TYPE Review
PUBLISHED 23 August 2022
DOI 10.3389/fgene.2022.876987
Comprehending the evolution of
OPEN ACCESS gene editing platforms for crop
EDITED BY
Jitendra Kumar, trait improvement
Indian Institute of Pulses Research
(ICAR), India
Priyanka Dhakate1, Deepmala Sehgal2, Samantha Vaishnavi3REVIEWED BY ,
Debjyoti Sen Gupta, Atika Chandra4, Apekshita Singh5, Soom Nath Raina5* and
Indian Institute of Pulses Research
(ICAR), India Vijay Rani Rajpal6*
Kumar Paritosh,
University of Delhi, India 1National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, India, 2International
Guoliang Yuan, Maize and Wheat Improvement Center (CIMMYT), México-Veracruz, Mexico, 3Department of Botany,
Oak Ridge National Laboratory (DOE), Central University of Jammu, Jammu, India, 4Department of Botany, Maitreyi College, University of
United States Delhi, New Delhi, India, 5Amity Institute of Biotechnology, Amity Institute of Biotechnology, Amity
Faiz Ahmad Joyia, University, Noida, India, 6Department of Botany, Hansraj College, University of Delhi, New Delhi, India
University of Agriculture, Pakistan
*CORRESPONDENCE CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas
Vijay Rani Rajpal,
vijayrani2@gmail.com (CRISPR-associated) system was initially discovered as an underlying
Soom Nath Raina, mechanism for conferring adaptive immunity to bacteria and archaea
soomr@yahoo.com
against viruses. Over the past decade, this has been repurposed as a
SPECIALTY SECTION genome-editing tool. Numerous gene editing-based crop improvement
This article was submitted to Plant
Genomics, technologies involving CRISPR/Cas platforms individually or in combination
a section of the journal with next-generation sequencing methods have been developed that have
Frontiers in Genetics revolutionized plant genome-editing methodologies. Initially, CRISPR/Cas
RECEIVED 16 February 2022 nucleases replaced the earlier used sequence-specific nucleases (SSNs),
ACCEPTED 29 June 2022
PUBLISHED 23 August 2022 such as zinc-finger nucleases (ZFNs) and transcription activator-like
effector nucleases (TALENs), to address the problem of associated off-
CITATION
Dhakate P, Sehgal D, Vaishnavi S, targets. The adaptation of this platform led to the development of concepts
Chandra A, Singh A, Raina SN and such as epigenome editing, base editing, and prime editing. Epigenome
Rajpal VR (2022), Comprehending the
evolution of gene editing platforms for editing employed epi-effectors to manipulate chromatin structure, while
crop trait improvement. base editing uses base editors to engineer precise changes for trait
Front. Genet. 13:876987. improvement. Newer technologies such as prime editing have now been
doi: 10.3389/fgene.2022.876987
developed as a “search-and-replace” tool to engineer all possible single-
COPYRIGHT
base changes. Owing to the availability of these, the field of genome editing
© 2022 Dhakate, Sehgal, Vaishnavi,
Chandra, Singh, Raina and Rajpal. This is has evolved rapidly to develop crop plants with improved traits. In this
an open-access article distributed review, we present the evolution of the CRISPR/Cas system into new-age
under the terms of the Creative
Commons Attribution License (CC BY). methods of genome engineering across various plant species and the
The use, distribution or reproduction in impact they have had on tweaking plant genomes and associated
other forums is permitted, provided the outcomes on crop improvement initiatives.
original author(s) and the copyright
owner(s) are credited and that the
original publication in this journal is
KEYWORDS
cited, in accordance with accepted
academic practice. No use, distribution CRISPR/Cas system, base editing, prime editing, epigenome editing, crop improvement
or reproduction is permittedwhich does
not comply with these terms.
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Dhakate et al. 10.3389/fgene.2022.876987
1 Introduction During the past decade, the term “CRISPR/Cas” has evolved
into a synonym for GE following which off-targeting instances
Over the past decade, the gene-editing platforms have shown with the use of CRISPR/Cas systems have reduced manifold
tremendous evolution to accommodate the dual concerns of (Modrzejewski et al., 2020). However, the goal of achieving “no
biosafety of edited crops and the efficiency of the platform used. off-target” remains elusive. In addition, with the involvement of
Efficient and rapid genomic sequencing platforms have facilitated a the NHEJ repair pathway, the efficiency of this platform has always
better understanding of plant genomes, particularly when used in been disputable. In the third phase of the evolution of GE
conjunction with genome editing (GE). Restructuring genomes via platforms, the CRISPR/Cas platform evolved to target the
introduction of heritable genomic changes for expressing desirable epigenome of an organism which was termed epigenome
quality traits in crops has been the focus of research for decades. The editing (Konermann et al., 2013). In epigenome editing,
primitive methods of genome restructuring involved the use of chromatin modification at specific genomic loci involves the
genotoxic agents to introduce random double-stranded breaks use of epi-effectors that are comprised of DNA recognition
(DSB) that were subsequently repaired by inherent non- domains (ZFNs, TALENs, or CRISPR/Cas system) and catalytic
homologous end joining (NHEJ) pathways resulting in random domains from a chromatin-modifying enzyme. Epigenome editing
mutations (Puchta, 2005). After decades of usage of these random has been slated to have promising results in numerous basic
mutations generating tools, GE platforms have gone through many sciences to decipher functions of chromatin structure and
phases of improvement over the years. For example, the discovery of associated modification in phenotypes.
sequence-specific nucleases (SSNs) such as zinc-finger nucleases In the fourth phase, the CRISPR/Cas system evolved into a new
(ZFNs) and transcription activator-like effector nucleases methodology called base editing, whereinRNA-guided endonucleases
(TALENs) helped to engineer the genome at intended loci by were employed to engineer all four possible transitions with increased
mediating the cleavage of dsDNA. The use of these nucleases precision (Komor et al., 2016). One of the major challenges that all of
induced the native NHEJ pathway for DNA repair (Salomon and the aforesaid techniques still face is to simultaneously engineer the
Puchta, 1998). This method of GE, however, is both cost- and labor- altered DNA at the intended target sites. These concerns were
intensive as it requires the development of sequence-specific addressed with the introduction of prime editing, marking the
nucleases/proteins. In addition, GE using these nucleases was fifth phase in the evolution of GE platforms. Prime editing is
inefficient as unintended off-target edits were introduced by the largely described as a “search-and-replace” technology that edits
induction of the error-prone NHEJ repair pathway. the intended genomic loci without generating DSBs (Anzalone
Given the obvious limitations of ZFNs and TALENs, the vacuum et al., 2019). This platform efficiently addresses the concerns of
was soon filled with the discovery of CRISPR (Clustered Regularly frameshift mutations that arise with the introduction of indels,
Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) further reducing off-target mutations. In addition, prime editing
nucleases. In prokaryotes, the CRISPR/Cas system exists as a can introduce all 12 possible nucleotide substitutions (including
means of endogenous small RNA-based adaptive defense transversions and transitions) (Anzalone et al., 2019).
mechanism that protects the host bacterial cell via sequence- The availability of all new-age GE strategies has not stolen the
specific recognition and targeted cleavage of viral DNA (Jinek thunder of the CRISPR/Cas platform owing to the ease of its use and
et al., 2012). With an approximate length of 32 bp, the length of relevance to editing genes in numerous crop plants. However, it is
CRISPR repeat sequences varies between 21 and 47 bp across only amatter of time before rapidly changingGEmethodswill replace
prokaryotes. Every CRISPR repeat sequence harbors a unique present-day CRISPR/Cas systems with more elegant and efficient
sequence that is specific to the bacterial species processing it and platforms.With every refinement of the platform, we are getting only
has, therefore, been conserved over the course of evolution (Karginov closer to generating precise introduced mutations/deletions with
andHanon 2010). CRISPRwas first discovered by a Japanese group in reduced off-target effects. In the present review, we evaluate the
1987while studying the iap gene from the E. coli genome (Ishino et al., evolution of GE platforms, such as CRISPR/Cas, epigenome editing,
1987). They identified CRISPR as homologous repeated sequences of base editing, and prime editing over the last decade to highlight the
only a few nucleotides interspersed by spacer sequences. Following paradigm shift in our understanding of GE strategies and the
this, CRISPRs were reported from the archaeal genome, Haloferax relevance of these platforms in present-day agriculture.
mediterranei (Mojica et al., 1993). However, the prodigious potential
of the CRISPR/Cas9 as a GE platform was discovered just a decade
ago (Jinek et al., 2012). To employ this tool, a customized small guide 2 Genome editing using zinc-finger
RNA (gRNA) is designed to identify the intended target and guide the nucleases and transcription
associated Cas9 protein to introduce DSBs in the target genomic activator-like effector nucleases
DNA. Indels are introduced at the target site as the repair pathway via
NHEJ is triggered. Over the course of evolution of the platform, new ZFNs and TALENs represent the first phase of the
variants of Cas proteins have beenmobilized to increase the efficiency development of GE platforms. Essentially GE is achieved via
of the CRISPR/Cas9-mediated GE. the introduction of DSBs followed by a homologous repair
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pathway or the NHEJ-DNA repair pathway. In the first phase of DNA cleavage with increased efficiency. Three major steps are
developing GE platforms, SSNs such as ZFNs and TALENs were involved in CRISPR/Cas mechanism. The first step is adaptation,
employed to introduce heritable genomic changes. ZFNs are wherein a small sequence from the mobile genetic elements
chimeric enzymes that work as a dimer. Each monomer has (MGEs) is harbored into the host CRISPR resulting in a novel
3–5 zinc-finger repeats along with a FokI cleavage domain. Each spacer sequence. This adaptive event helps the host bacterial cell
of the zinc fingers is capable of recognizing 3 bp of genomic evade the attack from the same virus in the future (Barrangou
DNA. Therefore, a ZFN dimer can effectively identify an et al., 2007). The selection of the target sequence to be
18–30 bp DNA with a gap of 5–7 bp (Kim et al., 2007). In incorporated into the CRISPR array is sequence-specific. In
plants, the first study involving ZFNs was reported in type I, II, and V CRISPR/Cas systems, a small sequence,
Arabidopsis, wherein heat shock was found to augment ZFN termed the protospacer adjacent motif (PAM), is found
expression. At least 10% of the transgenics obtained displayed the adjacent to the protospacer that is to be incorporated into the
mutations induced by ZFNs in future generations (Lloyd et al., CRISPR array. Therefore, PAM is cardinal to both acquiring the
2005). In maize, ZFNs were employed to introduce a DSB at ipk1, protospacer and bringing about the subsequent interference
and following this, a herbicide tolerance gene was inserted that (Datsenko et al., 2012; Zetsche et al., 2015; Fonfara et al.,
resulted in transgenics showing tolerance to herbicide (Shukla 2016). Although the acquisition mechanism of spacers is not
et al., 2009). One of the major disadvantages of ZFNs is that the yet fully deciphered, in almost all CRISPR/Cas systems, Cas1 and
zinc fingers could overlap and are largely dependent on the Cas2 proteins have been found tomaneuver the acquisition of the
sequence context around them and the intended DNA segment. spacer into the CRISPR array (Makarova et al., 2015; Shmakov
Therefore, employing ZFNs becomes both labor- and cost- et al., 2015). Both these proteins are found to be necessary for the
intensive as for every edit, the zinc-finger array is designed, acquisition of the spacer (Datsenko et al., 2012). The two proteins
and the sites available for the edits are limited (Boch and Bonas form a hetero-hexameric protein complex (Cas1–Cas2), which is
2010). Although many studies have reported ZFNs to edit genes, central to both excision and incorporation of the protospacer
its use as a tool of choice for GE now stands outdated. Another DNA into the CRISPR array (Nuñez et al., 2014). Barring a few
type of nucleases, TALENs, with DNA binding domains, was also exceptions, invariably the spacers are chronologically added to
employed to engineer genomic changes (Boch and Bonas 2010). the array (Shmakov et al., 2015). Cas1–Cas2 protein complex is
Thirty-four tandem repeats are typically present in the DNA central to protospacer acquisition across most type 1 and type II
binding domain along with repeat-variable di-residue (RVD) CRISPR/Cas systems. Therefore, this mode of spacer acquisition
comprised of two amino acids at positions 12 and 13, providing stands most well deciphered so far. In the second step, the
the TALENs with the ability to identify the intended target DNA CRISPR array is transcribed and processed. In addition, the
sequence (Cong et al., 2012; Streubel et al., 2012). Like ZFNs, associated Cas genes are also transcribed into crRNAs. This
TALENs also introduce DSBs in the intended genomic DNA step is subtype-specific, and therefore, subtype-specific
sequences, completely disrupting the gene and (or) introducing enzymes are employed. However, broadly across all CRISPR/
mutations. In comparison to ZFNs, TALENs can be designed for Cas systems, the CRISPR array is first transcribed into a
more target sites in the genomic DNA (Boch and Bonas 2010). In precursor crRNA (pre-crRNA). Different Cas proteins and
rice, TALENs were used to mutate theOsSWEET gene to develop ribonucleases cleave and process this in various types of
transgenic resistance to blight (Li et al., 2012). Similarly, in wheat, CRISPR/Cas systems to yield a mature crRNA. In the third
transgenic with increased resistance to powdery mildew was step, following infection, the mature crRNAs mediate subtype-
developed by employing TALENs induced mutations (Wang specific machinery driven mostly via Cas proteins to ensure
et al., 2014). In cabbage, early flowering plants were obtained effective cleavage of the MGE. The mechanism of different Cas
by employing TALENs (Sun et al., 2013). Like ZFNs, using proteins employed in various CRISPR/Cas systems has been well
TALENs is cost- and labor-intensive with limited success, and documented in many studies (Liu L et al., 2020; Talakayala et al.,
therefore, their use has now been largely suspended for 2022; Wada et al., 2022).
introducing genomic changes.
4 Classification of the clustered
3 Clustered regularly interspaced regularly interspaced short
short palindromic repeats/Cas palindromic repeats/Cas system
system-mediated genetic
modification The classification of the CRISPR/Cas systems identified so far
is primarily based on the presence of the effector Cas proteins
The CRISPR/Cas systems represent the second phase of that cleave the invading foreign nucleic acids. The primary
evolution in the development of GE platforms. CRISPR/Cas classification divides these systems into two classes: Class
systems are sequence-specific and, therefore, mediate targeted 1 and Class 2. Class 1 CRISPR/Cas systems employ a multi-
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protein complex, and Class 2 CRISPR/Cas systems recruit a prokaryotes nucleotide-binding (HEPN) domains with RNase
single effector protein. Further, classification of Class 1 and Class activity (Koonin and Makarova, 2019; Chaudhuri et al., 2022).
2 CRISPR/Cas systems into subtypes (I through VI) is dependent Cas13a was the first protein identified for type VI CRISPR/Cas
on their mechanism of action. The effector module of the systems (Chaudhuri et al., 2022). The evolution of type VI-B,
CRISPR/Cas system is divided into three stages: the such as Cas13b, is thought to have occurred from
adaptation stage, the expression and processing stage, and the transmembrane systems, making them unique from type VI
interference stage. In class 1 CRISPR/Cas systems (with types I, systems into a new subtype type VI-B (Chaudhuri et al.,
III, and IV), type I and type III systems employ a multi-protein 2022). Type VI systems only target RNAs, thus thought to
complex called the Cascade complex along with Cas3 nuclease- have lower instances of off-targeting and, in turn, do not
helicase and the Cmr complex for type I, type III-A, and type IIIB harm the host cell much. The extensive diversity of the
CRISPR/Cas systems, respectively (Koonin and Makarova, 2019; CRISPR/Cas system, as evident by their classification, reflects
Chaudhuri et al., 2022). However, class 2 CRISPR/Cas systems the evolution of the CRISPR/Cas-based defense mechanism in
(with types II, V, and VI) employ only one effector protein. In both archaea and bacteria. In addition, this diversity of CRISPR/
type II and type V CRISPR/Cas systems, the expression and Cas systems presents researchers with varied tools of GE to
processing of the crRNA are regulated by a single protein such as introduce precise changes with efficacy. Table 1 summarizes the
Cas9 and Cpf1, respectively (Makarova et al., 2015; Amitai and classification of the CRISPR/Cas systems identified so far.
Sorek, 2016). Type VI systems have been recently discovered and
are the only CRISPR/Cas systems to target RNA specifically
(Chaudhuri et al., 2022). In Class 1 CRISPR Cas systems, type 5 Repurposing native clustered
1 and type III are more prevalent than type IV in diverse bacterial regularly interspaced short
and archaeal populations. However, type II of the Class palindromic repeats/Cas9 for the
2 CRISPR/Cas system is found across all bacterial species development of genome-editing
(Koonin and Makarova, 2019). Depending on their function, platforms
Cas proteins can be primarily classified into four categories;
recombinases/nucleases that aid the acquisition of spacers, Class II CRISPR/Cas systems were found to be most suitable
ribonucleases that regulate the processing of crRNAs, for development into a tool for genetic manipulation owing to the
scanning complexes like the crRNP complex, and nucleases simplicity of their mechanism of action (Makarova et al., 2015).
that mediate the cleavage of the intended target sequences Type II CRISPR/Cas systems employ Cas9 protein that relies
(Van Der Oost et al., 2014). only on an RNA complex of crRNA:tracrRNA that is easy to
Class 1 CRISPR systems, types I and III, bear structural engineer into a single guide DNA (gDNA) molecule (Jinek et al.,
similarities suggesting evolution via a common ancestor 2012). These systems employ only two components: Cas9, a
(Chaudhuri et al., 2022). In addition, they employ DNA endonuclease, and a customizable gRNA. A single gRNA is
Cas9 endonuclease to process crRNA. Type I CRISPR/Cas sufficient to direct the cleavage of the intended sequences. The
systems are further divided into six subtypes, types I-A, I-B, gRNA molecules are customized to contain a sequence that
I-C, I-D, I-E, and I-F, depending on the distinct PAMs that the Cas9 recognizes and a target sequence that guides the
subunits require to regulate recognition and acquisition. The type complex to the intended locus (Anders et al., 2014). To
III systems are divided into four subtypes, type III-A, III-B, III-C, identify the intended target site, the Cas9-sgRNA complex
and III-D, based on variation in adaptation, recognition, and scans the targeted DNA for a PAM site, following this
interference modules of the effector protein complex. Chaudhuri 12 bases (seed region) of gRNAs proximal to PAM pair with
et al. (2022) discussed the further classification of type I and type the intended target sequence (Semenova et al., 2011).
III into subtypes at length. Class 2 CRISPR/Cas system is divided Mismatches in the seed region have been found to affect the
into three types, types II, V, and VI. Out of these, the type II activity of Cas9 adversely. However, mismatches in the 5’ PAM
system is the most dissected and well-understood system so far distal region are well-tolerated without affecting Cas9 nuclease
(Koonin and Makarova, 2019; Chaudhuri et al., 2022). This activity (Liu et al., 2016). Catalytic domains of Cas9, HNH, and
system employs the Cas9 endonuclease as the effector. Type V RuvC invariably result in a DSB in the DNA. Following this, DSB
system uses a single effector protein, Cas12. However, Cas12 has repair is initiated that is mediated either by homology direct
six subtypes, types V-A, V-B, V-C, V-D, V-E, and V-U, that repair (HDR) or the NHEJ pathway. The latter does not require a
identify distinct PAM sequences (Chaudhuri et al., 2022). Owing template for DNA repair and hence is error-prone. NHEJ is the
to obvious advantages such as smaller size, no dependency on active DNA repair mechanism in nature wherein Cas9-induced
tracr for target recognition, and asymmetric cleavage sites, DSBs are repaired (Moore and Haber 1996). NHEJ can, therefore,
Cas12 has now been actively replacing the Cas9 system for lead to small insertions or deletions that could yield a host of
GE in many animal and plant species. Type VI systems are mutations (Calvache et al., 2022; Wada et al., 2022). Such
characterized by the presence of higher eukaryotes and mutations are beneficial while knocking out a targeted gene
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TABLE 1 Classification of the identified CRSIPR-Cas systems.
Class Type Effector module Class Type Effector module
Class I I-A Cas8a2, Csa5 Class II V-B Cas12b
Class I I-B Cas8b Class II V-C Cas12c
Class I I-C Cas8c Class II V-D Cas12d
Class I I-D Cas10d Class II V-E Cas12e
Class I I-E Cse1, Cse2 Class II V-F Cas14
Class I I-F Csy1, Csy2, Csy3, Cas6f Class II V-G Cas12g
Class II II-A Csn2 Class II V-H Cas12h
Class II II-B Cas9 (Csx12 subfamily) Class II V-I Cas12i
Class II II-C N/A Class II V-J Cas12j
Class I III-A Csm2 (small subunit) Class II V-K Cas12k
Class I III-B Cmr5 (small subunit) Class II VI-A Cas13a
Class I IV DinG (Csf4) Class II VI-B Cas13b, along with proteins, Csx27, and Csx28
Class II V-A Cas12a (previously known as Cpf1) Class II VI-C Cas13c
Class II VI-D Cas13d
using CRISPR/Cas9 systems. However, being random and TaUbiL1. This study also validated the efficiency of using the
unpredictable makes this mode of DNA repair unsuitable for CRISPR/Cas9 system in combination with microspore
precise editing of intended genes. To this effect, HDR is a more technology in plants for both trait improvement and discovery
obvious choice of DSB repair mechanism for incorporation of (Bhowmik et al., 2018). In tomato, complete expression of the
desired sequences following cleavage by Cas9. In plants, GE HDR susceptibility gene SlyPMR4 was knocked down to generate
relies on a DNA template along with the gDNA and Cas9 for a tomato plants with resistance against powdery mildew
successful DSB repair (Calvache et al., 2022;Wada et al., 2022). In (Martínez et al., 2020). CRISPR/Cas9-based GE systems are
plants, through genetic engineering, many outstanding repairs now employed to improve multigenic traits such as biotic and
have been achieved via HDR, leading to gene replacement, DNA abiotic stresses in many crops. Table 2 summarizes studies
correction, and targeted knockouts. Figure 1 illustrates a wherein CRISPR/Cas has been used successfully for trait
diagrammatic representation of the adaptation to the CRISPR/ manipulation in crop plants. Figure 2 depicts schematic
Cas9 system in plants for gene editing. representation of the domains of crop sciences wherein
CRISPR/Cas platforms have largely contributed.
One of the most important applications of CRISPR/Cas9
6 Applications of clustered regularly platforms across the globe has been to engineer disease resistance
interspaced short palindromic in crop plants. Plant pathogens such as bacteria, viruses,
repeats/Cas9 system as a powerful nematodes, insects, and fungi are the most potent biotic stress
tool in crop improvement factors that impact the yield potential of crops across the globe.
Continuously evolving new strains of lethal pests make the battle
Present-day agriculture faces serious threats from both against the pathogens even more complicated and daunting
abiotic and biotic stresses. Rapidly changing climate and (Razzaq et al., 2019). Therefore, to protect and aid crops,
exponentially growing world population increase the pressure methodologies routed in concepts of genome engineering have
of ensuring food security for both present and future generations. been successfully developed (Jaganathan et al., 2018). Peng et al.
To mitigate agricultural losses and to aid crops in realizing their (2017) reported the development of varieties of Citrus sinensis
full potential, the only sustainable solution is to develop climate- (Wanjincheng orange) with increased resistance toXanthomonas
resilient crops. Since its discovery in 2012 as a potential tool for citri, which is responsible for the citrus canker disease in oranges.
genetic engineering, CRISPR/Cas9 system and its derivatives In this study, the expression of the gene, CsLOB1, which is
have rapidly replaced genome engineering methods in crop responsible for the development of the disease, was disrupted
improvement programs across the globe. In model crops such using the CRISPR/Cas9 system. Two alleles (cslob1g and cslob1)
as maize, a CRISPR/Cas9 mediated knocking and replacement in exist for the gene CsLOB1. The promoter region of both these
the liguleless-1 (LIG1) was reported (Svitashev et al., 2016). alleles inhibits an effector binding site (EBE) that is recognized by
Similarly, in wheat, CRISPR/Cas9 GE system was employed to the main effector PthA4 of Xcc to drive the expression of
introduce targeted mutations in two wheat genes, TaLox2 and cslob1 and results in the development of the disease. Five
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FIGURE 1
Schematic representation of steps involved in a CRISPR/Casmediated gene editing in plants. (A). Target gene selection and designing of sgRNA;
(B). Engineering the sgRNA in an appropriate binary vector. (C). CRISPR/Cas mediated cleavage via single/multiplex gene editing. (D). Transformation
in plants; (E). Screening and evaluation of the crops edited; (F). Evaluation of the plants for selecting transgene-free plant with edited gene(s)
regulating the trait of interest (adapted from Jaggannath et al. 2018).
independent constructs pCas9/CsLOB1sgRNAwere employed to the blast lesions. This work led to the development of a rice
modify the effector binding site EBE in the promoter region of cultivar with increased resistance against Magnaporthe oryzae
CsLOB1 alleles. Homologous mutants wherein the EBE was (Wang et al., 2016). In another study, blight-resistant plants were
completely disrupted were obtained, displaying no disease produced using CRISPR/Cas9 system-mediated targeted
development following infection with Xanthomonas citri mutagenesis of the SWEET13 gene (Zhou et al., 2015).
(Peng et al., 2017). In rice, an ethylene-responsive gene Management of diseases in crop plants is dominated by the
OsERF922 was knocked out using the CRISPR/Cas9 tool, frequent use of insecticides to curb yield losses. The development
which led to a marked reduction in the size and number of of crops resistant to viruses is, therefore, an efficient strategy to
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TABLE 2 CRISPR/Cas9-mediated improvement in major crop plants.
Plant species Target gene Trait of interest References
Rice (Oryza sativa) OsAAP6, OsAAP10 Reduced GPC Wang M et al. (2020)
OsBADH2 Fragrant rice Kumar et al. (2021)
eIF4G Resistance to tungro spherical virus Macovei et al. (2018)
OsGAD3 Increased GABA content Akama et al. (2020)
CrtI, PSY Increased β-carotene content Dong et al. (2020)
OsGS3, OsGW2, and OsGn1a Increased grain length and width Zhou et al. (2019)
OsDST Increased drought and salt tolerance Kumar et al. (2021)
OsPIN5b, GS3, and OsMYB30 Increased yield and cold tolerance Zeng et al. (2020)
OsPLDα1 Low phytic acid content Khan et al., 2019
Wheat (Triticum aestivum) TaGW7 Grain shape Wang et al. (2019)
EDR1 Resistant to powdery mildew Zhang et al. (2017)
TaGW2 Grain size Wang X et al. (2018)
α-Gliadin genes Low gluten content Sanchez et al. (2018)
TaBAK1-2, a-eIF4E, Ta-eIF(iso)4E Resistance to streak mosaic virus and yellow mosaic virus Hahn et al., 2021
TaSBEIIa Grain quality Li G et al. (2021)
TaNP1 Male sterility Li et al. (2020b)
Maize (Zea mays) SH2, GBSS Super sweet and waxy corn Dong et al. (2019)
Wx1 Waxy corn Gao et al. (2020)
ZmBADH2a, ZmBADH2b Aromatic maize Wang Z et al. (2021)
CLE genes Enhanced grain yield Liu et al. (2021)
GA20ox3 Semi-dwarf male plants Zhang C et al. (2020)
Tomato (Solanum lycopersicum) ANT1 Fruit color (purple) Čermák et al. (2015)
CLV3 Fruit size Zsögön et al., 2020
Psy1, CrtR-b2 Fruit color (yellow) D’Ambrosio et al. (2018)
OVATE, Fas, Fw2.2 Fruit size, oval fruit shape Zsogon et al. (2018)
ENO Fruit size Yuste-Lisbona et al. (2020)
CRTISO Fruit color (tangerine) Ben Shlush et al. (2021)
slyPDS Increased lycopene content Li J et al. (2018)
SlNPR1 Increased drought tolerance Li et al. (2019)
SlCBF1 Increased cold tolerance Li R et al. (2018)
SlMAPK3 Increased drought tolerance Wang et al. (2017)
miR482b and miR482c Resistance to Phytophthora infestans Hong et al. (2021)
SlyPMR4 Resistance against powdery mildew Martínez et al. (2020)
PL, PG2a, TBG4 Longer shelf life Wang et al. (2019)
SlLBD40 Enhanced drought tolerance Liu et al. (2020)
Rapeseed (Brassica napus) BnaFAD2 Improved fatty acid profile Huang et al. (2020)
BnaMAX1 Improved plant architecture and yield Zeng et al. (2020)
BnaA03.BP Compact plant architecture Fan et al. (2021)
yield a stable yet economically viable alternative (Wang W et al., eIF(iso)4E gene were found to impart complete resistance against
2021). To this effect, inducing deletions and introducing point the turnip mosaic virus (Pyott et al., 2016). Likewise, in
mutations in the genes using the CRISPR/Cas9 system is one of cucumber, eukaryotic translation initiation factor eIF(iso)4E
the most organic adaptations of the platform. The eukaryotic was engineered using the CRISPR/Cas9 system to generate
translation initiation factor genes such as eIF4E and eIF4G are an heritable homozygous point mutations that conferred
absolute requirement for the translation of RNA viruses (Shopan resistance to the mutants against zucchini yellow mosaic virus,
et al., 2020). Therefore, CRISPR/Cas9 technology has been papaya ringspot mosaic virus-W, and vein yellowing virus
employed in numerous plant species to engineer induced (Chandrasekaran et al., 2016). In Nicotiana benthamiana,
mutations in these genes. In Arabidopsis, point mutations in sgRNA/Cas9-mediated broad-spectrum immunity was
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FIGURE 2
Schematic representations of the domains of crops sciences wherein CRISPR/Cas platforms have largely contributed.
achieved against viruses such as beet curly top virus, Tomato leaf UTRof the target gene. Themutant plants were found to overexpress
curl Sardinia virus, Tomato yellow leaf curl virus, and Cotton leaf ARGOS8, which led to a stupendous increase in the yield in
curl Kokhran virus (Ali et al., 2016). In rice, the CRISPR/ comparison to the wild type under drought conditions during the
Cas9 system was used to generate eIF4G alleles that conferred flowering stage without any yield penalty under irrigated
resistance against the Rice tungro spherical virus (Macovei et al., environment (Shi et al., 2017). In rice, the CRISPR/Cas9 system
2018). Recently, Wang et al. (2021) employed the CRISPR/ was used to knock out gene OsRR2. The homozygous mutants
Cas9 system to generate novel eIF4G alleles to yield transgenic obtained displayed increased tolerance to salinity stress (Zhang
plants displaying complete resistance to rice black-streaked dwarf et al., 2019). In another study, three genes, OsPIN5b, GS3, and
virus. Engineering these mutations via the traditional OsMYB30, that determine panicle length, grain size, and cold
backcrossing would have taken years, but using the CRISPR/ tolerance, respectively, were simultaneously edited using the
Cas9 system expedited the process, and the goal was achieved in CRISPR/Cas9 system (Zeng et al., 2020). T2 generations of the
just a single generation. homozygous mutants of these genes displayed increased panicle
The CRISPR/Cas9 systemhas also been used extensively over the length, enlarged grain size, and increased cold tolerance,
past decade in generating climate-resistant cultivars in various crop respectively. The CRISPR/Cas9 tool has also been employed for
species such as cotton, maize, rice, wheat, potato, soybean, and the functional characterization of genes that regulate stress responses
tomato (Khan et al., 2021; Wang et al., 2021; Rahman et al., in plants. In Arabidopsis, three genes (CBF1, CBF2, and CBF3) have
2022). In wheat, two regulatory genes (i.e., TaDREB3 and been identified to confer cold acclimatization and tolerance.
TaDREB2) were mutated using the CRISPR/Cas9 system, which However, the underlying mechanism remained undeciphered
resulted in increased drought tolerance in the mutated plants in owing to the absence of any loss-of-function lines for these genes.
comparison to the wild cultivars (Kim et al., 2017). In maize, the Zhao et al. (2016) generatedmutants of the cbf gene family, cbf1, cbf2,
ZmARGOS8 gene that negatively regulates ethylene response was and cbf3. They generated cbf single, double, and triple mutants using
studied using the CRISPR/Cas9 system. The promoter of this gene the CRISPR/Cas9 platform. Interestingly, for the three genes, cbf
was knocked out and replaced with maize GOS2 promoter in 5′- triple mutants displayed compromised seedling development and
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reduced salt tolerance. However, both triple and double (cbf2cbf3) and 6 (GW2, GW5, and GW6), which are negative regulators of
mutants displayed increased sensitivity to feeding post-cold grain weight, was investigated in rice. A remarkable
acclimatization in comparison to the wild-type control. The cb1/ improvement was observed in grain weight and size (Xu et al.,
cb3 double mutants displayed increased resistance, indicating that 2016). The CRISPR/Cas9 system was also employed in rice to
accumulation of CBF2 is more important than CBF1 and CBF3 in knockout three heading date genes (i.e., Hd2, Hd4, and Hd5) (Li
regulating cold acclimation-dependent freezing tolerance. The et al., 2017). The mutants displayed early heading and higher
functional role of many other genes with a potential role in stress yield under drought stress conditions. Furthermore, a CRISPR/
tolerance was also investigated in the model system Arabidopsis. The Cas9 mediated disruption of the OsSWEET11 gene, known for
expression ofUGT79-B2 andB3 geneswas induced by abiotic stresses grain filling and sucrose transportation in rice, led to reduced
such as salinity, drought, and cold. Overexpression of these genes was sucrose concentration and grain weight, which suggested that
found to increase the resistance of the transgenics. However, gene overexpression of these genes would be beneficial in obtaining a
ugt79b2/b3 doublemutants generated using theCRISPR/Cas9 system better grain quality (Ma et al., 2017). In wheat, GASR7 was
were found to be susceptible to abiotic stresses compared to the wild- knocked out using the CRISPR/Cas9 tool, and the resulting
type control. The overexpression mutants accumulated mutants showed increased kernel weight (Zhang et al., 2016).
anthocyanins, but the ugt79b2/b3 double mutants that displayed In tomato, the use of CRISPR/Cas9 methods has also delivered
lower levels of anthocyanins were also found to be more susceptible seedless tomatoes (Ueta et al., 2017). In this study, a novel
to stresses than the wild-type control plants. These findings also sgRNA/Cas9 was employed, resulting in additional somatic
suggested that an array of anthocyanins impart resistance against mutation in SlIAA9, a key parthenocarpy gene. The mutation
abiotic stresses (Li et al., 2017). In rice, knockout mutants for the rate was 100%, and there were no off-target mutations. The
OsSAPK2 gene were developed for functional characterization of the mutants hence obtained displayed parthenocarpic fruit along
gene. Themutants showed insensitivity to abscisic acid and increased with an altered leaf shape.
sensitivity to drought and reactive oxygen species (ROS) during the
germination/seedling stage compared to the wild-type control plants.
These results suggested the active involvement of the OsSAPK2 gene 7 Evolution of clustered regularly
in mediating drought tolerance through increased stomatal closure interspaced short palindromic
(Lou et al., 2017). In another study,OsAnn3, a rice annexin gene, was repeats/Cas9 platform for precise
knocked out in rice using theCRISPR/Cas9 system. The survival ratio gene manipulation
of T1 mutant lines was found to be adversely affected, indicating that
the expression of OsAnn3 was central in imparting cold tolerance in CRISPR/Cas9 systems have evolved over the years, and many
rice (Shen et al., 2017). other approaches have also been routed in this technology. As
Drought stress in plants is governed by mitogen-activated discussed earlier, CRISPR/Cas9-mediated gene editing necessarily
protein kinases (MAPKs). In tomato, functional characterization introduces DSBs that are subsequently repaired by either NHEJ or
of MAPKs was achieved by knocking down SlMAPK3 using the HDR mechanisms (Kantor et al., 2020). This results in two major
CRISPR/Cas9 system (Wang et al., 2017). The resulting challenges in using CRISPR/Cas9 mechanisms. Firstly, although
slmapk3 mutants displayed severe wilting symptoms along with HDR promises insertion of only sequence-specific DNA, this
lower antioxidant enzymes, increased hydrogen peroxide, and pathway is synonymous with increased instances of indels and
increased membrane damage in comparison to the wild-type limited efficiency (Song et al., 2017). Secondly, reliance on the
control. In another study, a multiplex CRISPR/Cas9 system was HDR mechanism of gene repair restricts gene editing to only
used simultaneously to edit five tomato γ-aminobutyric acid dividing cells, adversely affecting the efficiency of this platform in
(GABA) shunt genes (CAT9, SSADH, GABA-TP1, TP2, and manipulating the disease resistance in plants (Bollen et al., 2018).
TP3). These genes are repressors of GABA metabolism. Hence, Many newer technologies that are primarily rooted in the CRISPR/
targeted mutagenesis of these genes led to a 19-fold increase in the Cas mechanism overcome some of these limitations and are more
accumulation of GABA in fruits and leaves (Li R et al., 2017). precise in achieving genome restructuring in plants. Some of these
The multiplex CRISPR/Cas9 system has proven to be technologies are detailed in the following sections.
beneficial in improving yield substantially in various cereal
crops. In rice, four genes [i.e., Grain Size 3 (GS3), Ideal Plant
Architecture 1 (IPA1), Grain Number 1a (Gn1a), and DENSE 7.1 Multiplex genome editing
AND ERECT PANICLE (DEP1)] were edited using the multiplex
CRISPR/Cas9 technique. The mutant plants displayed marked In plants, it is well documented that cellular processes are
improvement in all the aforesaid traits and resulted in better and orchestrated via the interplay of several redundant genes.
improved yields concerning tiller number and grain yield (Li Therefore, editing a single gene from a gene family has not
et al., 2016). Similarly, multiplex editing using the CRISPR/ been found to confer the desired phenotype as the redundant
Cas9 system of four genes, that is, GS3, Grain Widths 2, 5, genes from the same gene family compensate for the phenotype.
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In polyploid crop species, this presents an additional layer of attempts to increase the efficiency of Cas9 enzymes and, at the
complication due to multiple gene dosages or homolog effects. same time, curb any off-target silencing with the use of enzymes
Hence, a more efficient protocol for gene editing is required to such as dead cas9 (dcas9), SpCas9 Nickase (SpCas9n), and
aid multiplex gene editing. A single vector system has been used FokICas9 (fCas9) (Cong et al., 2013; Guilinger et al., 2014).
to design many sgRNA cassettes with single or multiple Other studies have reported the extraction of Cas9 proteins with
promoters in multiplex gene editing mediated via the increased sequence specificity owing to their novel PAM
CRISPR/Cas9 system (Liu et al., 2017). In Arabidopsis sequences. Nmecas9 was extracted from Neisseria meningitidis
thaliana, two sgRNAs were successfully employed to disrupt specific for PAM sequence 5′-NNNNGATT (Lee et al., 2016).
two homologs of CHLI (magnesium-chelatase subunit I) to SpCas9 is most commonly used for gene editing with a PAM
obtain an albino phenotype as both homologs have a function sequence 5′-NNGRRT (Ran et al., 2015). Modifications have
in the photosynthetic mechanism (Mao et al., 2013). In another been made for SpCas9 to identify shorter PAM sequences that
study in A. thaliana, multiplex gene editing was successfully not only increase the efficiency of the enzyme but also make the
employed to obtain quadruple mutants displaying dwarf delivery of the system easier (Hu et al., 2018). In plants, CRISPR/
phenotype by deploying three gRNAs (Wang et al., 2017). Cas9 mediated gene editing has been employed in many plant
Further, Čermák et al.(2015) developed a tool kit wherein species such as A. thaliana, rice, citrus, and tobacco (Jiang and
Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) was Doudna, 2017). Furthermore, St1Cas9 and St3Cas9 extracted
employed along with tRNA-processing enzymes to from Streptococcus thermophilus have also been employed in
simultaneously express multiple gRNAs. Using this method, CRISPR-mediated gene editing (Jiang and Doudna, 2017). These
they expressed 12 gRNAs from a single transcript to target Cas9 enzymes use different types of tracrRNA and crRNA for
deletions in six genes successfully. These Csy4 and tRNA identifying PAM sequences (Steinert et al., 2015). Out of all these
expression systems have been found almost twice as effective CRISPR systems employed so far, CRISPR/Cpf1, commonly
in introducing mutations. The use of this platform has been known as Cas13, is the most popular (Zetsche et al., 2015).
validated in tobacco (Nicotiana tabacum), tomato (Solanum Unlike Cas9, Cas13 requires only a sgRNA with 4–5 nucleotide
lycopersicum), wheat (Triticum aestivum), barley (Hordeum overhangs. In both animals and plants, the Cas13-mediated gene
vulgare), and Medicago truncatula (Čermák et al., 2015). editing has been found to target the desired genes with none or
Xie et al. (2015) reported an endogenous tRNA-processing very few off-targets (Endo et al., 2016). Due to their successes,
mediating gene editing by CRISPR/Cas9 in rice. Soon after, Tang type V CRISPR/Cpf1 has been popular in both plants and
et al. (2016) reportedly employed a single POL II promoter to drive animals to engineer gene editing (Zhang et al., 2017).
the expression of a hammerhead ribozyme and multiple gRNAs. Francisella novicida-derived FnCpf1 was used to achieve
The ribozyme cleaved distinct sgRNAs, and post-transcription targeted mutagenesis in both tobacco and rice. Similarly,
Cas9 processed functional Cas9 and gRNAs. In maize, the Lachnospiraceae-derived LbCpf1 has also been used to achieve
CRISPR/Cas9-based gene editing was successfully used to mutate targeted mutagenesis (Yin et al., 2017).
the homologs that determine genic male sterility (Liu et al., 2022).
Triple homozygous mutants were obtained that displayed complete
male sterility. Over the course of CRISPR/Cas evolution, multiplex 7.3 Epigenome editing
gene editing has emerged as an efficient tool to develop “multiple
genes-knock-out-cultivars.” Concomitantly, this methodology has Epigenome editing represents the third phase of plant GE,
enhanced our understanding of gene functions of desired traits that wherein changes are introduced to engineer the chromatin via
are governed by multiple genes, gene families, or even pleiotropic modification of epigenome at specific sites. It involves targeted,
genes. The technology has also opened vistas for investigating locus-specific, reversible, and heritable alterations of the
epistatic interactions/associations among genes or gene chromatin structure while bringing in no changes in the
complexes, especially for complex traits, whose genetic nucleotide sequences in the genomes by using epi-effectors.
architecture is largely influenced by epistasis. Epi-effectors are the epigenome engineering tools that
represent a programmable DNA binding/DNA recognition
domain in the genome. Additionally, the catalytic domains
7.2 New Cas variants to broaden the of chromatin-modifying enzymes (DNA methyltransferases
clustered regularly interspaced short and histone acetylases) represent components of an Epi-
palindromic repeats toolbox effector. Different epigenome editing tools are available for
creating, erasing, and reading various epigenetic codes in plants
Since the discovery of CRISPR/Cas9 mediated gene editing, (Jeltsch and Rots, 2018; Miglani and Singh, 2020; Miglani et al.,
numerous modifications have been incorporated into this 2020).
technology to address the issue of incompatible off-target Currently, epigenome editing has been performed through
sequences due to gRNA mismatches. There have been many three molecular platforms: zinc-finger proteins (ZFPs),
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transcription activator-like effectors (TALEs), and CRISPR and its modalities need to be standardized in crop plants for
dead CRISPR/Cas proteins. These act as DNA-binding domains commercial application.
(DBDs), and after interaction with epigenetic domains, they The first successful instance of epigenome editing was
modify the epigenetic marks at targeted sites in the genome to achieved in the model plant species A. thaliana (Johnson
bring about a restructuring of chromatin architecture and gene et al., 2014). A ZFN fused to RdDM (RNA-directed DNA
expression. The principle of epigenomic editing rests on the methylase) component SU(VAR)3-9 HOMOLOG 9 (SUVH9)
formation of fusion proteins between a designed DBD (ZFPs/ was involved in the recruitment of PolV during RdDMmediated
TALEs/nuclease null or dead Cas9) that targets an attached via methyl-DNA binding SUVH2 and SUVH9 proteins at the
enzymatic domain (chromatin modifiers; DNA FWA target to display DNA methylation induced gene silencing.
methyltransferases (DNMTs) or histone acetyltransferases Many other components of RdDM, such as SHH1, NRPD1,
(HATs) to define genomic target sites. Hence, the DNA RDR2, DMS3, and RDM, when joined with ZFs, have also been
sequences of the target genomic site are presented to DNA- shown to induce methylation at the FWA target in A. thaliana
binding protein domains that affect DNA function in the (Gallego-Bartolomé et al., 2019). A CRISPR dCas9-SunTag-
presence of an enzymatic effector domain. This way, based targeting system coupled with tobacco DRM
epigenome editing allows the precise modification of methyltransferase (NtDRMcd) was used to target DNA
individual chromatin marks at selected genomic sites methylation in A. thaliana (Zhong et al., 2014; Papikian et al.,
(Nakamura et al., 2021). 2019). It resulted in the induction of DNA demethylation at
Besides modulating gene expression, epigenome editing is an FWA and SUPERMAN promoters affecting gene transcription
appealing approach for understanding the mechanism of and triggering a developmental phenotype. Further, a repressive
chromatin modification, cellular reprogramming, and effect of H3K9me2 and non-CG DNA methylation on both
regulatory functions. It has applications in both basic research meiotic DSB and crossover formation in plant pericentromeric
involving gene expression studies and application-oriented heterochromatin resulted in manipulation of the rate and
epigenomic engineering of crop plants. The characterization of positions of crossing over. Increase in meiotic recombination
epialleles (i.e., alleles that are genetically alike but show variable in proximity to the centromeres (pericentromeric
genetic expression due to epigenomic modifications) is gradually recombination) and meiotic DNA double-strand breaks
picking up to be fully exploited in future crop improvement (DSBs) in Thale Cress (Papikian et al., 2019). Recently,
programs. Epigenome editing holds great promise in improving Gallego-Bartolomé et al. (2018), Gallego-Bartolomé et al.
crops by creating novel epiallelic diversity that can be exploited (2019), and Gallego-Bartolomé (2020) used ZF and CRISPR-
for future precision and smart crop epi-breeding (Gahlaut S K dcas9-SunTag systems fused with the catalytic domain of human
et al., 2020; Giudice et al., 2021; Kakoulidou et al., 2021). For demethylase TET1cd to test several RdDM components such as
epigenome editing, a modified CRISPR/dCas9 known as dead, RNA-dependent RNA polymerase 2 (RDR2), Microchidia 1 and
deactivated, null, or nuclease deficient Cas9 (dCas9) has been 6 (MORC1 and MORC6), RNA directed methylation 1 (RDM1),
created by silencing two mutations of the RuvC1 (D10A) and and defective in meristem silencing 3 (DMS3) to induce targeted
HNH (H841A) nuclease domains (Qi et al., 2013). The CRISPR- DNA methylation/demethylation at FWA locus in A. thaliana.
dCas 9 approach is attractive as it helps overcome the limitation ZF fusion with catalytic domain human demethylase TET1cd
of the DBD approach, wherein for targeting a different sequence, and SunTag-TET1cd system resulted in demethylation of the
a corresponding distinct protein is required, making it difficult to promoter of FWA (Flowering Wageningen) gene and
target a wide range of loci in the genomes. In this respect, CACTA1 transposon and activation of gene expression. While
CRISPR-dCas9 associated system offers flexibility as associated the fusion of ZF-RdDM and ZF-MORC6 enhanced targeted
gRNAs help the Cas proteins achieve genomic specificity FWA methylation, Microrchidia (MORC6) targeted DNA
(Nakamura et al., 2021). A single dCas protein can be methylation and triggered AGO- and DRM2-dependent
reoriented to target different loci simply by altering the methylation and gene silencing in A. thaliana (Gallego-
sequence of its associated gRNA. This way, the technology Bartolomé et al., 2019; Gallego-Bartolomé, 2020). These
offers a flexible platform for targeting almost any genomic studies provide important experimental evidence to design
sequence (Brocken et al., 2018). Epigenomic editing depends and utilize a highly targeted and heritable DNA methylation/
on inducing changes in chromatin architecture to influence gene demethylation system to modulate gene expression in crop
transcription and relies on primarily inducing reversible and plants.
heritable changes in epigenetic marks such as DNA and histones’ Fusion of CRISPR dCas9-HAT1 gene resulted in
methylation, acetylation, and phosphorylation. This results in hyperacetylation at AREB1 (abscisic acid-responsive element-
novel genetic variation in the form of epialleles and has binding protein 1) locus leading to activation of endogenous
tremendous potential for crop enhancement through epi- promoter of AREB1. This improved transcription of the AREB1
breeding. Although several publications have demonstrated gene involved in ABA perception improved chlorophyll content
the feasibility of epigenome editing in A. thaliana (Table 3), and drought tolerance due to the activation of bZIP TF, which
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TABLE 3 Epigenome editing in the model plant Arabidopsis thaliana.
DNB Domain/targeting system/ Epigenome editing/modification Response References
target
gene
ZFN fused to SUVH9 Recruitment of PolV during RdDM through DNA methylation and gene silencing Johnson et al. (2014)
methyl-DNA binding SUVH2 and
SUVH9 proteins
CRISPR dCas9-SunTag based targeting Manipulation of DNA methylation at FWA Modification of gene expression, induction Zhong et al. (2014), Papikian
system coupled with tobacco DRM promoter of DNA demethylation at FWA, and et al. (2019)
methyltransferase (NtDRMcd) SUPERMAN promoter affecting gene
transcription and triggering a developmental
phenotype
Mutation of the H3K9 methyl transferase Disruption of histone 3 di-methylation on Manipulation of the rate and positions of Underwood et al. (2018)
genes KYP/SUVH4 SUVH5, SUVH6, or the lysine 9 (H3K9me2) and non-CG DNA crossing over (CO). Increase in meiotic
CHG DNA methyl transferase gene CMT3 methylation via mutation of the recombination in proximity to the
H3K9 methyl transferase genes KYP/ centromeres (pericentromeric
SUVH4 SUVH5, SUVH6, or the CHG DNA recombination) and meiotic DNA double-
methyl transferase gene CMT3 strand breaks (DSBs). Repressive effect of
H3K9me2 and non-CG DNA methylation
on both meiotic DSB and crossover
formation in plant pericentromeric
heterochromatin
ZF fusion with catalytic domain human Demethylation of the promoter of FWA Targeted, complete, highly specific, and Gallego-Bartolomé et al.
demethylase TET1cd and SunTag-TET1cd (Flowering Wageningen) gene and CACTA1 heritable demethylation (removal of 5 mC at (2018), Gallego-Bartolomé,
system transposon specific loci in the genome) at FWA (2020)
promoter and activation of gene expression.
Reactivation and upregulation of the FWA
gene and a heritable late-flowering
phenotype. Targeted demethylation and
reactivation of heterochromatic TE-
CACTA1, although demethylation was
incomplete on this locus and remethylation
and resilience occurred once the trigger
construct was segregated out
ZF-RNA directed DNA methylase (RdDM); Co-targeting of both arms of the RdDM Enhanced targeted FWA methylation and Gallego-Bartolomé et al.
ZF-MORC6 pathway, siRNA biogenesis, and co-targeting silencing, microrchidia- (MORC6-) targeted (2019), Gallego-Bartolomé,
of Pol IV and Pol V synergistic recruitment DNA methylation. Trigger of AGO- and (2020)
DRM2-dependent methylation
CRISPR dCas9-HAT1 gene Hyperacetylation at AREB1 (Abscisic acid- Improved transcription of AREB1 gene Paixão et al. (2019)
responsive element-binding protein 1) locus involved in abscisic acid perception.
resulting in activation of endogenous Improved chlorophyll content and drought
promoter of AREB1 tolerance due to activation of bZIP TF that
can activate several stress tolerance-related
genes like RD29A
CRISPR dCas9-TET1 Essential requirement of methylated CG Induction of alternation between two epi- Li C et al. (2020)
(mCG) and mCHG (where H can be A, C, or allelic states at a specific locus
T) for targeting RdDM machinery to
remethylable loci. RdDm target loci to form
stable epialleles in the presence of specific
histone and DNA methylation marks
CRISPR-bacterial methyltransferase MQ1v De novo induction of CG methylation at Improved heritability of induced target- Ghoshal et al. (2021)
and CRISPR-SunTagMQ1v Systems different loci with varying efficiency with specific CG methylation and high specificity
CRISPR-MQ1v and CRISPR-SunTagMQ1v of CRISPR-based MQ1v systems
systems. CRISPR-SunTagMQ1v has shown
to be more potent than CRISPR-MQ1v.
Development of a CRISPR-based CG-
specific targeted DNA methylation system
can activate several stress tolerance-related genes such as RD29A loci were shown to form stable epialleles in the presence of
(Paixão et al., 2019). Further, Li et al. (2020a) showed essential specific histone and DNA methylation marks to induce
requirements of methylated CG (mCG) and mCHG by using alternation between two epiallelic states at a specific locus.
CRISPR dCas9-TET1 fusion (where H can be A, C, or T) for Recently, Ghoshal et al. (2021) used CRISPR-bacterial
targeting RdDM machinery to re-methylate loci. RdDm target methyltransferase MQ1v and CRISPR-SunTagMQ1v and
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FIGURE 3
Schematic representation of base editing in plants by using DNA and RNA base editors. (A). CRISPR/Cas9 system-mediated cytosine base
editing system (CBE). A sgRNA-dCas9 complex binds to the intended target sequence following this cytidine deaminase catalyses the deamination of
cytosine (C) resulting in a C-G to T-A conversion. (B)CRISPR/Cpf-1mediated CBE system. In this system, dCpf1 is fusedwith a cytidine deaminase, to
make C-G to T-A conversion in the non-targeted DNA strand. (C). CRISPR/Cas9-mediated adenine base editing system (ABE) employs an
Adenosine deaminase and catalytically impaired Cas9 fusion product to bind to the intended target site. The adenosine deaminase catalyses an A
(adenine) to I (inosine) change at the target site to introduce A-T to C-C conversion in the DNA strand (adapted from Bharat et al. 2020).
developed a CRISPR-based CG-specific targeted DNA marks and epigenetic target identification are among the current
methylation system to achieve de novo induction of CG thrust research areas. A few genetic elements controlled by DNA
methylation at different loci with varying efficiency. CRISPR- methylation and linked to desired plant traits have been
SunTagMQ1v was shown to be more potent than CRISPR- identified. For instance, naturally occurring epi-alleles that
MQ1v. These MQ1v-based tools appear to be attractive as accumulate high levels of vitamin E in tomatoes are associated
they offer flexibility to induce methylation at different levels at with differential methylation of a SINE retrotransposon located
different loci and show high specificity attributed to the Q147L in the promoter region of gene VTE3(1) (Quadrana et al., 2014).
mutation. Further, the study also demonstrated that for some In cotton, the COL2 epi-allele is associated with DNA
loci, CG methylation alone was enough to silence gene methylation changes and affects flowering time (Song et al.,
expression, and for these loci, CRISPR-MQ1v and CRISPR- 2017). It is important to accumulate epigenomic data in
SunTagMQ1v systems were likely to be more efficient than various crop species to help identify the potential candidate
the DRM2-based SunTag system developed by Papikian et al. editing targets. Information on genome-wide changes in DNA
(2019) described above. methylation in response to environmental stress has been
The above examples show the potential of epigenome editing gathered in crops such as rice (Guo et al., 2019; Rajkumar
technology inmodulating gene expression and showing observable et al., 2020), wheat (Kumar et al., 2017), soybean (Song et al.,
changes in the phenotypes by altering the DNAmethylation status 2012), and sesame (Komivi et al., 2018).
at various genetic loci in A. thaliana. Similar studies need to be
extended to crop species for exploiting the advantages of locus-
specific modulation of DNA methylation through epigenome 7.4 Base editing
editing. The new tier of epigenetic variability generated by
epigenome editing has significant potential in bringing about Base editing (BE) is a novel GE technology representing the
the genetic enhancement of crop species. fourth phase of the evolution of GE platforms wherein a single
Epigenome editing, as discussed here and in many other nucleotide in a DNA or RNA can be substituted irreversibly. The
reviews (Gahlaut V et al., 2020; Giudice et al., 2021; Kakoulidou process does not involve a double-stranded breaks (DSB) and
et al., 2021), offers opportunities for editing epigenetic codes in hence bypasses the undesirable effects of NHEJ and HDR
plant genomes globally or at selected loci to create novel genetic mechanisms. Of all the previous tinkering tools, BE is the
variability. To harness the benefits of epigenomic editing, most attractive for the simple reason that here the genome
however, it is important to define the specific epimark(s) modification is “base-pointed” and precise. It does not involve
linked with specific phenotypes and agronomic traits of additions or deletions in the genome (i.e., no change occurs in the
interest. In this context, genome-wide mapping of epigenomic DNA content of the organism). Neither does it involve the
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TABLE 4 Base editing mediated proof of concept and improvement studies in major crop plants.
Aim Editor Plant Genes targeted References
Proof of concept/ CBE Rice OsNRT1.1B,OzSLR1,OsCDC48,OsSPL14,OsSERK1, Lu and Zhu (2017), Zong et al. (2017), Ren
demonstration of editing OsSERK2, OsPi-ta, OsSBEIIb, OsPDS, OsALS, et al. (2017), Li P et al. (2017), Ren et al.
efficiency OsAOS1, OsJAR1, OsJAR2, OsCOI2, OsSNB, OsSPL7, (2018), Wang et al. (2019), Qin et al.
OsPMS3, OsSPL14, OsIPA1-T1, OsMKK6, OsEhd1, (2019), Sretenovic et al. (2021)
OsPi-d2, OsMPK3, OsROC
Wheat TaLOX2 Zong et al. (2017)
Maize ZmCENH3 Zong et al. (2017)
Arabidopsis LFY Choi et al. (2021)
Tomato SlALS1, SlCYC-B, SlDET1, SlDDB1, SlETR1, SlETR2, Hunziker et al. (2020), Kashojiya et al.
SlHWS, SlDELLA (2022)
Rapeseed BnaCLV3, BnaRGA, BnaA3.IAA7, BnaDA1, BnaALS Hu et al. (2020), Cheng et al. (2021)
ABE Rice OsACC-T1, OsALS-T1, OsCDC48-T3, OsDEP1, Hua et al. (2018), Hua et al. (2019), Wang
OsNRT1.1B-T1, OsIPA1, OsSLR1, OsMPK6, et al. (2019), Hua et al. (2020a), Sretenovic
OsMPK13, OsSERK2 and OsWRKY45, OsSPL14, et al. (2021)
OsSPL17, OsSPL16, OsSPL18, OsIDS1, OsTOE1,
OsSNB, OsPMS3, OsPMS1, OsSPL14, OsLF1,
OsIAA13, OsSPL7, OsSPL4, OsMADS5, OsWx, OsPi-
d3, OsGL2, OsGRF3, OsSLR1,
OsWSL5, OsZEBRA3 (Z3), OsROC
Wheat TaDEP1, TaGW2, TaALS, TaTub Li J et al. (2018), Han et al. (2022)
Tobacco NbPDS Wang W et al. (2021)
CGBE Rice OsALS, OsCGRS55 Sretenovic et al. (2021)
Tomato AGO7
Poplar PtPDS1, PtPDS2
DuBE Rice OsAAT, OsACC, OsCDC48, OsDEP1, BADH2-2, Li et al. (2020a), Xu R et al. (2021)
FSD2-1, LAZY1-2
Co-editing CBE Pear, apple PDS, ALS Malabarba et al. (2021)
Double CBE CBE Potato StDMR6-1, StGBSSI Veillet et al. (2020)
Simultaneous base CBE Rice OsSPL14, OsSPL17, OsSNB Hua et al. (2019)
editing and ABE
To introduce premature Poplar 4CL1, PII Li R et al. (2021)
stop codon
Resistance to biotic CBE Rice OsPi-d2, OsFLS2 Ren et al. (2017)
stress
Herbicide tolerance CBE Rice, wheat, watermelon, ALS1, ACC, GS1, TubA2 Chen et al. (2017), Tian et al. (2018),
foxtail millet, Arabidopsis, Zhang A et al. (2019), Veillet et al. (2019),
potato, pear, tomato, rapeseed Veillet et al. (2020), Cheng et al. (2021),
Kuang et al. (2020), Liu et al. (2020), Wu
et al. (2020), Zhang J et al. (2020),
Malabarba et al. (2021), Liang Y et al.
(2022)
Improved grain/fruit/ CBE Rice Waxy Li et al. (2020a), Xu et al. (2020), Tra et al.
seed quality (2021)
incorporation of DNA from another organism (i.e., the edited transversion editing. Figure 3 presents a schematic
organism does not become a GMO). It minimizes the chances of representation of the working mechanism of the base editing
unintended, unwarranted effects on the phenotype (Rees and Liu, methodology that has been employed for GE.
2018; Deb et al., 2022). With a perfect BE toolbox, one can
envisage generating desirable alleles for a trait by simply making 7.4.1 Cytosine base editors C to T
the required substitutions. All that is required is a base modifying GE has been revolutionized by engineering the CRISPR/
enzyme linked to a modified endonuclease, such as dCas9, which Cas9 to enable cytosine base editing (Komor et al., 2016). The
can target a desired region in the genome but not cause a DSB. first-generation cytosine base editors (BE1) comprised of
Since the advent of this technology in 2016, it has become catalytically dead dCas9 (D10A, H840A) fused with rat
possible to execute C to T and A to G transition and C to G apolipoprotein B mRNA editing enzyme (rAPOBEC1), a
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cytidine deaminase operating on ssDNA via a 16aa XTEN linker conversion of C to U and subsequently to G via base excision
at its N-terminus (rAPOBEC1-XTEN-dCas9). Although BE1 was repair (Chen et al., 2021) or by translesion polymerization (Liu
highly efficient in converting C:G to T:A in vitro, the same et al., 2016). The nicking of the opposite strand triggers the repair
decreased considerably when assessed within cells because of machinery of the cell, which converts C:G to G:C.
the base excision repair mechanism (BER). To bypass the in vivo
repair response and overcome decreased efficiency, second- 7.4.4 Dual-base editors
generation cytosine base editors (BE2) were formed by fusion Dual-base editors have recently been developed by merging
of Uracil DNA glycosylase inhibitor (UGI) to the C-terminal of the cytosine and adenine deaminases in a single editor termed
BE1. This inhibited the action of Uracil DNA glycosylase (UDG), variably as SPACE (synchronous programmable adenine and
which would otherwise have catalyzed the removal of U, resulting cytosine editor) (Grunewald et al., 2020), STEMEs (saturated
in reversion to C:G through BER. The C:G to T:A conversion targeted endogenous mutagenesis editors) (Li et al., 2020), ACBE
efficiency was sought to be further enhanced by generating a nick (adenine and cytosine base editor) (Xie et al., 2020), and DuBEs
on the non-edited DNA strand, thereby stimulating the cellular (dual-base editors) (Xu et al., 2021). Grunewald et al. (2020)
mismatch repair mechanism (MMR), which would replace the G fused the monomeric TadA of miniABEmax-V82G6 and
on the nicked strand opposite the U on the target strand by an A, pmCDA1 of Target-AID5 with the adenine deaminase at the
resulting in a U:A, which gets repaired to result in the desired T:A N-terminus and cytosine deaminase at the C-terminus of nCas9
substitution. This resulted in BE3, a BE2 with a dCas9 modified (D10A). Sakata et al. (2020) and Xie et al. (2020) also used the
to enable nicking activity (nCas9-H840A), resulting in much same architecture. Zhang et al. (2020) developed DuBEs (A&C-
more efficient C:G to T:A substitutions (Komor et al., 2016). BEmax) by fusing the two deaminases to the N-terminus and
found that hAID-TadA-TadA*linked to nCas9 (D10A) along
7.4.2 Adenine base editors A to G with two UGIs yielded higher editing efficiency compared to
Although CBEs use naturally occurring cytosine deaminases multiplexing with individual deaminase editors in human cells.
to convert cytosine to uracil or 5-methylcytosine to thymine, no Li et al. (2020) developed STEMEs by fusing both deaminases,
known adenine deaminases could deaminate the adenosine in APOBEC3A/ecTadA, to the N-terminus of nCas9 (D10A) and
DNA. In a significant breakthrough, Gaudelli et al. (2017) used tested them in rice. They reported better C to T and A to G
directed evolution to form a modified transfer RNA adenosine editing with the DuBE than that achieved using co-delivered
deaminase (TadA*), which could catalyze the deamination of deaminases and could generate herbicide resistance in rice.
deoxyadenosine in an ssDNA resulting in a deoxyinosine. TadA* Overall, DuBEs were more efficient in C to T edits than A to
was joined through the XTEN linked to the N-terminus of G. However, the plant DuBE version 1 (pDuBE1) developed by
Cas9 nickase with a nuclear localization signal (NLS) at its Xu et al. (2021) using TadA-8e and LjCDA1L-4 (Lethenteron
C-terminus (TadA*–XTEN–nCas9–NLS). The group japonicum CDA1-like 4) fused to the opposite termini of nCas9
engineered seven generations of ABEs to arrive at ABE7.10, (D10A) displayed highly efficient simultaneous A to G/C to T
which had high efficiency in converting A:T to G:C (Gaudelli edits (49.7%) in rice calli. Liang et al. (2022) furthered the scope
et al., 2017). of DuBEs by engineering an AGBE (fusing a CGBEwith an ABE),
which could render efficient C to G, C to T, C to A, and A to G
7.4.3 Cytosine to Guanosine base editor C to G editing possible in mammalian cells.
It had been observed that although the efficiency of C to T
transitions increased considerably by fusing UGI to BE1, in 7.4.5 Base editing in plants
absence of the glycosylase inhibitor, C to T conversions were Base editing (C to T transitions) in plants was demonstrated
not so clean and were accompanied by C to G and C to A for the first time in rice (Lu and Zhu, 2017; Ren et al., 2017; Zong
transversions (Komor et al., 2016). This action of glycosylase, et al., 2017; Li et al., 2017). Lu and Zhu (2017) formed a fusion
which sought to be inhibited in CBEs for improved recovery of protein, APOBEC1-XTEN-Cas9(D10A), as described by Komor
clean C to T substitutions, was tapped for accomplishing C to G et al. (2016), put it under the ubiquitin maize promoter, and used
transversion in CGBEs. Uracil DNA N-glycosylase (ecUNG) it for editing OsNRT1.1B and OsSLR1 in rice. Sequencing
from Escherichia coli (Kurt et al., 2021; Zhao et al., 2021) or confirmed C to T (1.4%–11.5%) and C to G (1.6%–3.9%)
rat XRCC1 (Chen et al., 2021) were linked to a nCas9 (D10A) and substitutions in both genes to be more in SLR1 than NRT1.1B.
further fused with a rat cytidine deaminase rAPOBEC1 (Chen Indels (10%) were much more than the <1% reported by Komor
et al., 2021; Zhao et al., 2021) or its engineered variant et al. (2016), probably because no uracil glycosylase inhibitor
rAPOBEC1 (R33A) (Kurt et al., 2021) or with human (UGI) was used. Zong et al. (2017) tailored the base editors by
activation-induced cytidine deaminase (h-AID) (Zhao et al., including UGI to form pnCas9-PBE (rAPOBEC1-nCas9-D10A-
2021). The resultant CGBEs or GBEs (glycosylase base UGI) and pdCas9-PBE (rAPOBEC1-dCas9-UGI) and found that
editors), UNG-nCas9-APOBEC1, XRCC1-nCas9-APOBEC1, these bring about C to T substitutions in three rice (cell division
UNG-APOBEC1-nCas9, and h-AID-nCas9-UNG, result in the cycle mutation 48 OsCDC48, nitrate transporter OsNRT1.1B,
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and a plant architecture gene OsSPL14), one wheat (TaLOX2), repertoire and performed better than the former at other
and one maize (ZmCENH3) gene with hardly any indels. PAM sites, whereas xCas9-BE4 (Hu et al., 2018) was the least
Cas9 nickase-based editor was more efficient than the one efficient in mammalian cells. Zhong et al. (2019) tested
with dCas9. In the same year, Li et al. (2017), while reporting xCas9(D10A)-rAPOBEC1, xCas9(D10A)-PmCDA1-UGI, and
greater than 40% substitutions, proposed that editing efficiency Cas9(D10A)-NG-PmCDA1-UGI in rice and concluded that
could vary depending on the target locus amongst three targeted xCas9(D10A)-based editors were comparable in efficiency to
loci (one on OsPDS and two on OsSBEIIb) of rice. those based on wtCas9(D10A). The former demonstrated
One of the limitations that were obvious in the initial period better fidelity concerning the protospacer, and Cas9-NG-based
of the use of this technology was the restriction imposed by the editors were more efficient among all three tested at relaxed PAM
availability or otherwise the canonical PAM sites in a genome. To sequences. Endo et al. (2019) used SpCas9-NGv1 nickase in rice.
overcome this challenge, Cas variants/orthologues with relaxed Veillet et al. (2020) used SpCas9NG-based CBE for editing
PAM sites both naturally occurring and engineered have been granule-bound starch synthase (StGBSS) and Downy Mildew
employed. Further, since the first reported use of rAPOBEC Resistant 6 (StDMR6-1) in potato. They also tested the
cytidine deaminase from a rat in BE1, deaminases sourced from performance of this editor in tomatoes by targeting two PAM
other organisms such as human apolipoprotein B mRNA editing sites in the acetolactate synthase (ALS) gene. GGT gave a lower
enzyme (hAPOBEC3A) (Gehrke et al., 2018; Wang W et al., efficiency (32%) than the canonical PAM NGN (64%).
2018), hAID (Hess et al., 2016), Petromyzon marinus cytidine Hua et al. (2018) adopted ABE7-10 (Gaudelli et al., 2017),
deaminase 1 (PmCDA1) (Nishida et al., 2016), and their mutated developed adenine base editor plant version 1, ABE-P1
forms with varying features vis-a-vis editing window, size, [TadA*7.10-SpCas9(D10A) nickase], and 2, ABE-P2
sequence preference, and so on have been reported (Cheng (TadA*7.10-SaCas9(D10A) nickase), and tested them on two
et al., 2019). rice genes: ideal plant architecture OsIPA1 and slender plants
Various proof of concept studies conducted in plants for base OsSLR1. In 2019, they made several new versions, ABE-P3, P4,
editing using natural and engineered variants of Cas in and P5, using SpCas9nVQR (D10A) and SpCas9-VRER (D10A)
combination with different cytidine/adenine deaminases have to increase target genome accessibility. They could successfully
been listed in Table 4. A SpCas-9 variant, SpCas9-VQR (D1135V edit at four loci: SPL14, SPL17, SPL16, and SPL18. With the same
+ R1335Q + T1337R), recognizes NGAN and NGNG PAM sites, set-up, they could demonstrate simultaneous cytosine and
broadening the reach within a genome (Kleinstiver et al., 2015). adenine editing using ABE-P2 and CBE-P1. Similar to reports
Ren et al. (2017) used this variant to develop two CBEs for rice, in mammalian systems, there were no indels or off-target or any
rBE3 (APOBEC1-XTEN-Cas9n-UGI-NLS) and rBE4 other unplanned base substitutions seen in rice. However, the
(APOBEC1-XTEN-Cas9nVQR-UGI-NLS), and successfully editing windows were larger in the target genes. Hua et al. (2019)
edited a blast susceptible protein and OsCERK1 (a receptor explored the use of SpCas9 and SaCas9 variants for widening the
kinase) with an efficiency of 17%. Steinert et al. (2015) and scope of the adenine base editing toolbox. They used nickases of
Kaya et al. (2017) recommended the use of Staphylococcus aureus VQR-, VRER-, and SAKKH-SpCas9 engineered variants to form
Cas9 (SaCas9) in plants because of its smaller size, longer target three ABEs, ABE-P3 (pRABEspVQR), ABE-P4 (pRABEsp-
sequence, different PAM, and somewhat higher efficiency than VRER), and ABE-P5 (pRABEsa-SaKKH), and two CBEs with
spCas9. A variant with three mutations E782K/N968K/R105H spCas9-VRER and saCas9-SAKKH, all of which were designed
(SaCas9-KKH SaKKH) has a relaxed PAM (NNNRRT) and tested in rice. The CBE and ABE formed with xCas9 were not
compared to the wild type (Kleinstiver et al., 2015). Qin et al. efficient. Wang et al. (2021) compared the capabilities of ABE8e
(2019) developed nSaCas9(D10A) and nSaKKH(D10A) nickase- and ABE7.10 in Nicotiana benthamiana and established that
based CBEs (Sa-BE3, SaKKH-BE3, Sa-eBE3, and SaKKH-eBE3) ABE8e (60.87%) was more efficient than ABE7.10 (20.83%).
and ABEs (Sa-ABE and SaKKH-ABE/ABE-P5) reporting up to Sretenovic et al. (2021) studied the applicability of CGBEs,
71.9% cytosine edited (nSaCas9, SLR1 gene) and 63.2% adenine for affecting transversions in plants for the first time. They
edited (nSaCas9, OsSPL17 gene) rice plants. Veillet et al. (2020) improvised the three CGBE platforms for successful use in
used the nickase SaCas9 (nSaCas9) with PmCDA1 to modify humans (Chen et al., 2021; Zhao et al., 2021; Kurt et al.,
granule-bound starch synthase (StGBSS) and Downy Mildew 2021) for use in three plant species: rice, tomato, and poplar.
Resistant 6 (StDMR6) in potato. It recognizes 5’--NNGGAT-3′ All three used the rat-derived rAPOBEC1 or its engineered
as a PAM site and has an editing window from −23 to −22. variant rAPOBEC1 (R33A). rAPOBEC1 in combination with
Nishimasu et al. (2018) engineered spCas9 to recognize NG ecUNG or rXRCC1 was fused with nCas9 (D10A), whereas
(spCas9-NG), a relaxed PAM, and used the nickase version rAPOBEC1 (R33A) was linked to rescuing and nCas9
fused with activation-induced cytidine deaminase (nSpCas9- (D10A). Three, four, and two target sites were chosen for
NG-AID/Target-AID-NG) to determine their editing editing in rice, tomato, and poplar, respectively. As compared
efficiencies. Although Target-AID had a better efficiency at to BE3, all three CGBEs induced better C to G conversions, but
the canonical PAM, Target-AID-NG had a wider PAM the overall efficiency of conversion was less than that reported in
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FIGURE 4
Diagrammatic representation of the Prime editing. sgRNA: single-guide RNA; Cas9n: Cas9 nickase; PAM: protospacer adjacent motif; PBS:
primer binding site; RT: reverse transcriptase; pegRNA: prime editing guide RNA; PE: prime editor (adapted from Hassan et al. 2020).
humans. The efficiency of editing using SpRY, which is not PAM (Malabarba et al., 2021), pear (Malabarba et al., 2021), oilseed
dependent, was also assessed. The authors achieved C to G rape (Wu et al., 2020; Cheng et al., 2021), Arabidopsis (Chen
editing, although the efficiency varied according to the system et al., 2017), foxtail millet (Liang et al., 2022), and wheat (Zhang
and target site. Because this was the first report, much needs to be et al., 2019). The eating and cooking quality (ECQ) is of utmost
done to improve the efficiency of plants. importance for all cereals, and it is primarily determined by the
Base editing is still an evolving technology, and many reports amylose content in the grain, determined by the Waxy (Wx)
primarily demonstrate the successful use of a base-editing gene-encoded granule-bound starch synthase I (GBSSI) (Li et al.,
toolbox in different plants. This technology can create random 2016). Xu et al. (2021) used CBEs to develop rice lines expressing
variations within genomes, which can be screened and selected a range of amylose content (0%–12%), which improved its ECQ
for advantageous traits. It also holds a great promise for considerably by making several substitutions near the soft rice
improvement in traits affected by SNPs. Applications of the allele site in Wx. Similarly, Li et al. (2020a) lowered the amylose
technology have been reported mainly as a gain of function content in rice grains. Veillet et al. (2020) incorporated base
for herbicide resistance and disease resistance and improvement substitutions in the GBSSI locus in potato, which could
in plant architecture, eating, and cooking quality (Table 4). eventually be used for controlling amylose content in the tubers.
Base editing of acetyl-CoA carboxylase (ACC) and Traditional methods of inducing mutations become
acetolactate synthase (ALS1) genes has been shown to confer especially difficult in polyploid species because they possess
herbicide resistance in rice (Li et al., 2020b; Liu et al., 2020; Zhang more than two copies of a gene. Base editing has successfully
et al., 2020), tomato (Veillet et al., 2019; Veillet et al., 2020), generated heritable substitutions in polyploid species such as
potato (Veillet et al., 2019), watermelon (Tian et al., 2018), apple oilseed rape, wheat, and cotton. Hu et al. (2020) used
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BnA3A1-PBE in rapeseed and demonstrated an editing RNaseH activity, and DNA–RNA substrate affinity. In
efficiency of up to 50.5%, much higher than 23.6% reported developing second-generation prime editors, PE2 an RT with
by Cheng et al. (2021) and 1.8% by Wu et al. (2020). Li et al. five mutations (D200N, L603W, T330P, T306K, and W313F),
(2018) demonstrated slight success (0.1%–1.1%) of PABE when fused with the nickase, was found to increase the efficiency
1–7 in affecting A to G transitions in the TaDEP1 and of the GE by 1.6–5.1 fold (Sretenovic and Qi 2022). The use of
TaGW2 wheat loci. PE2 was found to hinder the efficiency primarily due to two
It is quite evident that this technology has immense potential, factors. Firstly, the choice of single-stranded overhangs called
and once the challenges of discovering more efficient, PAM- “flaps” between unedited and edited to be paired with the native
independent DNA-binding proteins, better deaminases that can unmodified DNA strand. Secondly, choosing DNA strands as a
affect cleaner edits with zero off-targets, and engineering all template for DNA repair between unedited and edited was rather
possible substitutions are found, base editing can create a random (Gaudelli et al., 2017; Sretenovic and Qi, 2022). Many
revolution in the field of plant sciences in general and crop studies have shown that the introduction of nick in the
improvement in particular. unmodified strand enhanced the editing efficiency in both
plants and animal cells (Komor et al., 2016; Gaudelli et al., 2017;
Zong et al., 2017). Hence, to generate third-generation prime editors,
7.5 Prime editing PE3, nickase employed was used with an additional sgRNA to
simultaneously nick the other complementary strand (Anzalone
Prime editing marks the fifth phase of evolution in GE et al., 2019). This strategy enhanced the editing efficiency to
platforms. The technique was first developed and standardized introduce point mutations three-fold (Anzalone et al., 2019).
in human cells. Prime editing facilitates indels and all 12 possible With the use of the same protospacer, off-target instances were
base-to-base conversions, including transversions and found much lower for PEs in comparison to the use of Cas9 (Jiang
transitions, without triggering the error-prone repair pathways et al., 2021; Jiang et al., 2022). The increased efficiency of the prime
by the DSB (Anzalone et al., 2019). Briefly, in this technique, editor is attributed to multiple DNA hybridization events that occur
paired/coupled prime editing guide RNA (pegRNA) is composed with the use of PEs. At first, the intended genomic DNA and spacer
of single gRNA that is complementary to the one strand of the of the pegRNA hybridize. Next, hybridization occurs between the
targeted DNA along with a primer-binding site (PBS), and the target sequence in the genomic DNA and the PBS of the pegRNA,
customized sequences to be replaced at the target site fused with adding to the sequence specificity of the system. Finally, the target
Cas9 nickase are also present (Kumar et al., 2021). The PBS DNA also hybridizes with the edited DNA, which further adds
region primes to the second DNA strand to drive reverse another layer of sequence specificity to the system (Jiang et al., 2021;
transcriptase (RT) linked with the Cas9 nickase. RT Jiang et al., 2022). On the contrary, in a regular CRISPR/
transcribes and, in the process, copies the information Cas9 system, only one step of hybridization occurs between the
straightaway from pegRNA into the intended target site. sgRNA and the target genomic DNA occurs (Jiang et al., 2022;
Following this, 5′ and 3’ are the single-stranded overhangs Zhuang et al., 2022). Figure 4 presents a schematic representation of
integrated into the genomic DNA via endogenous DNA repair the working mechanism of the prime editing methodology that has
mechanisms (Anzalone et al., 2019). been employed for GE.
Research has successfully validated three generations of The success of prime editing protocols hinges on optimizing
primer editors (PEs), PE1, PE2, and PE3, in humans so far. In critical parameters such as transformation system, selection of
PE1, the first-generation PEs, wild-type reverse transcriptase suitable vectors, design of prime editor cassettes (nuclease/
from commercial Moloney murine leukemia virus (M-MLV) nickase), structure/sequence of, for example, pegRNA,
fused to the C terminus of the Cas9 (H840A) nickase was used, sgRNA, codon optimization of the vector constructs,
triggered by the expression of pegRNA in a distinct plasmid. As promoters, use of novel/engineered endonuclease, ribozymes,
mentioned earlier, pegRNA harbors a spacer sequence to reverse transcriptase, targeted genes, and method/s of
recognize and bind to the intended target site. In addition, detection. Agrobacterium-mediated transformation and floral
pegRNA carries an 8–15 nt of PBS and a template sequence to dip agroinfiltration are the preferred modes of gene transfer as
drive RT. However, the template sequence also contains a single copy inserts are efficiently achieved. However, other
customized, altered DNA sequence to be incorporated at the methods such as electroporation, PEG-mediated gene
intended site. The efficiency of this PE is largely determined by uptake, microinjection, and particle bombardment have
PBS length. Generally, 8–16 nt PBS length has been found to been tested in different plants and are now expanding
deliver results with increased efficiency (Anzalone et al., 2019). In rapidly to include monocots (rice and maize), dicots
an attempt to further increase the efficiency of this PE, numerous (Arabidopsis, Nicotiana benthamiana, potato, and tomato),
variants of M-MLV RT have been used. These variants were and even the bryophyte, Physcomitrium patens (Perroud
generated by inducing mutations in M-MLV RT. These et al., 2022) that is well known for incorporating DNA
mutations were found to alter processivity, thermostability, into specific genomic sites due to its innately high
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frequencies of homologous recombination (Rensing et al., Optimization of the melting temperature (Tm) of the PBS to
2020). around 30°C coupled with a dual-pegRNA strategy in plants (Lin
Researchers have been experimenting extensively with the et al., 2020) drastically increased the editing efficiencies by 17-
precise modeling of the molecular tool kit for high efficiency and fold in rice protoplasts, although stable expression and
specificity in several plants. As mentioned earlier, three versions transmission of the edits remain to be seen. Inclusion of the
of prime editors (PE1, PE2, and PE3) have been tested since t-RNA processing system (Xie et al., 2015) allows for the
2019 in human and plant cells. The versions vary in the use of generation of multiple gRNAs that allow for “multiplex GE.”
nickase, type of reverse transcriptase, position (C terminal or Detection of editing relies on the rates of transformation
N-terminal fusion with nickase), length of the prime binding site, coupled with the rate of editing. Several studies have reported the
and types of editing predicted (Jiang et al., 2022). Promoters co-transfection of T-DNA-containing vectors with the transgene
driving the expression of the prime editor apoprotein and the and the PE vectors harboring the editor and the edit. The targeted
gRNAs play an important role in the overall scheme of prime sites are usually PCR amplified from the genomic DNA isolated
editing in taxa and target gene of choice (Sretenovic and Qi from transformed plants and sequenced to identify the edits.
2022). Target sites have been categorized as type I and type II Most researchers have done Sanger’s sequencing, although the
based on the position of the edit concerning the nicking site. If HRM-High ResolutionMelting analysis has been included before
the edit is within 1–6 bp downstream of the pegRNA nicking site, sequencing by Perroud et al. (2022). Hi-TOM (high-throughput
then higher editing efficiencies are observed compared to the type tracking of mutations) was used by Xu et al. (2022) in maize
II targets, where the targeted edit position(s) are 7–17 bp and rice.
downstream of the pegRNA nicking site (Sretenovic and Qi, Different selection and counter-selection strategies have been
2022). The editing efficiencies of the same vectors thus vary with tested for the selection of transformed/edited cells. Perroud et al.
the target genes. This was reported in rice, where the prime editor (2022) have tested the use of APT/APRT (adenine phosphoribosyl
Sp-PE3 and gRNA were successful in introducing an S627N transferase) enzyme that catalyzes the conversion of adenine to AMP
mutation in the endogenousALS (acetolactate synthase) but were in Physcomitrium. This enzyme can convert 2-fluoroadenine (2FA)
unsuccessful in editing the APO1 (aberrant panicle organization) supplemented in the culture medium into a toxic 2-fluoro AMP
gene (Hua et al., 2020a). It was also successfully induced and counter selective compound. Thus, if the editing vectors are
present in regenerants. Three endogenous genes (GAI, ALS2, and successful, the APRT is mutated and the cells can grow and
PDS1) from tomato were tested for prime editing by PE3 strategy regenerate into plants on the 2FA medium. The DNA from these
using an optimized prime editor. Prime editing frequencies of plants is further analyzed to detect edited sequences. In potato, the
0.025%–1.66% were observed in four pegRNAs out of seven widely used acetolactate synthase (ALS) has been used for selection.
tested, comparable to rice editing frequencies (Lu et al., 2020). ALS confers resistance to several herbicides, particularly
Three genes (OsPDS,OsACC1, andOsWx) were used as targets to chlorsulfuron, and the specific amino acid change in StALS Pro-
test the pPE2 system. Using the t-RNA processing strategy was 187/186 to serine was targeted. In addition, the primary selection of
also used to target a rice endogenous 5-enolpyruvylshikimate-3- transgenics was on kanamycin. A PE-PE2 system was designed by
phosphate synthase (EPSPS) gene (OsEPSPS) for prime editing to fusing hygromycin phosphotransferase (Hpt) to the C-terminus of
confer glyphosate resistance. A peg RNA with gRNA (59 bp RT, the nSpCas9-M-MLV region with P2A, a self-cleaving 2A peptide,
13 nt PBS) and a second gRNA with the ability to nick at position driven byUbiquitin promoter ofmaize. PE-PE2 increased the editing
66 downstream were synthesized that could introduce triple efficiency by about threefold for three pegRNAs and gave improved
mutations. For this gene-editing, the prime editing efficiency editing frequencies (Perroud et al., 2022).
was 2.22% with both homozygous and heterozygous lines in rice The ability to introduce both transversions and transitions is
(Li et al., 2020c). The pPPEM construct was tested in rice by far the most significant attribute of prime editing technology.
protoplasts, targeting gene OsSULTR3, six at two different In addition, PEs have been found to successfully introduce
edits for the bacterial leaf streak disease susceptibility. The insertions, deletions, transitions, and transversions (Anzalone
editing efficiencies ranged from 0.7 to 2.2%. Besides editing et al., 2019). Perroud et al. (2022) reported that 0.06% of
endogenous genes, editing the transgenic reporter transformed protoplasts of Physcomitrium were edited, which
gene—fluorescent protein gene EGFP by SpPE2, SpPE3, and is less than the standard Cas9 mediated and base editing
SaPE3—was tested in rice calli. The inactive insert was edited to mutagenic strategies. However, the edit’s specificity is higher
active form successfully by SpPE3 at higher efficiencies than than CRISPR/Cas systems, and off-targets are few or none.
SpPE2, and none were observed with SaPE3, even though Sa Substitutions, insertions, and deletions have been observed in
compatible Cas9 and pegRNAs are required for efficient editing. the different taxa using the varied versions of prime editors.
The prime-editing gRNAs of diverse structures with varied The editing efficiency was similar in PE2- and PE3-based
PBS and RT lengths and nicking position of gRNAs have also vectors in Physcomitrium, whereas in potato, same
been reported to affect the prime editing efficiency (Xu et al., PE3 constructs failed to edit the ALS gene, which could be
2020; Hua et al., 2020a; Tang et al., 2020; Butt et al., 2020). edited by PE2-based vectors albeit at low frequencies. In rice,
Frontiers in Genetics 19 frontiersin.org
Dhakate et al. 10.3389/fgene.2022.876987
editing efficiencies were between 1.55% and 31.3% (Hua et al., to speed up the development of climate-resilient-high-yielding
2020b; Butt et al., 2020; Li et al., 2020d; Lin et al., 2020; Tang et al., cultivars. The application of molecular breeding approaches has
2020; Xu et al., 2020). The editing efficiencies ranged from 0.7% achieved great success in accelerating performance gains in
to 2.2%. Overall, the PE3 strategies were less efficient in plant various crops in the past decade. However, the need of the
cells than animal cells. However, further modifications and hour is to integrate new biotechnological methods and
adaptation of the technique would standardize prime editing technologies in the existing breeding programs to further
for more crop systems. Wang et al. (2021) have reported realize genetic gains. The unprecedented advances made in
insertion of up to 66 bases in Arabidopsis protoplasts, which GE technologies have shown great potential in genetic
is a four-fold increase over the 15-base insertion reported in rice. enhancement and boosting crop production. This review
For prime editing in dicots and monocots, easy-use vectors on highlights how newly evolved CRISPR/Cas systems have
PE2 and PE3 strategies have been created, named pPPED and successfully brought about a paradigm shift in crop
pPPEM (Wang et al., 2021). They have designed a pPEG cassette improvement programs. There has been a significant
for insertion of peg RNA or sgRNA, and then pPEG is inserted in advancement in understanding the functions of gene
the vectors PPEM or PPED. The pPPED vector was targeted in complexes underpinning complex traits, which was extremely
Arabidopsis. Editing efficiency is thus influenced by the length of daunting using the existing gene discovery approaches. The
reverse transcriptase and primer-binding site in the designed efficient use of GE tools in manipulating complex traits,
pegRNAs and sgRNAs. especially in polyploid crops, has now become feasible,
In addition to the biological parameters (plant taxa, especially when used in combination with the next-generation
molecular toolkit, transformation, and regeneration system), sequencing platforms.
the physical temperature parameter has a profound impact on Despite the substantial deployment of the CRISPR/
the editing frequencies. Because the efficiency of the M-MLV Cas platform in developing crops with desired traits, studies
reverse transcriptase is enhanced at higher temperatures, 32°C demonstrating the translation of the laboratory-based results into
and 37°C were tested, but no significant differences were the field have been anecdotal. In addition to being relevant at the
reported. However, the temperature variations were also tried genome level, the improved traits must also be realized in the
in prime editing (PPE) systems at 26°C and 37°C in rice, giving field without any trade-offs or counter effects on other traits of
significantly higher editing activity at 37°C (Lin et al., 2020). importance. Additionally, any genome strategy developed should
In summary, the modifications in the design of constructs, pose no threat to the environment and should be able to reduce
particularly to avoid by-products resulting from the scaffold of the the application of pesticides and fertilizers. One of the major
pegRNAs and reduction of off-targets, have been found to increase the challenges in developing cultivars by the GE route is rooted in
editing efficiencies. Gao (2015) suggested the shift from a knock-out low transformation and regeneration efficiencies. Numerous
strategy to a knock-in strategy by employing the homologous agronomically important crops such as sunflower, cotton, and
recombination process of DNA repair to increase targeted many others either have long transformation protocols with low
mutagenesis. This has been incorporated as a key attribute in the efficiencies or are outrightly recalcitrant. In addition, in crops
prime editing technology. Among the diverse strategies designed to where transformation protocols have been established,
achieve targeted mutagenesis, prime editing is a landmark regeneration efficiencies remain low, making the application
advancement in methods achieving increased efficiency and of GE strategies challenging.
reduced off-target effects. This method, for the first time, presented Furthermore, public acceptance of GE-modified crops has not
an efficient strategy to introduce all the 12-point mutations. With the come of age yet. A common misconception about these crops
availability of many diverse vectors (editors and pegRNAs) developed adversely affecting health and the environment has led many
by the different research groups and web-based design algorithms farmers to avoid reaping benefits from growing these crop
available (Peg-finder, PE-Designer /PE-Analyzer, pegIT, PrimeDesign, cultivars. This bias automatically trickles down to the
and PlantPegDesigner), the deployment of this technique is at the consumers and, in turn, results in limited acceptance of these
threshold of revolutionizing precision breeding of crop plants. Asmost crops for public consumption. Therefore, we believe, scientists
of the genes of importance rely on altering a fewand specific nucleotide across the globe need to ensure a healthy flow of information using
changes to confer traits rather than large-scale alteration of genes, present-day outreach tools, including social media, to educate the
prime editing presents an opportunity to drive the development of consumers about the differences between transgenic approaches
gene editing platforms that are precise, effective, and elegant. and the risks and benefits of using modern GE-modified crops.
Although GE platforms are radically different, precise, and
superior to traditional transgenic approaches, at the moment,
8 Conclusion these methods still go through governmental scrutiny and
assessment in many countries. Nonetheless, in the foreseeable
Under the scenario of ever-rising food demands and climate future, new-age GE platforms in plants are contemplated to be
change, there is tremendous pressure on scientists and breeders employed as a tool for efficiently engineering the majority of crop
Frontiers in Genetics 20 frontiersin.org
Dhakate et al. 10.3389/fgene.2022.876987
plants. We expect and hope that these methods can be integrated respect to cost and labor. Lastly, at present, we need more
into breeding programs globally with relatively lesser regulatory dynamic regulatory measures in place to ease the development
procedures compared to conventional transgenic approaches. and use of these platforms in crop improvement programs.
The development of these measures will need comparable
attention and consistent research efforts to continually assess
developed crop varieties on various climatic and genomic Author contributions
parameters, especially in our present-day rapidly changing
climate and pest pressure. VRR conceptualized and finalized the manuscript. PD, DS,
SV, AS, AC and VRR participated in preparing and curating the
manuscript and the revision. PD, SNR, and VRR helped in
9 Future directions preparing and finalizing the draft of the MS. All authors read
and approved the MS.
The evolution of various GE platforms has made it possible
for molecular biologists to precisely target gene(s) of interest.
Primarily, only CRISPR/Cas has been used for gene editing. Only Conflict of interest
recently, techniques such as epigenome editing, prime editing,
and base editing have been used for gene editing. These The authors declare that the research was conducted in the
techniques are powerful alternative strategies that have been absence of any commercial or financial relationships that could
developed for gene editing in plants. However, glaring be construed as a potential conflict of interest.
challenges still exist that continue to impede the goals of The reviewer KP declared a shared affiliation with the
achieving sustainable crop production. These challenges stem author(s) AC, VRR to the handling editor at the time of review.
from the complexity of both endogenous and exogenous cues in
plant development, making it nearly impossible for any single GE
platform to deliver efficiently. Present-day advances in GE Publisher’s note
protocols need to be primed toward generating platforms that
are more precise, efficient, accurate, and, most importantly, All claims expressed in this article are solely those of the
feasible. At first, no off-target silencing should result from authors and do not necessarily represent those of their affiliated
using these methods. Secondly, the delivery and results organizations or those of the publisher, the editors, and the
obtained in crop plants should not vary from species to reviewers. Any product that may be evaluated in this article, or
species. In addition, the genomic changes should be traceable claim that may be made by its manufacturer, is not guaranteed or
in future generations with precision and also remain feasible with endorsed by the publisher.
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