Plant Physiology and Biochemistry 196 (2023) 724–730

Contents lists available at ScienceDirect

Plant Physiology and Biochemistry

journal homepage: www.elsevier.com/locate/plaphy

CRISPR/Cas genome editing in plants: Dawn of Agrobacterium

transformation for recalcitrant and transgene-free plants for future
crop breeding
S. Antony Ceasar a, *, S. Ignacimuthu b
a
Division of Plant Molecular Biology & Biotechnology, Department of Biosciences, Rajagiri College of Social Sciences, Cochin, 683 104, Kerala, India
b
Xavier Research Foundation, St. Xavier’s College, Affiliated to the Manonmaniam Sundaranar University, Palayamkottai, 627 002, Tamil Nadu, India

A R T I C L E I N F O A B S T R A C T

Keywords: Genome editing tools based on CRISPR/Cas system have been posed to solve many issues in agriculture and
Genetic transformation improve food production. Genetic engineering by Agrobacterium-mediated transformation has helped to impart
Genome editing specific traits straightaway in many crops. Many GM crops have also reached the field for commercial cultiva­
Transgenic plants
tion. Genetic engineering requires mostly a transformation protocol often mediated by Agrobacterium to insert a
Rice
CRISPR/Cas system
specific gene at a random locus. Genome editing with CRISPR/Cas system is a more precise technique for the
targeted modification of genes/bases in the host plant genome. Unlike the conventional transformation system,
wherein elimination of marker/foreign gene was possible only post-transformation, CRISPR/Cas system could
generate transgene-free plants by delivering CRISPR/Cas reagents such as the Cas protein and guide RNAs gRNA
(s) preassembled to form ribonucleoproteins (RNPs) into plant cells. CRISPR reagent delivery might be helpful to
overcome issues with plants that are recalcitrant to Agrobacterium transformation and the legal hurdles due to the
presence of the foreign gene. More recently, the grafting of wild-type shoots to transgenic donor rootstocks
developed by the CRISPR/Cas system has reported transgene-free genome editing. CRISPR/Cas system also re­
quires only a small piece of gRNA besides Cas9 or other effectors to target a specific region in the genome. So this
system has been projected to be a key contributor to future crop breeding. In this article, we recap the main
events of plant transformation, compare the difference between genetic transformation and CRISPR/Cas-
mediated genome editing, and draw insights into the future application of the CRISPR/Cas system.

1. Introduction past decades (Aldemita et al., 2015; Lucht, 2015). However, the pres­
ence of marker genes and candidate genes of foreign origin continues to
Improving crop production has been the main focus of plant science fuel the debate on transgenic plants, which has prevented many coun­
researchers in recent decades. While conventional and molecular tries from approving these plants for commercial cultivation. The pres­
marker-assisted breeding helped to breed better crops, these techniques ence of multiple copies of genes also caused problems silencing genes in
still need to pass through the time-consuming process for new variety future generations of transgenic plants (Kamthan et al., 2016). A few
development. Plant gene transfer offered a straightforward solution for efforts have also shown that the marker genes could be eliminated from
transferring candidate genes from any source to any plant, thus the transgenic plants by subsequent transformation cycles (Gleave et al.,
imparting the desired trait quickly (Ashraf, 2010; Jouanin et al., 1998; 1999; Tuteja et al., 2012). However, integrating foreign genes with the
Wahid et al., 2007; Wang et al., 2003). Although initially met with a lot host chromosome is essential for transgenic plants developed by Agro­
of resistance from NGOs, the public, and politicians, a sizeable number bacterium tumefaciens’s T-DNA-based gene transfer methods. Due to its
of transgenic plants reached the field for commercial cultivation (Bates low cost and ease of handling, the Agrobacterium-mediated trans­
et al., 2005; Halpin, 2005; Klümper and Qaim, 2014). Many countries formation system was frequently used for plant transformation in many
have cultivated edible and non-edible transgenic crops conferring labs. However, many plant species and even specific genotypes of the
tolerance to various biotic and abiotic stresses for human use during the same species are highly recalcitrant for Agrobacterium-mediated

* Corresponding author. Department of Biosciences, Rajagiri College of Social Sciences, Cochin, 683 104, Kerala, India.
E-mail addresses: antony_sm2003@yahoo.co.in, saceasar@rajagiri.edu (S. Antony Ceasar).

https://doi.org/10.1016/j.plaphy.2023.02.030
Received 4 January 2023; Received in revised form 15 February 2023; Accepted 16 February 2023
Available online 18 February 2023
0981-9428/© 2023 Elsevier Masson SAS. All rights reserved.
S. Antony Ceasar and S. Ignacimuthu Plant Physiology and Biochemistry 196 (2023) 724–730

transformation, thus limiting the application of genetic transformation. transgenic plants (Gleave et al., 1999; Matthews et al., 2001; Corneille
Targeted genome editing of plants has opened new opportunities for et al., 2001), the presence of the foreign gene in the host chromosome of
plant science researchers to study the gene function and improve the transgenic plants caused concern for commercial cultivation.
plants for specific traits. Genome editing tool has been widely applied to Genome editing is a more precise tool for altering organisms’ genes,
plants for precision breeding and crop improvement programs. Espe­ allowing researchers to modify plant genomes more precisely and effi­
cially the clustered regularly-interspaced short palindromic repeats ciently. Genome editing tools introduce breaks at the targeted genome
(CRISPR)/CRISPR-associated protein 9 (Cas9) system rapidly adopted location and help remove, insert, or replace the base(s). In the early
by many labs worldwide. CRISPR/Cas9 system offered a more efficient stages of genome editing, to induce specific DSBs at a targeted site,
and user-friendly plant genome editing tool. Scores of research and re­ mega-nucleases (MNs) (Stoddard, 2005), zinc finger nucleases (ZFNs)
view articles have been published on the status and prospects of targeted (Sander et al., 2007), and transcription activator-like effectors nucleases
genome editing in model and non-model plants by the CRISPR/Cas (TALENs) (Reyon et al., 2012) were used. These tools require extensive
system (reviewed in Ceasar et al., 2022; Zhu et al., 2020; Montecillo plasmid construction with a costly and labor-intensive procedure
et al., 2020; Puchta, 2017; Manghwar et al., 2019; Bao et al., 2019). (Juillerat et al., 2014). CRISPR/Cas system offers user-friendly and
CRISPR/Cas system offered transgene-free genome editing by intro­ cost-effective steps for efficient genome editing in plants (Doudna and
ducing preassembled Cas protein and gRNA(s) as ribonucleoproteins Charpentier, 2014; Jinek et al., 2012). CRISPR-based genome editing
(RNPs) into plant cells for certain applications, which could help evade may or may not require the genetic transformation of plants with foreign
all stringent regulatory hurdles for quicker field cultivation. CRISPR genes (Box 1). Thus CRISPR/Cas system might help to engineer plants
reagent delivery would also help to transform the recalcitrant species that the Agrobacterium-mediated system cannot transform.
that are difficult to transform by the Agrobacterium. In the article, we In traditional genetic transformation, integrative vectors were used
quickly review the achievements of genetic transformation and genome most frequently to insert the gene/trait. The T-DNA-based integrative
editing and highlight the advantages of genome editing for better crop vectors were used for the Agrobacterium-mediated transformation of the
breeding to conserve food security in the future. plants (Fig. 2). However, the CRISPR/Cas-based system works in
creating mutant (transgenic) plants without the need to integrate the
2. Gene transfer vs genome editing foreign DNA with the host chromosome since it has the option of
delivering the transformation reagents such as gRNA and Cas9 (Veillet
The ability to transfer foreign genes into plant cells (protoplasts) at et al., 2019; Woo et al., 2015; Ghogare et al., 2021). Both traditional
the end of the 20th century caused excitement among plant science re­ T-DNA-based transformation and certain types of CRISPR/Cas-based
searchers (Krens et al., 1982; Lörz et al., 1985; Baba et al., 1986). The genome editing (Such as CRISPRa and CRIPSRi) still require efficient
genetic transformation was followed by the development of fertile genetic transformation system. In contrast, genetic transformation effi­
transgenic plants in rice and maize (Shimamoto et al., 1989; Gordon-­ ciency is dependent on plant species and genotype. Progress made with
Kamm et al., 1990), thus setting the platform for the transgenic era CRISPR/Cas-based reagent delivery could help overcome this hurdle. In
(Fig. 1). The discovery of A. tumefaciens’s Ti plasmids, T-DNA, and en­ conventional genetic transformation-mediated knockout/knockdown of
gineering of this plasmid to make binary plasmids for custom gene genes, RNAi-based vectors were used predominantly. In the RNAi
transfer helped many labs around the world to create several trans­ strategy, the T-DNA bearing the inverted repeat sequence of the target
formation experiments (Bevan, 1984; Horsch et al., 1985). Agro­ gene was used to downregulate the gene post-transcriptionally, which
bacterium-mediated and physical methods like biolistic gun transformed was ineffective in bringing about the gene knockdown. But the
many crop plants like maize, wheat, rice, barley, soybean, millets, etc. gRNA-based strategy with Cas9 nuclease helps knockout all alleles of the
(Gelvin, 2003; Ramkumar et al., 2020; Rivera et al., 2012). Genetic gene more efficiently.
transformation has helped to create several transgenic plants such as Bt
corn/maize, Bt cotton, Bt potatoes, herbicide-resistant cotton, soybeans, 3. Genome editing by CRISPR/Cas system
viral-resistant papaya, ‘Flavr Savr tomato’ with delayed ripening, canola
with modified oil composition, etc. (reviewed in Ramkumar et al., In 2012, Jennifer Doudna and Emmanuelle Charpentier reported
2020). Most transgenic plants are cultivated in the US and South CRISPR/Cas system as a programmable genome-editing tool (Jinek
America (Moeller and Wang, 2008). Countries like India have approved et al., 2012). They revealed that Cas9 nucleases could work when tar­
only non-edible plants like Bt cotton for commercial cultivation. getted with the help of small gRNA, which shortened the procedure and
Although tools were introduced to eliminate the marker gene from preparation for genome editing (Jinek et al., 2012). The CRISPR/Cas9

Fig. 1. Major milestones of genetic transformation and genome editing in plants. Key events related to genetic transformation and genome editing of various
plants are illustrated with the year.

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Box 1
Comparison of plant genetic transformation and genome editing properties. Various factors involved in plant genetic transformation and
genome editing are listed and compared.

Properties Genetic transformation CRISPR/Cas genome editing

Gene addition/gene transfer Non-targeted Targeted by gRNA
Gene integration Essential Non-essential
Construct delivery Agrobacterium, Biolistic & PEG/Ca2+ transfection Agrobacterium, Biolistic & PEG/Ca2+ transfection
Genome sequence Not required Required for gRNA designing
Transgene-free reagent delivery No Yes; Cas9 & gRNA with protoplast
Targeting molecule No gRNA, pregRNA, etc
Gene silencing Post-transcriptional -RNAi (knock down) Pre-transcriptional- DNA breaks (knock out)
Gene knockout By T-DNA insertion mutations By gRNA and Cas9
Transcriptional regulation No CRISPRi (activation) (Qi et al., 2013)
CRISPRa (repression) (Qi et al., 2013)
Targeted base editing/SNP No Adenosine base editor (ABE) (Gaudelli et al., 2017)
Cytosine base editor (CBE) (Komor et al., 2016)
Long sequence editing No Prime editing (~80 bases) (Anzalone et al., 2019)

Fig. 2. Comparative illustration of plant genetic transformation and genome editing. Key steps involved in genetic transformation and genome editing are
illustrated. In genetic transformation, the T-DNA-based gene insertion was predominately followed by the Agrobacterium-, Biolistic-, and PEG/Ca2+-mediated
transformation methods. In CRISPR/Cas-mediated genome editing, gRNA-based targeted editing of genes was attempted. Targeting the genome by gRNA help
achieve various tasks such as transcriptional control, gene knockout, gene knockin, base editing, mRNA editing, etc.

system is associated with Cas9 nucleases and the sgRNA (Lintner et al., Cas9/gRNA complex searches the PAM (5′ -NGG-3′ for S. pyogenes Cas9)
2011). The Cas9 nucleases contain HNH and RuvC domains as molecular and then examines the base paring between DNA and gRNA with a
scissors. The gRNA complexed with Cas9 nucleases to create confor­ matching sequence and results in the cleavage of target DNA sequence
mational changes in the targeted genome of interest by a designed guide (Jinek et al., 2012) (Sternberg et al., 2015). The generated DSB is
sequence (20 nucleotides) via base pairing to the targeted region. The repaired either by non-homologous end joining (NHEJ) or

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homology-directed repair (HDR) methods. CRISPR/Cas9 system was decade. A quick Scopus database search (dated 4th January 2023) for the
quickly adopted for gene knockout studies in various organisms, articles with various plant names for the mention of CRISPR is presented
including plants (reviewed in Ceasar et al., 2016; Nadakuduti et al., (Fig. 4). Most mention of CRISPR has occurred with rice, a model, and
2018). The CRISPR/Cas9 system has been successfully applied in plants cereal food crop. This was followed by the Arabidopsis. Being a model
to improve various traits (Fig. 3). These include not only model plants, plant with a small genome and short regeneration time, most CRISPR
such as Arabidopsis, but also crops, such as rice, tobacco, sorghum, tools have been applied to Arabidopsis to study the function of several
wheat, maize, soybean, tomato, potato, poplar, apple, and banana genes and signaling events. Apart from these model plants, CRISPR has
(Reviewed in Ansari et al., 2020; Bao et al., 2019; Ceasar et al., 2022; S A also been used in other food crops and tubers such as maize, wheat,
Ceasar et al., 2016; Kamburova et al., 2017; Montecillo et al., 2020; Xing soybean, tobacco, tomato, potato, etc. (Fig. 4). These studies paved the
et al., 2014). Following CRISPR/Cas9, several other variants of way to understanding the molecular genetic mechanism of various traits
CRISPR/Cas were developed and also applied in diverse plants. These and improve upon them, such as tolerance to abiotic and biotic stresses
include CRISPR/dCas9 for transcriptional control for gene activation and improved growth and yield.
and repression (Qi et al., 2013), CRISPR-cytosine base editor (CBE) Many attempts were also made to develop DNA-free genome-edited
(Komor et al., 2016), CRISPR-adenosine base editor (ABE) (Gaudelli plants (reviewed in Laforest and Nadakuduti, 2022). In DNA-free
et al., 2017) for single base changes, CRISPR-prime editor for long base genome editing, RNP complex consisting of particular Cas protein for
changes (Anzalone et al., 2019), PAM-less Cas9 (Satish et al., 2016), specific editing and in vitro transcribed gRNA for targeting are delivered
Cas13 (Abudayyeh et al., 2017), and Cas14 (Burstein et al., 2017) into plant cells. Apart from protoplasts of rice, tobacco, Arabidopsis, and
systems. lettuce transfected by the polyethylene glycol-calcium (PEG-Ca2+) with
RNPs (Woo et al., 2015), embryonic maize and wheat cells were also
4. Progress made in plant genome editing with CRISPR/Cas delivered with RNPs by particle bombardment to produce DNA-free
system plants (Toda et al., 2019). More recently, a Germany-based group
introduced the graft-mobile gene editing system that produces
CRISPR/Cas system has been widely applied in plants during the past transgene-free offspring in one generation (Yang et al., 2023). In this

Fig. 3. Application of CRISPR/Cas9 system in various plants to improve drought tolerance, fungal resistance, salinity tolerance, and bacterial resistance.
The genes targeted for developing resistance in each plant are mentioned for each trait in the boxes. Fungal resistance is developed by targeting OsERF922 (Wang
et al., 2016) and OsPi21 (Nawaz et al., 2020) genes in rice and BnWRKY70 in canola (Nawaz et al., 2020), SlPMR4, SlPelo and SlMlo1 genes (Pramanik et al., 2021) in
tomato, and AtERF019 in Arabidopsis (Lu et al., 2020). Bacterial resistance was developed by CRISPR/Cas9-mediated disruption of OsWEET (Oliva et al., 2019) and
OsMPK5 (Xie and Yang, 2013) genes in rice, SlDMR6 (Thomazella et al., 2021) and SlJAZ2 (Ortigosa et al., 2019) genes in tomato. Salinity tolerance was achieved by
CRISPR/Cas9 targetting of OsVDE (X. T. Wang et al., 2021), OsRR22 (Zhang et al., 2019), OsNAC041 (Bo et al., 2019) genes in rice, GmAITR (T. X. Wang et al., 2021)
and GmNACO (M. Li et al., 2021) genes in soybean, TaHAG1 (Zheng et al., 2021) in wheat, SlHyPRP1 (Tran et al., 2021) and SlARF4 (Chen et al., 2021) genes in
tomato. Drought tolerance was achieved by disrupting OsADR3 (J. Li et al., 2021), OsNCED3 (J. Li et al., 2021), OsDST (Santosh Kumar et al., 2020), OsABA8ox2
(Zhang et al., 2020), OsSRL1,2 (Liao et al., 2019) genes in rice, ARGOS8 (Shi et al., 2017) gene in maize, GmMYB118 (Du et al., 2018), GmDrb2a and GmDrb2b (Curtin
et al., 2018) genes in soybean, BnaRGA (Yang et al., 2018), BnaA6.RGA (Yang et al., 2018) genes in canola, TaERF3 (Kim et al., 2018) and TaDREB2 (Wu et al., 2020)
genes in wheat, Ca4CL, and CaRVE1 (Badhan et al., 2021) genes in chickpea, SlMAPK3 (Badhan et al., 2021), SlNPR1 (Li et al., 2020), SlLBD40 (Li et al., 2020) genes
in tomato, HSFA6a and HSFA6b (Li et al., 2020) genes in Arabidopsis.

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Fig. 4. CRISPR applications in plants are on the rise. Results of a Scopus database search dated 04-01-2023 (9.30 a.m., IST) with the keywords CRISPR AND
respective plant name). The number of articles mentioning CRISPR is captured from 2016 to 2022 for rice, Arabidopsis, maize, wheat, soybean, tomato, and potato.

work, they have designed the tRNA-like sequence motifs for Cas9, and rice and wheat (Ceasar, 2021). Altering the texture and appearance of
gRNA transcripts that move RNAs from transgenic rootstocks to grafted fruits and tubers could also be possible by applying CRISPR/Cas tools.
wild-type shoots, which helped for heritable gene editing demonstrated Increasing the shelf life of fruits like a tomato would be the priority with
in Arabidopsis thaliana and Brassica rapa (Yang et al., 2023). This the CRISPR/Cas tools.
approach could help engineer the recalcitrant genotypes within the
species and yield transgene-free crops for safer cultivation. DNA-free 6. Conclusion
genome editing is considered a promising technique in biotechnology
due to its potential to avoid some of the safety concerns associated with Since the green revolution, introducing every new tool and technique
foreign DNA into cells, and such efforts would help create more plants in has created much hype and has been predicted to benefit crop breeding.
the future. These include conventional mutation, genetic transformation, genome
sequencing, molecular marker-assisted breeding, etc. CRISPR/Cas-
5. The future direction of breeding better crops with the mediated genome editing is a recent addition to the breeding toolbox
CRISPR/Cas system for crop improvement. As with other tools, the CRISPR/Cas system has
also been projected to solve many agricultural issues. It has already been
The CRISPR/Cas system has been predicted to solve ongoing issues in applied to many crops, and scores of reports are available to successfully
agriculture production and help strengthen food security by improving develop genome-edited crops through CRISPR/Cas system. Rice is the
crop plants for various traits. As in the recent past, rice might continue to single crop that has attracted so much attention for CRISPR application.
receive more attention for CRISPR-mediated crop improvements. Several traits have already been imparted and improved upon by
Although several trait improvements, such as fungal resistance applying the CRISPR/Cas system. CRISPR/Cas reagent delivery and the
(Reviewed in Paul et al., 2021), drought and salinity tolerance creation of transgene-free crops have been major advantages of CRISPR-
(reviewed in Romero and Gatica-Arias, 2019), and enhancement of seed edited plants, which could help escape the stringent rules for GM crop
quality and quantity (reviewed in Liu et al., 2021) have already been commercialization. Although CRISPR has been applied in many model
attempted in rice, this crop continues to get more attention for genome crops like rice and Arabidopsis, orphan crops like millets have not been
editing research. Improving rice’s nutrient transport and seed nutrient paid much attention for CRISPR-mediated genome editing. Prudent
quality could continue to be major thrust areas of research in the coming application of CRISPR in all crops, including orphan crops, could help
decades. Efforts made with genetic engineering strategy to improve the improve crop production and conserve food security.
nutrient contents like golden rice (Beyer et al., 2002) could be acceler­
ated by the CRISPR/Cas tool since it can edit the specific and targeted Authors contribution
region of the genome. For example, genes for specific nutritional com­
ponents could be added at the targeted region, and genes involved with SAC planned and designed the article; SAC and SI wrote the article.
anti-nutritional properties could be removed from the genome by SAC drew the images. SAC and SI edited and revised the article.
CRISPR/Cas9 system. Apart from resistance to biotic and abiotic
stresses, the management of fertilizers seems to be the key to meeting Declaration of competing interest
food security. CRISPR/Cas tools have also been predicted to improve
nutrient use efficiency with less dependency on synthetic fertilizers The authors declare that they have no known competing financial
(Ceasar et al., 2022; Sathee et al., 2022). CRISPR/Cas tool also might interests or personal relationships that could have appeared to influence
help improve the nutrient use efficiency in rice since it is the only crop the work reported in this paper.
that demands much fertilizer, especially nitrogen, for its production.
Similarly, CRISPR/Cas application could extend seed quality enrichment Data availability
to other cereals, such as maize, wheat, barley, sorghum, etc. Millets had
not focussed much on modern genetic and genomic studies in the past No data was used for the research described in the article.
decades. They are often called orphan crops due to less application of
modern tools and limited resources for crop improvement purely due to Acknowledgments
their cultivation in low-income countries (Ceasar and Maharajan, 2022).
However, orphan crops like millet are also believed to strengthen food SAC thanks the Rajagiri College of Social Sciences for the research
security (Chapman et al., 2022). The application of precise CRISPR tools support.
could help unravel the mechanism of climate resilience and nutrient
enrichment in millets and transfer such traits to non-millet cereals like

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