MINI REVIEW

published: 24 January 2022

doi: 10.3389/fgeed.2022.830178

Advances in Delivery Mechanisms of

CRISPR Gene-Editing Reagents in
Plants
Larissa C. Laforest 1 and Satya Swathi Nadakuduti 1,2*
1
Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, United States, 2Department of Environmental
Horticulture, University of Florida, Gainesville, FL, United States

Gene-editing by CRISPR/Cas systems has revolutionized plant biology by serving as a

functional genomics tool. It has tremendously advanced plant breeding and crop
improvement by accelerating the development of improved cultivars, creating genetic
variability, and aiding in domestication of wild and orphan crops. Gene-editing is a rapidly
evolving field. Several advancements include development of different Cas effectors with
increased target range, efficacy, and enhanced capacity for precise DNA modifications
with base editing and prime editing. The existing toolbox of various CRISPR reagents
facilitate gene knockouts, targeted gene insertions, precise base substitutions, and
multiplexing. However, the major challenge in plant genome-editing remains the
efficient delivery of these reagents into plant cells. Plants have larger and more
Edited by: complex genome structures compared to other living systems due to the common
Jimmy R. Botella,
The University of Queensland,
occurrence of polyploidy and other genome re-arrangements. Further, rigid cell walls
Australia surrounding plant cells deter the entry of any foreign biomolecules. Unfortunately, genetic
Reviewed by: transformation to deliver gene-editing reagents has been established only in a limited
Yanpeng Wang, number of plant species. Recently, there has been significant progress in CRISPR reagents
Institute of Genetics and
Developmental Biology (CAS), China delivery in plants. This review focuses on exploring these delivery mechanisms categorized
Phanikanth Jogam, into Agrobacterium-mediated delivery and breakthroughs, particle bombardment-based
Kakatiya University, India
delivery of biomolecules and recent improvements, and protoplasts, a versatile system for
*Correspondence:
Satya Swathi Nadakuduti
gene-editing and regeneration in plants. The ultimate goal in plant gene-editing is to
s.nadakuduti@ufl.edu establish highly efficient and genotype-independent reagent delivery mechanisms for
editing multiple targets simultaneously and achieve DNA-free gene-edited plants at scale.
Specialty section:
This article was submitted to Keywords: gene-editing, CRISPR-Cas9, gene targeting, agrobacterium-mediated transformation, biolistics,
Genome Editing in Plants, protoplasts, nanoparticles
a section of the journal
Frontiers in Genome Editing
INTRODUCTION
Received: 06 December 2021
Accepted: 05 January 2022
CRISPR/Cas9 derived from Streptococcus pyogenes (SpCas9) is the most used gene-editing reagent in
Published: 24 January 2022
plants. Unlike its predecessors, zinc finger nucleases (Gao et al., 2010; Osakabe et al., 2010; Zhang
Citation: et al., 2010) and Transcription Activator-like Effector Nucleases (TALENs) (Cermak et al., 2011; Li
Laforest LC and Nadakuduti SS (2022)
et al., 2012), which rely on protein-based DNA recognition mechanisms, CRISPR/Cas systems are
Advances in Delivery Mechanisms of
CRISPR Gene-Editing Reagents
RNA-guided endonucleases. The resulting versatility, simplicity, and cost-effectiveness brought
in Plants. about by CRISPR led to significant advances in plant genome engineering. In the CRISPR/Cas9
Front. Genome Ed. 4:830178. system, a chimeric single guide RNA (sgRNA), formed by fusion of CRISPR RNA (crRNA) and a
doi: 10.3389/fgeed.2022.830178 trans-activating crRNA (tracrRNA), directs the SpCas9 nuclease to generate blunt double-strand

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Laforest and Nadakuduti Delivery of CRISPR Reagents into Plants

breaks (DSBs) at the genomic DNA target site three bases González et al., 2020, 2021; Sidorov et al., 2021; Zhang et al., 2021).
upstream of Protospacer Adjacent Motif (PAM) sequence of This review will focus on various advances in CRISPR delivery
‘NGG’ (Jinek et al., 2012). The DSBs are repaired either by mechanisms in plants categorized into Agrobacterium-mediated
error-prone non-homologous end joining (NHEJ) resulting in delivery and breakthroughs for efficient and heritable mutagenesis
insertion-deletion mutations (InDels) leading to gene knock-out and gene targeting (GT) in plants; particle bombardment mediated
or by precise, albeit inefficient, homology-directed repair (HDR) delivery of DNA, RNA, and protein biomolecules for plant gene-
through which DNA insertions are achieved by providing an editing, and protoplast transfection and regeneration of transgene-
external donor repair template (DRT) (Atkins and Voytas, 2020; free gene-edited plants. The ultimate goal in plant gene-editing is to
Dong and Ronald, 2021). In addition to Cas9, multiple other Cas establish highly efficient and species non-specific reagent delivery
variants with alternative PAM requirements have been identified mechanisms for editing multiple targets simultaneously and
and successfully utilized in plants expanding the range of DNA achieve DNA-free gene-edited plants at scale.
recognition (Kaya et al., 2016; Jia et al., 2017; Steinert et al., 2017;
Zhang Y. et al., 2019; Veillet et al., 2020). Furthermore, base Breakthroughs in Agrobacterium-Mediated
editors (BEs), including cytosine, adenine, and glycosylase BEs
can precisely convert one target DNA base to another without a
Delivery of CRISPR Reagents for Efficient
DSB. BEs rely on base excision repair, facilitating both transition and Heritable Mutagenesis and Gene
and transversion mutations, and are increasingly being used in Targeting
plant systems (Shimatani et al., 2017; Zong et al., 2017; Shan and Agrobacterium-mediated genetic transformation remains the
Voytas, 2018; Zhang R. et al., 2019; Li et al., 2020; Zhao et al., principal means of delivering gene-editing reagents including
2020). In addition, prime editing (PE), a versatile “search-and- CRISPR/Cas variants, base editing and prime editing reagents,
replace” strategy, was also developed (Anzalone et al., 2019) and into plants (Lin et al., 2020). This method typically involves
optimized in plants (Butt et al., 2020; Lin et al., 2020; Tang et al., inoculating the explants with Agrobacterium expressing gene-
2020; Xu et al., 2020). PEs copy desired edits incorporated into the editing cassettes integrated into its T-DNA (Figure 1A). Upon
PE gRNA (PegRNA) directly into the genomic DNA by target infection of plant cells, the T-DNA containing the CRISPR
primed reverse transcription. With this existing toolbox of cassette likely gets integrated into the host plant genome
various CRISPR reagents, the biggest challenge in plant leading to stable genetic transformation. Transgene-free gene-
genome-editing remains to be the efficient delivery of these editing has been achieved by transient expression of CRISPR
reagents into plant cells. reagents by regenerating events without employing selection
Several plant species have larger and more complex genome (Chen et al., 2018). This is important for generating edited
structures compared to other living systems. Polyploidy and plants with no foreign DNA to avoid regulatory oversight and
genomic rearrangements are common in plants, and rigid cell for vegetatively propagated plants, where segregating out the
walls surrounding the plant cells deter the entry of any foreign integrated transgene by making crosses is not feasible.
biomolecules. Furthermore, genetic transformation to deliver Agrobacterium has a limited host range, and several plant
transgenes has only been established in a limited number of species are recalcitrant to Agrobacterium-mediated
plant species and genotypes within each species. This is transformation. Furthermore, the regeneration process
currently considered the biggest bottleneck in plant genome involving tissue culture leads to undesirable somaclonal
engineering. Gene-editing reagents are delivered into plants, variations in edited lines. Floral dip method of transformation,
most commonly as plasmid DNA constructs and predominantly only amenable to Arabidopsis thaliana and some related species
by Agrobacterium-mediated transformation or particle (Clough and Bent, 1998; Lu and Kang, 2008) can generate
bombardment are summarized in tables recently (Sandhya transformed seeds, bypassing the need for regeneration. Other
et al., 2020; Ghogare et al., 2021). In both methods, the plasmid means of avoiding regeneration process include, the use of A.
DNA with CRISPR/Cas expression cassette is likely to get rhizogenes, which can drastically reduce time between reagent
integrated into a random genomic site(s), leading to continued delivery and mutation evaluation, as well as widening the range of
expression in host genomes. With the revision of the regulatory species transformed (Yoshida et al., 2015; Triozzi et al., 2021).
landscape of gene-edited lines in the US (USDA press release1) and
across the world (Nadakuduti et al., 2018; Lassoued et al., 2021), Co-delivery of Developmental Regulators
developing gene-edited lines without integrating foreign genomic
DNA into the host plant is gaining prominence. DNA-free delivery
with CRISPR Reagents via Agrobacterium
of in vitro transcripts (IVTs), pre-assembled ribonucleoprotein to Expedite and Improve Gene-editing
complexes (RNPs), or transient expression of plasmid DNA Efficiency in Plants
constructs delivered into protoplasts, and subsequent Developmental regulators (DRs) are genes involved in dictating
regeneration of gene-edited plants have been successful in meristem identity in plants. Ectopic expression of DRs in plants
several plant species (Liang et al., 2017; Andersson et al., 2018; has resulted in somatic embryogenesis, formation of embryos
from somatic tissues (Lowe et al., 2016). Overexpression of DRs
such as Baby Boom (Bbm) and Wuschel2 (Wus2) enhanced
1
https://www.usda.gov/media/press-releases/2018/03/28/secretary-perdue-issues- regeneration and transformation frequency in both dicot and
usda-statement-plant-breeding-innovation. monocot plants (Srinivasan et al., 2007; Deng et al., 2009; Lowe

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Laforest and Nadakuduti Delivery of CRISPR Reagents into Plants

FIGURE 1 | Agrobacterium mediated delivery of CRISPR gene-editing reagents in plants. (A) Conventional Agrobacterium-mediated transformation consisting of
T-DNA carrying expression cassette for Streptococcus pyogenes Cas9 and kanamycin resistance gene NptII, both driven by cauliflower mosaic virus 35S promoter
(CaMV 35S), and a single guide RNA (sgRNA) driven by the U6 promoter targeting the phytoene desaturase (PDS) gene. Explants are infected and co-cultivated with
agrobacterium cultures, then placed on selective media for callus induction and regeneration. The resulting gene-edited lines are transgenic and have
photobleaching phenotype. (B) A. tumefaciens T-DNA harboring sgRNA targeting PDS along with plant developmental regulators (DRs) Wuschel2 (Wus2) driven by
nopaline synthase (nos) promoter, and isopentenyl synthase (ipt) driven by 35S promoter are injected in Cas9 expressing soil grown plants after meristem removal. DRs
induce new meristems at the wounded site and pds phenotype is visible in edited meristems. Offspring from seeds produced on de novo meristems show segregation
for photobleaching phenotype. Maher et al. (2020) found that de novo meristems with bi-allelic mutations did not set viable seeds, and edited offspring are only recovered
from meristems exhibiting mosaicism. (C) Tobacco rattle virus (TRV) is a bipartite RNA virus: TRV1 encodes replicases RNA-dependent RNA polymerase (RDRP), a
movement protein (MP), a 16 KDa cysteine rich protein, and a ribozyme (RZ) and can independently replicate itself and move within the plant during infection. TRV2,
encodes a coat protein (CP) and, a sgRNA targeting PDS fused to Flowering locus T (FT) driven by a pea early browning virus promoter (PeBv). FT is a mobile RNA which
increases infection spread by reaching the shoot apical meristem (SAM). TRV1 and TRV2 are introduced into T-DNA regions of agrobacterium and infiltrated into 35S:
Cas9 transgenic plants. Systemic infection of the plant leads to editing of somatic and germline cells thereby increasing heritability. Infected plants exhibit photobleaching
and pds phenotype segregates in progeny. (D) Sonchus yellow net rhabdovirus (SYNV) is a negative-strand RNA virus encoding the core structural proteins
nucleoprotein (N), phosphoprotein (P), and the large RNA polymerase (L), and Sc4 protein, matrix protein (M), glycoprotein (G) which are involved in cell-to-cell
(Continued )

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Laforest and Nadakuduti Delivery of CRISPR Reagents into Plants

FIGURE 1 | movement. The viral cassette is manipulated to express a Cas9 nuclease and a tRNA-gRNA-tRNA (tgtRNA) which is processed to release the sgRNA
targeting the PDS gene by tRNA processing enzymes. Soil grown plants are infiltrated with agrobacterium harboring the SYNV plasmid. Explants from systemically
infected leaves are prepared and placed on non-selective regeneration medium. Regenerants are then transferred to soil. Since Cas9 is delivered virally and SYNV does
not integrate into the host genome nor have a DNA-phase, the resulting plants are non-transgenic.

et al., 2016). This phenomenon was leveraged to induce de novo Enhancing Gene Targeting by
meristems in somatic tissues by injecting Agrobacterium cultures Agrobacterium-Mediated Delivery of
co-delivering DRs and gene-editing cassettes directly into soil-
grown plants (Figure 1B). Wus2 and Isopentenyl transferase (Ipt),
CRISPR Reagents
GT includes precise DNA modifications based on HDR using a
when co-delivered with gene-editing reagents by Agrobacterium
DRT with homology to the host target DNA on both ends. DSBs
injections into dicot plants generated meristems in somatic
generated by CRISPR/Cas reagents initiate the cell repair
tissues with edits, enabling tissue culture free gene-editing
process. However, NHEJ is the predominant repair
(Maher et al., 2020). This can potentially be a high throughput
mechanism in plants cells to repair these DSBs as HDR is
and less tedious approach when Cas9 expressing plants are
not active throughout the cell cycle. This, in combination
generated. Alternatively, Growth Regulating Factor (GRF) and
with inefficient delivery of DRT to facilitate HDR, make GT
GRF-interacting Factor (GIF) cofactor when expressed as GRF4-
very inefficient in plants. To increase GT frequencies, viral
GIF chimera increased the speed and efficiency of regeneration
replicons including Bean Yellow Dwarf Virus (BeYDV)
(Debernardi et al., 2020). Co-delivery of GRF4-GIF chimera and
(Baltes et al., 2014; Butler et al., 2015; Čermák et al., 2015;
CRISPR-Cas9 on the same T-DNA increased the regeneration
Cermak et al., 2017; Wang et al., 2017; Vu et al., 2020) or wheat
efficiency in both monocots and dicots and produced fertile
Dwarf virus (WDV) (Gil-Humanes et al., 2017) have been
edited plants (Debernardi et al., 2020). The expression of DRs
successfully used in several dicot and monocot plants. These
is extremely beneficial in plant species that are recalcitrant to
viral replicons carrying the CRISPR expression cassette and
regeneration or ones with long regeneration periods to reduce the
DRT undergo rolling-circle replication in the host cells thereby
time and cost of plant gene-editing.
increasing the abundance of nuclease and availability of DRT for
HDR (Baltes et al., 2014). The GT event is not heritable if it
Viral Vectors and Mobile RNAs for Systemic doesn’t occur in the germline cells. To increase the heritability of
Delivery of CRISPR Reagents for Heritable GT, germline-specific promoters including the egg-cell, early
Gene-Editing embryo-specific promoter and pollen-specific promoters or
Recently, viral vectors showed promise for efficient delivery of promoters active in the shoot apical meristems (SAM) have
CRISPR reagents into germline cells to achieve heritable and DNA- been employed to drive Cas9 expression (Wang et al., 2015; Yan
free gene-editing (Ali Z. et al., 2015; Ellison et al., 2020; Ma et al., et al., 2015; Mao et al., 2016). Furthermore, to improve the
2020; Kujur et al., 2021; Li et al., 2021). Traditionally, heritable efficiency of heritable in-frame gene insertions and amino acid
modifications are accomplished by stable expression of the CRISPR substitutions by HDR, plants expressing Cas9 from germline-
cassettes and generating transgenic lines through regeneration. specific promoters are used for sequential transformation with
Autonomously replicating viral vectors delivered into plants via HDR constructs containing DRT and sgRNA targeting the gene
Agrobacterium offer an alternative for heritable gene-editing in of interest. This led to an increase in GT efficiency of up to 9%
plants. RNA viruses don’t integrate into the plant genome but have (Miki et al., 2018). Since GT is a rare phenomenon, even with all
lower cargo capacity impeding their use for Cas9 delivery. Tobacco the advances to improve efficiency, selection must still be
rattle virus (TRV), a bipartite positive-strand RNA virus is widely employed to detect positive GT events. A piggyBac
used in plants. TRV mediated sgRNA delivery into Cas9 transposition system from T-DNA has been used to
overexpressing lines by agroinfiltration has been optimized in eliminate the GT selection marker from host plant genome.
dicots, albeit with low heritability of edits (Ali et al., 2015; Cody In this method, a transposon integrates into the host genome at
et al., 2017). To improve heritability, the endogenous mobile RNA TTAA element and excises without a footprint (Nishizawa-
Flowering Locus T (FT) has been fused to sgRNA to enhance Yokoi et al., 2015). Recently, a novel marker elimination system
mobility and facilitate systemic distribution within plant to reach was developed wherein the excision is based on I-SceI
germline cells (Figure 1C) (Ellison et al., 2020). Barley stripe recognition site. By overlapping this recognition site on 5′
mosaic virus (BMSV) has been engineered to deliver sgRNAs and 3′ homology arms of the DRT, seamless marker
into wheat to achieve heritable genome editing. Furthermore, by elimination and precise GT have been achieved (Endo et al.,
co-infiltration of a pool of BMSV vectors harboring different 2021). To this end, the same research group also developed a
sgRNAs resulted in multiplexed mutagenesis in the progeny (Li piggyBac-mediated transgenesis system to temporarily express
et al., 2021). Sonchus yellow net rhabdovirus (SYNV), a negative- CRISPR and selection marker cassettes from T-DNA with
strand RNA virus with higher cargo capacity, has been engineered subsequent excision of piggyBac via transposase after
to carry both Cas9 and sgRNA for DNA-free in planta editing successful editing and selection had occurred (Nishizawa-
(Figure 1D) (Ma et al., 2020). Yokoi and Toki, 2021).

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Laforest and Nadakuduti Delivery of CRISPR Reagents into Plants

FIGURE 2 | Biomolecules delivered via biolistics and protoplast transfections for regenerating gene-edited plants. (A) Biomolecules used for gene-editing are
delivered into plants cells in a variety of forms including plasmid DNA, ssDNA, mRNA or ssRNA, prepared via in vitro transcription (IVT), and preassembled ribonucleic
proteins (RNPs) using IVTs and recombinant proteins. Targeted mutagenesis and gene targeting (GT) can be enhanced by various mechanisms. For example, fusion of
Cas9 to VirD2, one component of the agrobacterium relaxosome complex integral to the cleavage of T-DNA from the Ti plasmid, as well as its localization and
integration in the plant genome, has been shown to increase homology-directed repair (HDR) mediated GT using a donor repair template (DRT). DRT in this case is a
single stranded DNA (ssDNA) harboring the desired edits (red) and the canonical 25 bp right border (RB) sequence (green), and is delivered to the plant cell along with the
Cas-VirD2 fusion protein. VirD2 will covalently bind the template, thus bringing it in close proximity to the DSB induced by Cas9. Delivering Trex2 exonuclease has also
been shown to increase HDR as well as the efficiency of multiplex editing when sgRNA are co-delivered and processed by t-RNA system, illustrated by green boxes
between sgRNA. p = phosphorylation. (B) Particle bombardment or biolistics, rely on the physical disruption of plant cell walls by metal particles, often gold, coated with
ssDNA or dsDNA, IVTs or RNPs, which are introduced to the cell. Bombarded explants can be regenerated in tissue culture with or without selection to recover gene
(Continued )

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Laforest and Nadakuduti Delivery of CRISPR Reagents into Plants

FIGURE 2 | edited plants. Au = gold particles. (C) Protoplast transfection and regeneration is shown. polyethylene glycol (PEG) mediated transfection is the most
common way to deliver biomolecules for gene-editing to protoplasts. Post transfection, protoplasts are immobilized on culture media where protoplasts undergo cell
divisions to form microcalli, followed by shoot and root formation and finally resulting in regeneration of entire gene-edited plants. Editing at the target site is confirmed by
sequencing represented in the chromatogram * = deletions.

Biolistics for Delivery of CRISPR Reagents Gene Insertion or Replacement by Intron

Into Plants as DNA, RNA, or Proteins Targeting and Determining Genomic Safe
Biolistics or particle-bombardment, is a common alternative for Harbors
transforming plants recalcitrant to Agrobacterium-infection. It To leverage the relatively more efficient NHEJ compared to
relies on physically breaching the plant cell wall and membrane HDR for targeted insertions, DNA fragments were inserted in
with gold or tungsten microprojectiles coated with biomolecules selected introns such that any mutations by NHEJ would not
accelerated to very high velocities. Biolistics offers the possibility of affect protein-coding sequences of either endogenous or
delivering a variety of cargo including plasmid DNA, ssDNA, RNA, inserted genes. By bombarding calli with plasmids
or ribonucleic proteins (RNPs) assembled from IVTs and expressing pairs of sgRNA targeting adjacent introns of
recombinant proteins (Figure 2A). Major drawbacks of biolistic target genes and DRT harboring desired mutations flanked
delivery include random integration of cargo at multiple genomic by the same intronic sgRNA sites, replacement of endogenous
sites when delivered as DNA and labor-intensive preparation of gene has been achieved at 2% frequency. Additionally, the
explants such as calli or immature embryos with the capability to gene replacement events were heritable (Li et al., 2016).
regenerate. Enhancers and promoters up to 2 Kb were introduced into
the target site using these modified DRTs (Lu et al., 2020).
Advances in Biolistic Delivery for DNA-free Another strategy for targeted insertion by NHEJ is to
determine the genomic safe harbors (GSH) in the host
Gene-Editing and Chromosome plant genome, within which integrations of transgenes do
Engineering not cause any genic disruptions or adverse morphological
Instead of plasmid DNA, bombarding RNPs was successfully effects. A 5.2 Kb carotenoid biosynthesis cassette was inserted
demonstrated to produce transgene-free gene-edited lines in at targeted GSH to generate marker-free rice with high
cereal crops (Svitashev et al., 2016; Liang et al., 2017; Banakar carotenoid containing seeds and no-off target mutations
et al., 2019, 2020; Zhang et al., 2021) (Figure 2B). In addition, observed (Dong et al., 2020).
when a single base pair mismatch was present in the protospacer of
sgRNA targeting homeologs, a dramatic decrease in off-target
editing was observed with RNPs as compared to plasmid DNA
delivery indicating high specificity of RNPs (Liang et al., 2017).
Enhancing HDR by Delivery of
Furthermore, RNPs also facilitated large heritable inversion of Transcript-Donor Templates or by
75.5 Mb in maize chromosome 2, when assembled with guide VirD2 Relaxase-Cas9 Fusion
RNAs flanking the junctions of the desired inversion (Schwartz Recent advances in HDR by particle bombardment include
et al., 2020). Such precise chromosomal engineering in invaluable to delivery of ssDNA, including a canonical 25 bp right border
crop breeding. To avoid labor-intensive preparation of explants, in (RB) sequence of T-DNA, as DRT co-delivered with a plasmid
planta biolistic delivery using SAM as a target tissue (Hamada et al., expressing Cas9-VirD2 fusion protein (Ali et al., 2020)
2017) for germline transmission was employed as an alternative (Figure 2A). Achieving a 20.8% HDR efficiency, this method
(Hamada et al., 2018; Imai et al., 2020). Embryonic SAM exposed relies on the ability of the VirD2 protein, an Agrobacterium
mature wheat seeds were bombarded with plasmid DNA expressing virulence factor, to covalently bind the RB of DRT, thus
CRISPR cassettes to generate gene-edited lines (Hamada et al., 2018; bringing it in close proximity to the DSB induced by Cas9
Imai et al., 2020). Alternatively, when pollen was used as a target (Figure 2A) (Ali et al., 2020). Other attempts to improve
tissue to bombard plasmid DNA for gene-editing in Nicotiana HDR include the delivery of DRT as transcripts. RNA-DRT
benthamiana, the bombarded pollen retained fertility and was shown to result in higher HDR efficiency than DNA-DRT
delivered the cargo into the ovules (Nagahara et al., 2021). when delivered to rice calli, possibly due to the high stability of
Furthermore, technical improvements have also been made to RNA:DNA complexes, resulting in edited rice with two desired
overcome variability between bombardments. A double-barreled point mutations in the ALS gene conferring herbicide tolerance
gene gun in combination with cell counting software was (Li et al., 2019). This transcript-templated HDR (TT-HDR),
developed to scale bombardment experiments with an internal approach improves not only HDR efficiency but also creates a
standard, thereby reducing standard deviation between DNA-free path to HDR-mediated gene-editing, which may avoid
bombardments by half (Miller et al., 2021). regulatory hurdles.

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Laforest and Nadakuduti Delivery of CRISPR Reagents into Plants

Protoplasts Provide a Versatile System for Future Aspects of Delivering Plant-Gene

DNA-free Genome Editing in Plants Editing Reagents
Protoplasts are plant cells devoid of cell walls, which offer a Relying on tissue culture-based plant genetic transformation methods
versatile platform for DNA-free GE and a good transient system and inefficient reagent delivery mechanisms are the major bottle
to evaluate the activity of gene-editing reagents before moving necks to overcome before we realize the full potential of gene-editing
into a more-labor intensive transformation pipeline in plants. Current advancements in delivery mechanisms, including
(Nadakuduti et al., 2019; Lin et al., 2020). Polyethylene glycol de novo meristem induction or use of viral vectors to circumvent
(PEG)-mediated transfection and electro-transfection are two tissue culture, rely on Agrobacterium for delivery and have been
common methods to deliver plasmid DNA, IVTs, or RNPs into demonstrated only in dicots and need to be expanded to monocots.
protoplasts for transient expression of CRISPR cassettes. Delivering repair templates for HDR through these innovative
Subsequently, edited plants can be regenerated from methods is also a future possibility. Furthermore, smaller sized
transfected protoplasts by tissue culture procedures Cas9 alternatives would overcome the cargo capacity of some of
(Figure 2C). Plasmid DNA may integrate into the host these viral vectors. Agrobacterium, however, has a narrow host range
genome randomly as filler DNA during protoplast for infection and several species are recalcitrant to Agrobacterium
transfection (Gorbunova and Levy, 1997; Kim and Kim, transformation. Particle bombardment has been shown to be better
2016). However, IVTs or RNPs offer DNA-free gene-editing equipped for co-delivery of cargo for simultaneous editing than
by immediately editing the target site, bypassing transcription Agrobacterium and is universally applicable to all plant species
and translational machinery respectively in the cell and rapidly and cell types (Kuang et al., 2020). Chromosomal inversions
degrade (Liang et al., 2017, 2018; Andersson et al., 2018; achieved via bombardment could revolutionize breeding by
González et al., 2020, 2020, 2020; Lee et al., 2020; Sidorov unlocking regions for chromosomal cross overs, creating novel
et al., 2021; Zhang et al., 2021). However, plant regeneration linkage groups and facilitating targeted recombination to
from protoplast remains unestablished in many plant species. In maximize genetic gain in crops. However, complex segregation
addition, somaclonal variations and genome instability is patterns of DNA integrated in bombarded plant genomes might
reported in regenerated lines (Fossi et al., 2019). Once complicate downstream uses of transformed plants. Agrobacterium
efficient protoplast isolation, transfection, and regeneration and biolistic transformation of pollen also bypasses regeneration but
have been established in a plant species, it could be a high often results in pollen with lower viability (Wang et al., 2008; Zhao
throughput platform by combining with flow cytometry and et al., 2017). In addition, pollen-tube transformations may result in
omic analyses for optimizing gene-editing. Furthermore, chimerism (Ali A. et al., 2015). While pollen magnetofection has
multiplexing, editing multiple genes at a time has been improved on these drawbacks (Zhao et al., 2017), its application
achieved using protoplasts (Klimek-Chodacka et al., 2021; remains constrained to dicots (Vejlupkova et al., 2020). The prospects
Nicolia et al., 2021; Yu et al., 2021; Zhang et al., 2021). By of nanoparticles as delivery engines for plant genome editing are also
co-delivering Three Prime Repair exonuclease 2 (TREX2) and encouraging (Demirer et al., 2021) and further advances are essential
CRISPR/Cas9 into protoplasts, targeted mutagenesis using a to facilitate plant gene-editing.
multiplexing strategy was further improved (Weiss et al., 2020)
(Figure 2A).
AUTHOR CONTRIBUTIONS
Nanocarrier-Mediated Delivery of CRISPR/ SN conceived the idea; SN and LL wrote the manuscript. All
Cas Reagents in Plants authors read and approved the manuscript.
Nanotechnology has evolved in the past decade in the field of
plant genetic engineering. Nanomaterials including carbon
nanotubes (CNTs), carbondots, mesosporous silicon FUNDING
nanoparticles (MSNs) etc have been used to deliver
biomolecules such as DNA, RNA, RNPs and proteins etc., Funding for SN was provided by the Biotechnology Risk
discussed in recent reviews (Kumari and Singh, 2021; Mujtaba Assessment Grant Program competitive grant no. 2018-33522-
et al., 2021). Nanoparticle-mediated delivery of DNA and 28736 from the USDA National Institute of Food and Agriculture
proteins into both nuclear and chloroplast genomes has been and start-up funds from the Environmental Horticulture
achieved in plants (Demirer et al., 2019, 2020; Kwak et al., 2019). Department, University of Florida.
Furthermore, Cre protein was previously delivered via MSNs for
maize GE via loxP site demonstrating the feasibility of gene-
editing (Martin-Ortigosa et al., 2014). Gene-editing using RNPs ACKNOWLEDGMENTS
delivered by nanoparticles has been achieved in human cells
(Wang et al., 2016; Lee et al., 2017; Mout et al., 2017). However, it We thank Dr. Felix Enciso-Rodriguez from the Colombian
has yet to be achieved in plants mainly due to high delivery Corporation for Agricultural Research for reviewing the
efficiencies required for GE. manuscript and editorial suggestions.

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Laforest and Nadakuduti Delivery of CRISPR Reagents into Plants

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Frontiers in Genome Editing | www.frontiersin.org 10 January 2022 | Volume 4 | Article 830178