plasmids are arguably the most common cargo of interest; DNA loading capacities are a useful metric
for the upper limit for cargo sizes each method can sustain.

b While biolistio particle-mediated delivery can theoretically be utilized in unlimited target species, the ability to target species depends on the target tissue (by extension, cell wall structural strength) and
capability of available equipment.

“The use of protoplasts as target tissue necessitates regeneration protocols and progeny segregation that are time-consuming and are challenged by the limited plant species amenable to protoplast
regeneration.

d Progress has been made on increasing transformation efficiency in recalcitrant monooots [9].

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Box 2. Nanoparticles

Nanoparticles (1-100 nm in at least one dimension) can be engineered with varied compositions, morphologies, sizes,
and charges, enabling tunable physical and chemical properties. Ranging from zero to three dimensional, NPs are novel
tools that have a wide range of applications, including but not limited to energy storage, sensing devices, and biomedical
applications [109,110].

In addition to their high degree of tunability, NPs possess several advantages that validate their recent widespread use,
with particular emphasis in the biomedical industry. Most NPs can be prepared with consistent properties for low batch-
to-batoh variability, and can be designed to target biological systems, tissues, cells, or subcellular structures with high
specificity [52], Moreover, NP-mediated gene and drug delivery can overcome common issues faced with viral vectors;
NPs are often less immunogenic and oncogenic and can carry diverse and larger cargo, although the increased NP
sizes when biomolecules are surface-loaded raise the challenge of bypassing biological barriers [111], Furthermore, the
effects of NP use have yet to be thoroughly studied, though existing research points to nanoparticle chemistry, size, and
dose as tunable parameters to control cytotoxioity[1 12,113],

NPs are typically classified based on morphology and chemical properties. The most common categories include
polymeric [114], lipid [115], magnetic [116], metallic [117], and carbon-based NPs [118], NPs can be synthesized with
either a top-down or bottom-up approach using techniques such as lithography [119], deposition [120], and self-
assembly [121],

In NP-based delivery, a variety of strategies are employed to load NPs with the desired cargo. Physical techniques such
as encapsulation or entrapment are commonly used in drug delivery to ensure the progressive release of drugs.
Chemical techniques where the NP surface is modified for cargo grafting are in development, including noncovalent
conjugation (electrostatic interaction [122], ir-ir stacking [123]) and covalent conjugation [23].

superior performance over conventional methods. For instance, Torney and colleagues’ MSN
study achieved transgene expression with 1000 x less DNA than the tens to hundreds of
micrograms of DNA typically required for conventional PEG-transfection in protoplasts [45].

A few recent examples show promise for NP-mediated passive delivery to plants in vitro [64-66]
and in vivo [51,67] in, for example, N. tabacum protoplasts [66] and Arabidopsis thaliana roots
[51,67], respectively (Table 3). Demirer and colleagues have recently achieved passive delivery
of DNA plasmids and protected siRNA using functionalized CNT NPs for transient GFP
expression in Eruca sativa (arugula) leaves and transient silencing of constitutively expressed
GFP in transgenic Nicotiana benthamiana leaves [68]. This study also demonstrates CNT-
mediated transient GFP expression in Triticum aestivum (wheat), indicating the potential for
passive NP delivery in both model and crop species with high efficiency and low toxicity. While
many more studies are needed to optimize NP properties and functionalization, these early
results are promising for further exploration of NPs as a plant biomolecule delivery platform that
addresses the shortcomings of conventional methods. Furthermore, with the advent of nucle¬
ase-based gene editing technologies (Box 3), it is of great interest to optimize the delivery of
these revolutionary genome engineering tools by exploring NP-based delivery strategies for
diverse biomolecular cargoes.

Genome Editing has Enabled a New Era of Plant Science

Engineered Nucleases for Plant Genome Editing

Engineered nuclease systems, namely ZFNs, TALENs, and CRISPR-Cas, have emerged as
breakthrough genome editing tools owing to their high genetic engineering specificity and
efficiency (Box 3), whereby CRISPR-Cas has demonstrated increased simplicity, affordability,
and multiplexing capabilities over TALENs and ZFNs in plants [69,70]. Since 2012, CRISPR-Cas
has shown success for genome editing in both model and crop species, including A. thaliana, N.
benthamiana, N. tabacum (tobacco), Oryza sativa (rice), T. aestivum (wheat), Zea mays (corn),
Solanum lycopersicum (tomato), and Sorghum bicolor, among others [71 ,72]. Notably, CRISPR-
Cas mutations as small as 1 bp have been conserved through three plant generations [73,74],

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Table 2. Challenges in Plant Genetic Engineering and Proposed Advantages of NP Delivery

Desired outcome

Nonheritable a

(somatic/transient expression)

Heritable

(germline/stable transformation)

Targeted tissue

Leaves

Roots

Protoplasts Zygotic embryo

Somatic embryogenic calli

Tissue-specific biological and
experimental challenges

• Cell wall

• Inefficient cellular uptake

• Epidermal barrier

• Cell wall

• Inefficient cellular uptake

• Cell wall degradation
protocol

• Inefficient cellular uptake

• Cell wall

• Inefficient cellular uptake

• Embryo collection/calli
induction

• Calli regeneration

• Cell wall

• Inefficient cellular uptake

• Totipotency/calli induction

• Calli regeneration

Proposed advantages of NP
delivery

• NP-cell wall permeability

• NP-stomata permeability
[57]

• Anionic NPs root-to-shoot
vascular translocation [46]

• Passive uptake or direct
mesophyll injection
without gene gun or
protoplasts

• Tunable NP properties
and ligands for subcellular
targeting

• NP-cell wall permeability

• Cationic NP root
accumulation [46]

• Passive uptake without
gene gun or protoplasts

• Tunable NP properties
and ligands for subcellular
targeting

• Tunable NP properties
and ligands for subcellular
targeting

• NP-oell wall permeability

• Tunable NP properties
and ligands for subcellular
targeting

• NP-cell wall permeability

• Tunable NP properties
and ligands for subcellular
targeting

a While these somatic tissues (leaves, roots, protoplasts) are most commonly targeted for transient expression experiments, heritable outcomes may be derived through somatic embryogenesis
(dedifferentiation of somatic tissue).

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Table 3. Select Summary of NP-Mediated Genetic Engineering in Plants

NP type

Cargo

Plant species; cell/tissue type

Delivery method

Comments

Year

Refs

With external aid

Gold capped MSNs

GFP plasmid; chemical
expression inducer

N. tabacum cotyledons; Z.
mays embryos

Biolistic

Co-delivery and controlled release of
DNA and chemicals

2007

[45]

Poly-L-lysine coated
starch NPs

GFP plasmid

Dioscorea zingiberensis C.H.
Wright calli suspension

Sonoporation

5% transient expression efficiency;
some integration occurs

2008

[60]

Gold-plated MSNs

GFP and mCherry plasmids;
GFP protein

Allium cepa epidermis tissue

Biolistic

DNA and protein co-delivery

2012

[59]

Magnetic gold NPs

(3-glucuronidase (GUS)
plasmid

Brassica napus protoplasts
and walled cell suspension

Magnetic field

Transient GUS expression

2013

[61]

Gold-plated MSNs

AmCyanl and DsFted2
plasmids; Cre protein

Z. mays embryos

Biolistic

DNA and protein co-delivery; both
transient and stable expression

2014

[58]

Dimethyl aminoethyl
methacrylate (DMAEM)
polymer NPs

Yellow fluorescent protein
(YFP) and GFP plasmids

N. tabacum and Ceratodon
purpureus protoplasts

PEG transfection

Both transient and stable expression

2017

[62]

Magnetic Fe 3 0 4 NPs

Selectable marker gene
plasmids

Gossypium hirsutum pollen

Magnetic field

~1 % efficiency for generating stable
transgenic seeds

2017

[63]

In vitro without external aid

Polyamidoamine
(PAMAM) dendrimer NPs

GFP plasmid

Agrostis stolonifera L. calli

Passive

48.5% cells showed transient
expression

2008

[65]

Calcium phosphate NPs
(CaPNPs)

GUS plasmid

Brassica juncea hypocotyl

explants

Passive

80.7% stable transformation
efficiency

2012

[64]

Organically
functionalized CNTs

YFP plasmid

N. tabacum protoplasts and
leaf explants

Passive

Both transient and stable expression

2015

[66]

In vivo without external aid

Organically
functionalized MSNs

mCherry plasmid

A. thaliana roots

Passive

46.5% transient expression efficiency

2013

[51]

PAMAM dendrimer NPs

Double-stranded DNA for

RNA interference

A. thaliana roots

Passive

Developmental gene silencing led to
systemic phenotypes

2014

[67]

Polymer functionalized
CNTs

GFP plasmid; siRNA for
transgenic GFP silencing

E. sativa, N. benthamiana,

and T. aestivum leaves

Passive

95% transient silencing efficiency;
transient expression in mature leaves

[68]

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Box 3. Traditional Genetic Engineering versus Nuclease-Enabled Genome Editing
Genetic engineering refers broadly to manipulating a cell’s genome and gene expression profile. Techniques for genetic
engineering may cause recombinant protein expression, up/downregulation of a gene, permanent gene knockout,
targeted mutations in the host gene, or insertion of large foreign DNA segments into the host genome. Genome
modifications may be transient, permanent, or heritable and involve many types of biomolecules (most commonly RNA,
DNA, and proteins) which are sometimes taken up passively by cells but often require enhanced delivery techniques,
such as gene guns, microinjection, electroporation, sonoporation, nanoparticle-assisted delivery, and engineered
bacteria or viruses. In plants, genetic engineering is hindered by the cell wall, requiring delivery methods that are highly
host-specific or limited by challenges in plant regeneration.

Nuclease-enabled genome editing refers to techniques where genes are removed or changed with engineered
nucleases, a class of enzymes that perform targeted double-stranded breaks (DSBs) at specific locations in the host
genome. When nucleases perform DSBs, the cell undergoes homology-directed repair (HDR) or nonhomologous end¬
joining (NHEJ) to repair the cut. NHEJ is a random, error-prone repair process that involves realignment of a few bases,
such that the high error frequency provides a simplistic pathway for gene knockout. HDR is a nonrandom repair process
requiring large stretches of sequence homology, allowing for precise edits by introducing customized homologous
recombination sequences for gene knockout, knock-in, and targeted mutations. Prominent tools in genome editing are
zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly
interspaced short palindromic repeat)-Cas (CRISPR associated) systems. In the 1990s, ZFNs became the first nuclease
system engineered for selectable genome editing in bacteria |124], TALEN and CRISPR-Cas genome editing systems
were developed for bacteria and eukaryotes more recently, around 2009 and 2012, respectively [125-128]. Composed
of protein complexes containing a DNA-binding domain and a DNA-cleaving domain, ZFNs and TALENs rely on protein/
DNA recognition to induce endogenous DNA repair. CRISPR-Cas systems are composed of a nuclease protein (Cas)
and a guide RNA (gRNA) with sequence homology to the genomic target, and therefore rely on the formation of a
ribonucleoprotein (RNP) complex to induce HDR or NHEJ. While all three systems have their drawbacks, CRISPR-Cas
has revolutionized the field of genome editing owing to its relatively superior simplicity, efficiency, and multiplexing ability
(i.e., simultaneous editing of different genes) over ZFNs and TALENs.

which is promising for stable transgene-free modified crops. As with traditional genetic engineer¬
ing of plants, many of the limitations for implementing gene editing tools in plants (low editing
efficiency, tissue damage, species limitations, cargo-type limitations) originate in biomolecular
transport into plant cells. As such, NP-based biomolecule delivery to plants stands to enable
higher-throughput plant genome editing via DNA, single guide RNA (sgRNA), and RNP delivery,
and thus warrants a discussion on the state of the plant genome editing field.

Global Landscape of Regulatory Uncertainty towards Genetically Engineered Crops
Genetic engineering of crops has evolved to overcome limitations in traditional breeding, as
breeding is slow, laborious, and lacks precise control over plant genotype and phenotype
generation. Modern biotechnology enables rapid development of crop variants with disease
and pest resistance, stress tolerance, higher yield, and enhanced nutritional value. Since 1996,
global genetically modified organism (GMO) cultivation has increased 110-fold to 185 mega¬
hectares in 2016 [75] (Figure 2). The US is a leader in GMO production but highly regulates
production of modified crops, which poses, among other challenges, significant financial barriers
to commercialization of new crop variants [76]. The US GMO pipeline is product-based but
sensitive to plant pests, such that Agrobacterium automatically triggers regulation, while other
methods of gene delivery are often deregulated if the product is nontransgenic [76,77]. European
Union GMO regulation is process-based and affects any organism whose genome has been
modified other than by mating or natural recombination [78], but includes exceptions for certain
types of mutagenesis that will likely exempt modern gene editing [79], The advent of nuclease-
based gene editing (Box 3) has set forth a global reevaluation of the legislation surrounding
genetically engineered crops, wherein several leading GMO cultivators have exempted non¬
transgenic genome-edited plants from regulation (Figure 2). Recently, the USDA officially stated
that there are no future plans to include genome-edited plants under the current US regulatory
umbrellafor GMOs [131]. However, due to differences in regulatory philosophy and public opinion,
several countries oppose deregulation of nontransgenic genome-edited plants and it remains

892 Trends in Biotechnology, September 2018, Vol. 36, No. 9

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Area of GMO cultivation worldwide in 2016 (millions of hectares) 3

Brazil

49.1

Argentina

Canada

India

Paraguay

Pakistan

China

23.8

Strict policy in Europe: 'Opt-out' model allows EU member states prohibit
cultivation of EU-approved GMOs within their own territory. Most states opt-out GMO
cultivation but allow imports for animal feed.

Genome editing could bypass strict policy: In January 2018, European Court of
Justice Advocate General states that plant mutagenesis by modern gene editing
techniques may qualify for regulatory exemption in the EU. b

South Africa Ml.?
Uruguay! 1.3
Bolivia 11.2
Australia 10.9
Philippines! 0.8
Myanmarl 0.3
Spainl 0.1
Sudani 0.1
Mexicol 0.1
ColombialO.l

Nontransgenic genome edited plants are:

0 Subject to GMO regulation
# Not subject to GMO regulation
0 Currently undergoing regulatory review
0 Not yet explicitly addressed

Sweden: CRISPR/Cas edited A. thaliana granted non-GMO status in 2015. c

Regulatory review underway in China: Chinese
government strongly supports GM crops, National
Biosafety Committee still developing regulations for
plant genome editing as of january 2018. d

Canadian regulations differ
from the rest: Canadian law
addresses 'plants with novel
traits' (PNTs) rather than GMOs,
existing legislation adequately
xovers plant genome editing!

U.S. GMO regulation and pipeline cost: 6+

years, $50 million+ regulatory pipelines for GMOs,
as many as 10 nuclease-edited plants bypassed
regulation in the US as of January 2018. C 'S

Argenetina pioneers plant gene editing
legislation: In 2017, Argentina passed the first
legislation specific to modern genome editing;
nontransgenic gene edited plants are exempt from
regulation^ As of 2018, similar resolutions were
V^passed in Chile and Brazil!-'

• Active GMO cultivation/trials
0 Not restrictive to GMOs

• Restrictive to GMOs
No data available

Regulatory review coming to an end in
Australia: As of january 2018, following
12-month review, Australian policy expected
to loosen up for gene editing in plants. e

Trends in Biotechnology

Figure 2. Genetically Modified Organism (GMO) Cultivation and Regulatory Attitudes Worldwide. Despite a long, expensive regulatory pipeline, the US is a
leader for GMO cultivation worldwide, followed by Brazil and Argentina, with Argentina being the first to directly address modern genome editing techniques in GMO
legislation. European and Australian regulatory attitudes are strict but have recently evolved as of January 2018, suggesting that regulations for genome-edited plants
will soon be relaxed in these regions. Nuclease-based edits without transgene integration escape regulation, even in countries with large agricultural GMO industries and
complex regulatory systems. Globally, GMO regulation and commercial use is heterogenous and uncertain due to economic, ecological, and sociopolitical
complexities. This map is a simplification of the convoluted global landscape regarding genetically engineered crops. ‘Restrictive to GMOs’ indicates a complete
or partial ban on GMOs and GMO-derived products for commercial or research purposes. a [75], b [79], °[80] , d [1 03], e [104], *[105], 9 [106], h [132], '[133].

unclear how enforcement of GMO status will proceed worldwide in the future [80]. Despite the
heterogenous and dynamic global regulatory landscape, nuclease-based genome editing cur¬
rently plays a critical role in overcoming regulatory restrictions and ensuring scientific progress, as
well as commercial implementation of engineered crop variants.

Nanocarriers Hold Promise for Nuclease-Based Plant Genome Editing
Genome editing tools may increase the throughput of plant molecular biology and genetic studies,
and as such could shift the paradigm in regulatory oversight of transgenic plants. Species,
amenable tissue, expression strategy (DNA, RNA, or protein), and delivery method contribute

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to the efficacy of transgene expression or modification and to the propensity of transgene
integration into the host genome. ‘DNA-free’ genome editing techniques are increasingly attrac¬
tive, especially from a regulatory perspective, to eliminate all risk of transgene integration. Recently,
RNP delivery has been demonstrated in A. thaliana and O. sativa protoplasts via PEG-transfection
[81 ] and Z. mays embryos via gene gun delivery [82] ; the methods used in both of these studies are
primarily throughput-limited by challenges in progeny regeneration. The challenge to realizing
efficient, stable gene editing in plants is twofold. First, plant germline cells cannot be transformed
by any current method (with the exception of Arabidopsis floral dip [83]) and therefore progeny
must be regenerated from embryogenic calli. Second, the cell wall imposes a rigid transport barrier
to biomolecule delivery, such that conventional delivery in plants is either destructive and ineffi¬
cient, or host-specific. Thus, the foremost limitation for broad-scale implementation of plant
genome editing originates from an inability to target germline cells, and the absence of an efficient
and species-independent bio-cargo delivery strategy. While engineered nuclease systems have
begun to reveal remarkable potential for the future of plant genome engineering, novel carriers are
required to overcome the restrictions of conventional delivery methods, but could also begin to
pave the way for efficient progeny regeneration or direct germline editing in plants.

NPs have begun to facilitate and enhance genome editing through efficient and targeted
delivery of plasmids, RNA, and RNPs [84], In mammalian cells, NPs are routinely used for
efficient, direct cytosolic/nuclear delivery of Cas-RNPs in many cell types [85], and RNP delivery
has been shown to greatly reduce off-target effects in comparison with plasmid-based CRISPR
systems [84], However, in plants, the cell wall has hindered the development of an analogous
system that can passively deliver genome editing cargo to mature plants and across species.
Thus, there remains much potential for designing NP carriers with diverse cargo loading
capabilities (DNA, RNA, proteins) and optimal geometry/chemistry to efficiently bypass the
cell wall and membranes in dense plant tissues without external aid. Previous work [51,67,68]
shows that some NP formulations are capable of passive internalization in planta with DNA,
RNA, or protein cargo. These NP scaffolds, namely CNTs, MSNs, and polymeric NPs, should
be further explored for delivering engineered nuclease systems to plants.

Concluding Remarks and Future Perspectives

Genetic engineering of plants has greatly accelerated scientific progress and paved the way for
crop variants with improved growth characteristics, disease and pest resistance, environmental
stress tolerance, and enhanced nutritional value. In parallel, advances in site-specific genome
editing technologies have optimized the precision with which genetic engineering of organisms
can be accomplished. However, conventional methods of plant genetic engineering and genome
editing are limited in scope. This is primarily due to the cell wall that imposes a barrier to efficient
delivery of biomolecules, which could potentially be overcome by NPs. Agrobacterium is a
preferred method for plant genetic transformation, but is only effective in a limited range of host
species and is an automatic trigger for regulatory oversight in the United States. Biolistic particle
delivery and PEG-transfection are effective, host-independent transformation methods, but
difficulties in regenerating healthy plant tissue and low-efficiency editing are severe drawbacks
to their broad-scale and high-throughput implementation. NPs have recently emerged as a novel
method of targeted biomolecule delivery in mammalian cells, especially for clinical applications.
However, exploration of nanocarriers for biomolecule delivery in plants remains a nascent field,
with much potential for the future of plant biotechnology and genome editing (see Outstanding
Questions). Preliminary studies show that NPs with proper surface chemistry and physical
properties analogous to those developed for animal systems are capable of delivering biomo¬
lecules to plants in vivo and in vitro with improvements over conventional methods. However, as of
yet, most nanocarriers in plants still require assistance from conventional methods (i.e., gene gun),

Outstanding Questions

Are there nanoparticle varieties yet to
be discovered for efficient biomolecule
delivery in plants, or do we lack knowl¬
edge of, or control over, optimal nano¬
particle modifications for applications
in plant systems?

Can we narrow the current design
space to a single nanoparticle type
with tunable functionalization for pas¬
sive delivery in plants, regardless of
cargo type, plant species, and tissue
variety?

How might we gain a better mechanis¬
tic understanding of nanoparticle inter¬
nalization into plant cells, and how can
we harness this knowledge towards
rational design of nanoparticles for a
range of biological delivery
applications?

Will challenges in biomolecule delivery
and progeny regeneration always
remain decoupled, or will nanoparticle
delivery enable significant increase in
throughput and efficiency of genetic
studies on plant regenerative biology
and stable transformation?

While genome editing by induced non-
homologous end-joining does not
invoke regulatory oversight in many
countries, how will genome edits intro¬
duced by homology-directed repair
(where integration of a repair template
is necessary) be classified from a leg¬
islative standpoint?

How can scientists, the public, and
regulatory bodies create a space for
open communication to address the
risks of introducing crop variants to
the environment, while continuing to
enable scientific progress and com¬
mercialization of sustainable and resil¬
ient crop variants?

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or are limited to in vitro studies. To our knowledge, the field of plant bioengineering has yet to fully
demonstrate a reliable strategy for NP-mediated passive biomolecule delivery to plants. To realize
the full scientific and humanitarian potential in genetic engineering of both model and crop species,
especially with the advent of nuclease-based genome editing, a promising focus will be to optimize
NPs as efficient and ubiquitous delivery vessels of diverse biomolecules, tunable across cargo
types, species, and tissues, for both transient and stable genetic engineering. However, because
germline transformation is currently limited to only one model plant species (Arabidopsis), even a
ubiquitous delivery strategy for precise genome editing would be limited by the success of
regenerating progeny from somatic tissue. A remarkable, yet conceivable, future accomplishment
of NP delivery in plants could be enablement of unprecedented, highly parallel genetic studies that
elucidate the precedents for success in tissue regeneration, and the direct manipulation of
germline plant cells.

References

1. Ray, D.K. et al. (2013) Yield trends are insufficient to double
global crop production by 2050. PLoS One 8, e66428

2. Zhao, C. et al. (2017) Temperature increase reduces global
yields of major crops in four independent estimates. Proc. Natl.
Acad. Sci. U. S. A. 114, 9326-9331

3. Abdallah, N.A. et al. (2015) Genome editing for crop improvement:
challenges and opportunities. GM Crops Food 6,183-205

4. Azencott, H.R. et al. (2007) Influence of the cell wall on intracel¬
lular delivery to algal cells by electroporation and sonication.
Ultrasound Med. Biol. 33, 1805-1817

5. Nyaboga, E. et al. (2014) Agrobacterium - med iated genetic
transformation of yam ( Dioscorea rotundata ): an important tool
for functional study of genes and crop improvement. Front. Plant
Sci. 5, 463

6. Gelvin, S.B. (2017) Integration of Agrobacterium T -DNA into the
plant genom e.Annu. Rev. Genet. 51, 195-217

7. Gleba, Y. et al. (2005) Magnifection—a new platform for
expressing recombinant vaccines in plants. Vaccine 23,
2042-2048

8. Stoddard, T. et al. Cellectis. Agrobacterium- mediated genome
modification without t-dna integration, WO2016125078A1

9. Lowe, K. et al. (2016) Morphogenic regulators baby boom and
Wuschel improve monocot transformation. Plant Cell 28,1998-2015

10. Zeng, X. et al. (2014) Nanoparticle-directed sub-cellular locali¬
zation of doxorubicin and the sensitization breast cancer cells by
circumventing GST-mediated drug resistance. Biomaterials 35,
1227-1239

11. Dekiwadia, C.D. et al. (2012) Peptide-mediated cell penetration
and targeted delivery of gold nanoparticles into lysosomes. J.
Pept. Sci. 18, 527-534

12. Mout, R. et al. (2017) Direct cytosolic delivery of CRISPR/Cas9-
ribonucleoprotein for efficient gene editing. ACS Nano 11,
2452-2458

13. Zhong, J. et al. (2015) A smart polymeric platform for multistage
nucleus-targeted anticancerdrug delivery. Biomaterials 65,43-55

14. Mao, H.-Q. et al. (2001) Chitosan-DNA nanoparticles as gene
carriers: synthesis, characterization and transfection efficiency.
J. Control Release 70, 399-421

15. Davis, M.E. et al. (2010) Evidence of RNAi in humans from
systemically administered siRNA via targeted nanoparticles.
Nature 464, 1067-1070

16. Yan, M.etal. (2010) A novel intracellular protein delivery platform
based on single-protein nanocapsules. Nat. Nanotechnol. 5,
48-53

17. Sengupta, S.etal. (2005) Temporal targeting of tumour cells and
neovasculature with a nanoscale delivery system. Nature 436,
568-572

18. Swyer, T. et al. (2014) Nanoparticle oxygen delivery to the
ischemic heart. Perfusion 29, 539-543

19. Haham, M. et al. (2012) Stability and bioavailability of vitamin D
nanoencapsulated in casein micelles. Food Fund. 3, 737

20. Karimi, M. et al. (2016) Smart micro/nanoparticles in stimulus-
responsive drug/gene delivery systems. Chem. Soc. Rev. 45,
1457-1501

21. Ta, T. et al. (2014) Localized delivery of doxorubicin in vivo from
polymer-modified thermosensitive liposomes with MR-guided
focused ultrasound-mediated heating. J. Control. Release 194,
71-81

22. Mu, Q. etal. (2015) Stable and efficient paclitaxel nanoparticles
for targeted glioblastoma therapy. Adv. Healthc. Mater. 4,
1236-1245

23. Svenson, S. et al. (2016) Tumor selective silencing using an
RNAi-conjugated polymeric nanopharmaceutical. Mol. Pharm.
13, 737-747

24. Hou, X.-F. et al. (2015) Enzyme-responsive protein/polysaccha¬
ride supramolecular nanoparticles. Soft Matter 11, 2488-2493

25. Wang, M. et al. (2016) Efficient delivery of genome-editing
proteins using bioreducible lipid nanoparticles. Proc. Natl. Acad.
Sci. U. S. A 113, 2868-2873

26. Hoshino, A. et al. (2004) Quantum dots targeted to the assigned
organelle in living cells. Microbiol. Immunol. 48, 985-994

27. Lai, C.Y. et al. (2003) A mesoporous silica nanosphere-based
carrier system with chemically removable CdS nanoparticle
caps for stimuli-responsive controlled release of neurotransmit¬
ters and drug molecules. J. Am. Chem. Soc. 125, 4451-4459

28. Vijayaraghavan, K. and Ashokkumar, T. (2017) Plant-mediated
biosynthesis of metallic nanoparticles: a review of literature,
factors affecting synthesis, characterization techniques and
applications. J. Environ. Chem. Eng. 5, 4866-4883

29. Baker, S. et al. (2016) Nanoagroparticles emerging trends and
future prospect in modern agriculture system. Environ. Toxicol.
Pharmacol. 53, 10-17

30. Tripathi, D.K. et al. (2017) An overview on manufactured nano¬
particles in plants: uptake, translocation, accumulation and
phytotoxicity. Plant Physiol. Biochem. 110, 2-1 2

31. Zuverza-Mena, N. et al. (2017) Exposure of engineered nano¬
materials to plants: insights into the physiological and biochem¬
ical responses-a review. Plant Physiol. Biochem. 1 10, 236-264

32. Hussain, H.l. et al. (2013) Mesoporous silica nanoparticles as a
biomolecule delivery vehicle in plants. J. Nanoparticle Res. 15, 1 676

33. Liu, Q. etal. (2009) Carbon nanotubes as molecular transporters
for walled plant cells. Nano Lett. 9, 1007-1010

34. Koo, Y. et al. (2015) Fluorescence reports intact quantum dot
uptake into roots and translocation to leaves of Arabidopsis
thaliana and subsequent ingestion by insect herbivores. Environ.
Sci. Technol. 49, 626-632

35. Kurepa, J. et al. (2010) Uptake and distribution of ultrasmall
anatase Ti0 2 alizarin red S nanoconjugates in Arabidopsis thali¬
ana. Nano Lett. 10, 2296-2302

Trends in Biotechnology, September 2018, Vol. 36, No. 9

895

Trends in Biotechnology

Cell

REVIEWS

36. Gonzalez-Melendi, P. etal. (2008) Nanoparticles as smart treat¬
ment-delivery systems in plants: assessment of different tech¬
niques of microscopy for their visualization in plant tissues. Ann.
Bot. 101, 187-195

37. Larue, C. et al. (2012) Accumulation, translocation and impact of
Ti02 nanoparticles in wheat ( Triticum aestivum spp.): influence
of diameter and crystal phase. Sci. Total Environ. 431,1 97-208

38. Wang, P. et al. (2016) Nanotechnology: a new opportunity in
plant sciences. Trends Plant Sci. 21, 699-712

39. Eichert, T. and Goldbach, H.E. (2008) Equivalent pore radii of
hydrophilic foliar uptake routes in stomatous and astomatous
leaf surfaces - further evidence for a stomatal pathway. Physiol.
Plant 132, 491-502

40. Eichert, T. et al. (2008) Size exclusion limits and lateral hetero¬
geneity of the stomatal foliar uptake pathway for aqueous sol¬
utes and water-suspended nanoparticles. Physiol. Plant 134,
151-160

41. Schwab, F. et al. (2015) Barriers, pathways and processes for
uptake, translocation and accumulation of nanomaterials in
plants - critical review. Nanotoxicology 10, 1-22

42. Ma, X. et al. (2010) Interactions between engineered nanopar¬
ticles (ENPs) and plants: phytotoxicity, uptake and accumula¬
tion. Sci. Total Environ. 408, 3053-3061

43. Larue, C. et al. (2014) Foliar exposure of the crop Lactuca sativa
to silver nanoparticles: evidence for internalization and changes
in Ag speciation. J. Hazard. Mater. 264, 98-106

44. Albanese, A. et al. (2012) The effect of nanoparticle size, shape,
and surface chemistry on biological systems. Annu. Rev.
Biomed. Eng. 14, 1-16

45. Torney, F. et al. (2007) Mesoporous silica nanoparticles deliver
DNA and chemicals into plants. Nat. Nanotechnol. 2, 295-300

46. Zhu, Z.-J. et al. (2012) Effect of surface charge on the uptake
and distribution of gold nanoparticles in four plant species.
Environ. Sci. Technol. 46, 12391-12398

47. Fan, L. et al. (2015) Endocytosis and its regulation in plants.
Trends Plant Sci. 20, 388-397

48. Serag, M.F. et al. (2013) Nanobiotechnology meets plant cell
biology: carbon nanotubes as organelle targeting nanocarriers.
RSC Adv. 3, 4856

49. Serag, M.F. et al. (2011) Trafficking and subcellular localization
of multiwalled carbon nanotubes in plant cells. ACS Nano 5,
493-499

50. Selby, L.l. et al. (2017) Nanoescapology: progress toward under¬
standing the endosomal escape of polymeric nanoparticles. Wiley
Interdiscip. Rev. Nanomed. Nanobiotechnol. 9, el 452

51. Chang, F.-P. et al. (2013) A simple plant gene delivery system
using mesoporous silica nanoparticles as carriers. J. Mater.
Chem. B 1, 5279

52. Zhao, H. et al. (2016) Polymer-based nanoparticles for protein
delivery: design, strategies and applications. J. Mater. Chem. B
4060, 4060-4071

53. Lallana, E. et al. (2012) Click chemistry for drug delivery nano¬
systems. Pharm. Res. 29, 1-34

54. Lee, K. et al. (201 7) Nanoparticle delivery of Cas9 ribonucleo-
protein and donor DNA in vivo induces homology-directed DNA
repair. Nat. Biomed. Eng. 1, 889-901

55. Slowing, l.l. et al. (2008) Mesoporous silica nanoparticles as
controlled release drug delivery and gene transfection carriers.
Adv. Drug Deliv. Rev. 60, 1278-1 288

56. Bimbaum, K.D. and Sanchez Alvarado, A. (2008) Slicing across
kingdoms: regeneration in plants and animals. Cell 132,697-710

57. Raliya, R. et al. (2016) Quantitative understanding of nanoparti¬
cle uptake in watermelon plants. Front. Plant Sci. 7, 1288

58. Martin-Ortigosa, S. et al. (2014) Mesoporous silica nanoparticle-
mediated intracellular Cre protein delivery for maize genome
editing via loxP site excision. Plant Physiol. 164, 537-547

59. Martin-Ortigosa, S. etal. (2012) Gold functionalized mesoporous
silica nanoparticle mediated protein and DNA codelivery to plant
cells via the biolistic method. Adv. Funct. Mater. 22, 3576-3582

60. Liu, J. etal. (2008) Preparation of fluorescence starch-nanopar-
ticle and its application as plant transgenic vehicle. J. Cent.
South Univ. Technol. (Engl. Ed.) 15, 768-773

61. Hao, Y. et al. (201 3) Magnetic gold nanoparticles as a vehicle for
fluorescein isothiocyanate and DNA delivery into plant cells.
Botany 91, 457-466

62. Finiuk, N. et al. (2017) Investigation of novel oligoelectrolyte
polymer carriers for their capacity of DNA delivery into plant
cells. Plant Cell. Tissue Organ Cult. 1 31, 27-39

63. Zhao, X. et al. (2017) Pollen magnetofection for genetic modifi¬
cation with magnetic nanoparticles as gene carriers. Nat. Plants
3, 956-964

64. Naqvi, S. et al. (2012) Calcium phosphate nanoparticle medi¬
ated genetic transformation in plants. J. Mater. Chem. 22, 3500

65. Pasupathy, K. etal. (2008) Direct plant gene delivery with a poly
(amidoamine) dendrimer. Biotechnol. J. 3, 1078-1082

66. Burlaka, O.M. et al. (2015) Plant genetic transformation using
carbon nanotubes for DNA delivery. Cytol. Genet. 49, 349-357

67. Jiang, L. etal. (2014) Systemic gene silencing in plants triggered
by fluorescent nanoparticle-delivered double-stranded RNA.
Nanoscale 6, 9965

68. Demirer, G.S. et al. (2018) High aspect ratio nanomaterials
enable biomolecule delivery and transgene expression or silenc¬
ing in mature plants. bioRxiv Published online January 30,2018.
http://dx.doi.Org/1 0.1101 /1 79549

69. Ma, X. et al. (2015) A robust CRISPR/Cas9 system for conve¬
nient, high-efficiency multiplex genome editing in monocot and
dicot plants. Mol. Plant 8, 1274-1 284

70. Osakabe, Y. and Osakabe, K. (2015) Genome editing with
engineered nucleases in plants. Plant Cell Physiol. 56, 389-400

71. Bortesi, L. and Fischer, R. (201 5) The CRISPR/Cas9 system for
plant genome editing and beyond. Biotechnol. Adv. 33, 41-52

72. Arora, L. and Narula, A. (2017) Gene editing and crop improve¬
ment using CRISPR-Cas9 system. Front. Plant Sci. 8, 1932

73. Feng, Z. et al. (2014) Multigeneration analysis reveals the inheri¬
tance, specificity, and patterns of CRISPR/Cas-induced gene
modifications in Arabidopsis. Proc. Natl. Acad. Sci. U.S. A. Ill,
4632-4637

74. Jiang, W. et al. (2014) Efficient CRISPR/Cas9-mediated gene
editing in Arabidopsis thaliana and inheritance of modified genes
in the T2 and T3 generations. PLoS One 9, e99225

75. James, C. (2016) Global Status of Commercialized Biotech/GM
Crops: 2016. ISAAA Brief No. 52, ISAAA

76. Camacho, A. et al. (2014) Genetically engineered crops that fly
under the US regulatory radar. Nat. Biotechnol. 32,1087-1091

77. Jones, H.D. (201 5) Regulatory uncertainty over genome editing.
Nat. Plants 1, 1 4011

78. Parliament, E.U. (2001) Directive 2001/18/EC of the European
Parliament and of the Council of 12 March 2001 on the deliberate
release into the environment of genetically modified organisms and
repealing Council Directive. Off. J. Eur. Comm. L. 106, 1-39

79. Bobek, M. (2018) Opinion of Advocate General Bobek in Case
C-528/16, Court of Justice of the European Union

80. Ishii, T. and Araki, M. (2017) A future scenario of the global
regulatory landscape regarding genome-edited crops. GM
Crops Food 8, 44-56

81. Woo, J.W. et al. (2015) DNA-free genome editing in plants with
preassembled CRISPR-Cas9 ribonucleoproteins. Nat. Biotech¬
nol. 33, 1162-1164

82. Svitashev, S. et al. (2016) Genome editing in maize directed by
CRISPR-Cas9 ribonucleoprotein complexes. Nat. Commun. 7,
13274

83. Zhang, X. et al. (2006) Agrobacterium- mediated transformation
of Arabidopsis thaliana using the floral dip method. Nat. Protoc.
1, 641

84. Liu, C. et al. (2017) Delivery strategies of the CRISPR-Cas9
gene-editing system for therapeutic applications. J. Control.
Release 266, 17-26

896 Trends in Biotechnology, September 2018, Vol. 36, No. 9

Trends in Biotechnology

Cell

REVIEWS

85. Mout, R. etal. (2017) Direct cytosolic delivery of CRISPR/Cas9-
ribonucleoprotein for efficient gene editing. ACS Nano 11,
2452-2458

86. Antonelli, N.M. and Stadler, J. (1990) Genomic DNA can be
used with cationic methods for highly efficient transformation of
maize protoplasts. Theor. Appl. Genet. 80, 395-401

87. Ragelle, H. et al. (2014) Chitosan nanoparticles for siRNA deliv¬
ery: optimizing formulation to increase stability and efficiency. J.
Control. Release 176, 54-63

88. Zuris, J.A. et al. (2015) Cationic lipid-mediated delivery of pro¬
teins enables efficient protein-based genome editing in vitro and
in vivo. Nat. Biotechnol. 33, 73-80

89. Pantarotto, D. etal. (2004) Functionalized carbon nanotubes for
plasmid DNA gene delivery. Angew. Chem. Int. Ed. Engl. 43,
5242-5246

90. Kam, N.W. et al. (2005) Functionalization of carbon nanotubes
via cleavable disulfide bonds for efficient intracellular delivery of
siRNA and potent gene silencing. J. Am. Chem. Soc. 127,
12492-12493

91. Kneuer, C. et al. (2000) A nonviral DNA delivery system based on
surface modified silica-nanoparticles can efficiently transfect
cells in vitro. Bioconjug. Chem. 11, 926-932

92. Chen, A.M. et al. (2009) Co-delivery of doxorubicin and Bcl-2
siRNA by mesoporous silica nanoparticles enhances the effi¬
cacy of chemotherapy in multidrug-resistant cancer cells. Small
5, 2673-2677

93. Boussif, O. et al. (1995) A versatile vector for gene and oligonu¬
cleotide transfer into cells in culture and in vivo: polyethyleni-
mine. Biochemistry 92, 7297-7301

94. Negrutiu, I. et al. (1987) Hybrid genes in the analysis of transfor¬
mation conditions. Plant Mol. Biol. 8, 363-373

95. Aigner, A. et al. (2002) Delivery of unmodified bioactive ribozymes
by an RNA-stabilizing polyethylenimine (LMW-PEI) efficiently down-
regulates gene expression. Gene Ther. 9,1700-1707

96. Silva, A.T. et al. (2010) Conjugated polymer nanoparticles for
effective siRNA delivery to tobacco BY-2 protoplasts. BMC
Plant Biol. 10, 291

97. Sgolastra, F. et al. (2017) Sequence segregation improves non-
covalent protein delivery. J. Control. Release 254, 131-136

98. Mizutani, O. etal. (2012) Modified Cre-loxP recombination in
Aspergillus oryzae by direct introduction of Cre recombinase
for marker gene rescue. Appl. Environ. Microbiol. 78, 4126-
4133

99. Sun, W. et al. (2015) Self-assembled DNA nanoclews for the
efficient delivery of CRISPR-Cas9 for genome editing. Angew.
Chem. Int. Ed. Engl. 54, 12029-12033

100. Sandhu, K.K. et al. (2002) Gold nanoparticle-mediated trans¬
fection of mammalian cells. Bioconjug. Chem. 13, 3-6

101. Lee, J.-H. et al. (2009) All-in-one target-cell-specific magnetic
nanoparticles for simultaneous molecular imaging and siRNA
delivery. Angew. Chem. Int. Ed. Engl. 48, 4174-4179

102. Mout, R. et al. (2017) General strategy for direct cytosolic protein
delivery via protein-nanoparticle co-engineering. ACS Nano 11 ,
6416-6421

103. Gao, W. et al. (2018) Risk analysis for genome editing-derived
food safety in China. Food Control 84, 128-137

104. McCarthy, M. (2018) Genetic modification laws set for shake-up,
with health and agriculture research industries to benefit (inter¬
view with Raj Bhula). ABC Australia, http://www.abc.net.au/
news/rural/2018-01 -19/gene-tech-regulator-proposes-
shakeup-for-genetic-modification/9341354

105. Schuttelaar & Partners (2015) The regulatory status of New
Breeding Techniques in countries outside the European Union.
http://www.nbtplatform.org/background-documents/
rep-regulatory-status-of-nbts-oustide-the-eu-june-2015.pdf

106. Waltz, E. (2018) With a free pass, CRISPR-edited plants reach
market in record time. Nat. Biotechnol. 36, 6-7

107. Bevan, M. (1984) Binary Agrobacterium vectors for plant trans¬
formation. Nucleic Acids Res. 12, 8711-8721

108. Klein, T.M. et al. (1987) High-velocity microprojectiles for deliv¬
ering nucleic acids into living cells. Nature 327, 70-73

109. Tiwari, J.N. et al. (2012) Zero-dimensional, one-dimensional,
two-dimensional and three-dimensional nanostructured materi¬
als for advanced electrochemical energy devices. Prog. Mater.
Sci. 57, 724-803

110. Khan, I. etal. (2017) Nanoparticles: properties, applications and
toxicities. Arab. J. Chem. Published online May 18,2017. http://
dx.doi.org/1 0.1 01 6/J.ARABJC.2017.05.01 1

111. Wang, L. et al. (2016) In vivo delivery systems for therapeutic
genome editing. Int. J. Mol. Sci. 17, E626

112. Kim, l.-Y. etal. (2015) Toxicity of silica nanoparticles depends on
size, dose, and cell type. Nanomedicine 11,1 407-141 6

113. Shang, L. etal. (2014) Engineered nanoparticles interacting with
cells: size matters. J. Nanobiotechnol. 1 2, 5

114. Petersen, M.A. et al. (2013) Bioresorbable polymersomes for
targeted delivery of cisplatin. Bioconjug. Chem. 24, 533-543

115. Tekedereli, I. et al. (2013) Therapeutic silencing of Bcl-2 by
systemically administered siRNA nanotherapeutics inhibits
tumor growth by autophagy and apoptosis and enhances the
efficacy of chemotherapy in orthotopic xenograft models of ER
(-) and ER (+) breast cancer. Mol. Ther. Nucleic Acids 2, el 21

116. Yin, P.T. et al. (2014) Combined magnetic nanoparticle-based
microRNA and hyperthermia therapy to enhance apoptosis in
brain cancer cells. Small 10, 4106-4112

117. Zhang, K. et al. (2012) Antibody-linked spherical nucleic acids
for cellular targeting. J. Am. Chem. Soc. 134, 16488-16491

118. Smith, B.R. et al. (2014) Selective uptake of single-walled car¬
bon nanotubes by circulating monocytes for enhanced tumour
delivery. Nat. Nanotechnol. 9, 481-487

119. Chen, P.-C. et al. (2016) Polyelemental nanoparticle libraries.
Science 352, 1565-1569

120. Lu, J. et al. (2014) Toward atomically-precise synthesis of sup¬
ported bimetallic nanoparticles using atomic layer deposition.
Nat. Commun. 5, 3264

121. Hu, X. et al. (2013) Polyprodrug amphiphiles: hierarchical
assemblies for shape-regulated cellular internalization, traffick¬
ing, and drug delivery. J. Am. Chem. Soc. 135, 17617-17629

122. Agudelo, D. etal. (2016) tRNA conjugation with chitosan nanopar¬
ticles: an AFM imaging study. Int. J. Biol. Macromol. 85,150-156

123. Shi, Y. et al. (2015) Complete regression of xenograft tumors
upon targeted delivery of paclitaxel via it-it stacking stabilized
polymeric micelles. ACS Nano 9, 3740-3752

124. Kim, Y.G. et al. (1996) Hybrid restriction enzymes: zinc finger
fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. U. S. A.
93, 1156-1160

125. Jinek, M. etal. (2012) A programmable dual-RNA-guided DNA
endonuclease in adaptive bacterial immunity. Science 337,
816-821

126. Cong, L. et al. (2013) Multiplex genome engineering using
CRISPR/Cas systems. Science 339, 819-823

127. Boch, J. etal. (2009) Breaking the code of DNA binding speci¬
ficity of TAL-type III effectors. Science 326, 1509-1512

128. Moscou, M.J. and Bogdanove, A.J. (2009) A simple cipher
governs DNA recognition by TAL effectors. Science 326, 1 501

129. Wong, M.H. et al. (2016) Lipid exchange envelope penetration
(LEEP) of nanoparticles for plant engineering: a universal locali¬
zation mechanism. Nano Lett. 16, 1161-1172

130. Giraldo, J.P. et al. (2014) Plant nanobionics approach to aug¬
ment photosynthesis and biochemical sensing. Nat. Mater. 13,
400-408

131. USDA (2018) Secretary Perdue Issues Usda Statement on Plant
Breeding Innovation, Press Release No. 0070.18

132. National Technical Biosafety (2018) Normative Resolution No.
16/2018, CTNBio

133. Servicio Agricola y Ganadero (2017) Applicability of Resolution
No. 1523/2001 on Propagation Material Developed by New
Plant Breeding Techniques, SAG

Trends in Biotechnology, September 2018, Vol. 36, No. 9

897