Over the past decade, the gene-editing platforms have shown tremendous evolution to accommodate the dual concerns of biosafety of edited crops and the efficiency of the platform used. Efficient and rapid genomic sequencing platforms have facilitated a better understanding of plant genomes, particularly when used in conjunction with genome editing (GE). Restructuring genomes via introduction of heritable genomic changes for expressing desirable quality traits in crops has been the focus of research for decades. The primitive methods of genome restructuring involved the use of genotoxic agents to introduce random double-stranded breaks (DSB) that were subsequently repaired by inherent non-homologous end joining (NHEJ) pathways resulting in random mutations (Puchta, 2005). After decades of usage of these random mutations generating tools, GE platforms have gone through many phases of improvement over the years. For example, the discovery of sequence-specific nucleases (SSNs) such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) helped to engineer the genome at intended loci by mediating the cleavage of dsDNA. The use of these nucleases induced the native NHEJ pathway for DNA repair (Salomon and Puchta, 1998). This method of GE, however, is both cost- and labor-intensive as it requires the development of sequence-specific nucleases/proteins. In addition, GE using these nucleases was inefficient as unintended off-target edits were introduced by the induction of the error-prone NHEJ repair pathway.

Given the obvious limitations of ZFNs and TALENs, the vacuum was soon filled with the discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) nucleases. In prokaryotes, the CRISPR/Cas system exists as a means of endogenous small RNA-based adaptive defense mechanism that protects the host bacterial cell via sequence-specific recognition and targeted cleavage of viral DNA (Jinek et al., 2012). With an approximate length of 32 bp, the length of CRISPR repeat sequences varies between 21 and 47 bp across prokaryotes. Every CRISPR repeat sequence harbors a unique sequence that is specific to the bacterial species processing it and has, therefore, been conserved over the course of evolution (Karginov and Hanon 2010). CRISPR was first discovered by a Japanese group in 1987 while studying the iap gene from the E. coli genome (Ishino et al., 1987). They identified CRISPR as homologous repeated sequences of only a few nucleotides interspersed by spacer sequences. Following this, CRISPRs were reported from the archaeal genome, Haloferax 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 RNA (gRNA) is designed to identify the intended target and guide the associated Cas9 protein to introduce DSBs in the target genomic 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 variants of Cas proteins have been mobilized to increase the efficiency of the CRISPR/Cas9-mediated GE.

During the past decade, the term “CRISPR/Cas” has evolved into a synonym for GE following which off-targeting instances with the use of CRISPR/Cas systems have reduced manifold (Modrzejewski et al., 2020). However, the goal of achieving “no off-target” remains elusive. In addition, with the involvement of the NHEJ repair pathway, the efficiency of this platform has always been disputable. In the third phase of the evolution of GE platforms, the CRISPR/Cas platform evolved to target the epigenome of an organism which was termed epigenome editing (Konermann et al., 2013). In epigenome editing, chromatin modification at specific genomic loci involves the use of epi-effectors that are comprised of DNA recognition domains (ZFNs, TALENs, or CRISPR/Cas system) and catalytic domains from a chromatin-modifying enzyme. Epigenome editing has been slated to have promising results in numerous basic sciences to decipher functions of chromatin structure and associated modification in phenotypes.

In the fourth phase, the CRISPR/Cas system evolved into a new methodology called base editing, wherein RNA-guided endonucleases were employed to engineer all four possible transitions with increased precision (Komor et al., 2016). One of the major challenges that all of the aforesaid techniques still face is to simultaneously engineer the altered DNA at the intended target sites. These concerns were addressed with the introduction of prime editing, marking the fifth phase in the evolution of GE platforms. Prime editing is largely described as a “search-and-replace” technology that edits the intended genomic loci without generating DSBs (Anzalone et al., 2019). This platform efficiently addresses the concerns of frameshift mutations that arise with the introduction of indels, further reducing off-target mutations. In addition, prime editing can introduce all 12 possible nucleotide substitutions (including transversions and transitions) (Anzalone et al., 2019).

The availability of all new-age GE strategies has not stolen the thunder of the CRISPR/Cas platform owing to the ease of its use and relevance to editing genes in numerous crop plants. However, it is only a matter of time before rapidly changing GE methods will replace present-day CRISPR/Cas systems with more elegant and efficient platforms. With every refinement of the platform, we are getting only closer to generating precise introduced mutations/deletions with reduced off-target effects. In the present review, we evaluate the evolution of GE platforms, such as CRISPR/Cas, epigenome editing, base editing, and prime editing over the last decade to highlight the paradigm shift in our understanding of GE strategies and the relevance of these platforms in present-day agriculture.

ZFNs and TALENs represent the first phase of the development of GE platforms. Essentially GE is achieved via the introduction of DSBs followed by a homologous repair pathway or the NHEJ-DNA repair pathway. In the first phase of developing GE platforms, SSNs such as ZFNs and TALENs were employed to introduce heritable genomic changes. ZFNs are chimeric enzymes that work as a dimer. Each monomer has 3–5 zinc-finger repeats along with a FokI cleavage domain. Each of the zinc fingers is capable of recognizing 3 bp of genomic DNA. Therefore, a ZFN dimer can effectively identify an 18–30 bp DNA with a gap of 5–7 bp (Kim et al., 2007). In plants, the first study involving ZFNs was reported in Arabidopsis, wherein heat shock was found to augment ZFN expression. At least 10% of the transgenics obtained displayed the mutations induced by ZFNs in future generations (Lloyd et al., 2005). In maize, ZFNs were employed to introduce a DSB at ipk1, and following this, a herbicide tolerance gene was inserted that resulted in transgenics showing tolerance to herbicide (Shukla et al., 2009). One of the major disadvantages of ZFNs is that the zinc fingers could overlap and are largely dependent on the sequence context around them and the intended DNA segment. Therefore, employing ZFNs becomes both labor- and cost-intensive as for every edit, the zinc-finger array is designed, and the sites available for the edits are limited (Boch and Bonas 2010). Although many studies have reported ZFNs to edit genes, its use as a tool of choice for GE now stands outdated. Another type of nucleases, TALENs, with DNA binding domains, was also employed to engineer genomic changes (Boch and Bonas 2010). Thirty-four tandem repeats are typically present in the DNA binding domain along with repeat-variable di-residue (RVD) comprised of two amino acids at positions 12 and 13, providing the TALENs with the ability to identify the intended target DNA sequence (Cong et al., 2012; Streubel et al., 2012). Like ZFNs, TALENs also introduce DSBs in the intended genomic DNA sequences, completely disrupting the gene and (or) introducing mutations. In comparison to ZFNs, TALENs can be designed for more target sites in the genomic DNA (Boch and Bonas 2010). In rice, TALENs were used to mutate the OsSWEET gene to develop transgenic resistance to blight (Li et al., 2012). Similarly, in wheat, transgenic with increased resistance to powdery mildew was developed by employing TALENs induced mutations (Wang et al., 2014). In cabbage, early flowering plants were obtained by employing TALENs (Sun et al., 2013). Like ZFNs, using TALENs is cost- and labor-intensive with limited success, and therefore, their use has now been largely suspended for introducing genomic changes.

The CRISPR/Cas systems represent the second phase of evolution in the development of GE platforms. CRISPR/Cas systems are sequence-specific and, therefore, mediate targeted DNA cleavage with increased efficiency. Three major steps are involved in CRISPR/Cas mechanism. The first step is adaptation, wherein a small sequence from the mobile genetic elements (MGEs) is harbored into the host CRISPR resulting in a novel spacer sequence. This adaptive event helps the host bacterial cell evade the attack from the same virus in the future (Barrangou et al., 2007). The selection of the target sequence to be incorporated into the CRISPR array is sequence-specific. In type I, II, and V CRISPR/Cas systems, a small sequence, termed the protospacer adjacent motif (PAM), is found adjacent to the protospacer that is to be incorporated into the CRISPR array. Therefore, PAM is cardinal to both acquiring the protospacer and bringing about the subsequent interference (Datsenko et al., 2012; Zetsche et al., 2015; Fonfara et al., 2016). Although the acquisition mechanism of spacers is not yet fully deciphered, in almost all CRISPR/Cas systems, Cas1 and Cas2 proteins have been found to maneuver the acquisition of the spacer into the CRISPR array (Makarova et al., 2015; Shmakov et al., 2015). Both these proteins are found to be necessary for the acquisition of the spacer (Datsenko et al., 2012). The two proteins form a hetero-hexameric protein complex (Cas1–Cas2), which is central to both excision and incorporation of the protospacer DNA into the CRISPR array (Nuñez et al., 2014). Barring a few exceptions, invariably the spacers are chronologically added to the array (Shmakov et al., 2015). Cas1–Cas2 protein complex is central to protospacer acquisition across most type 1 and type II CRISPR/Cas systems. Therefore, this mode of spacer acquisition stands most well deciphered so far. In the second step, the CRISPR array is transcribed and processed. In addition, the associated Cas genes are also transcribed into crRNAs. This step is subtype-specific, and therefore, subtype-specific enzymes are employed. However, broadly across all CRISPR/Cas systems, the CRISPR array is first transcribed into a precursor crRNA (pre-crRNA). Different Cas proteins and ribonucleases cleave and process this in various types of CRISPR/Cas systems to yield a mature crRNA. In the third step, following infection, the mature crRNAs mediate subtype-specific machinery driven mostly via Cas proteins to ensure effective cleavage of the MGE. The mechanism of different Cas proteins employed in various CRISPR/Cas systems has been well documented in many studies (Liu L et al., 2020; Talakayala et al., 2022; Wada et al., 2022).

The classification of the CRISPR/Cas systems identified so far is primarily based on the presence of the effector Cas proteins that cleave the invading foreign nucleic acids. The primary classification divides these systems into two classes: Class 1 and Class 2. Class 1 CRISPR/Cas systems employ a multi-protein complex, and Class 2 CRISPR/Cas systems recruit a single effector protein. Further, classification of Class 1 and Class 2 CRISPR/Cas systems into subtypes (I through VI) is dependent on their mechanism of action. The effector module of the CRISPR/Cas system is divided into three stages: the adaptation stage, the expression and processing stage, and the interference stage. In class 1 CRISPR/Cas systems (with types I, III, and IV), type I and type III systems employ a multi-protein complex called the Cascade complex along with Cas3 nuclease-helicase and the Cmr complex for type I, type III-A, and type IIIB CRISPR/Cas systems, respectively (Koonin and Makarova, 2019; Chaudhuri et al., 2022). However, class 2 CRISPR/Cas systems (with types II, V, and VI) employ only one effector protein. In type II and type V CRISPR/Cas systems, the expression and processing of the crRNA are regulated by a single protein such as Cas9 and Cpf1, respectively (Makarova et al., 2015; Amitai and 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 1 and type III are more prevalent than type IV in diverse bacterial and archaeal populations. However, type II of the Class 2 CRISPR/Cas system is found across all bacterial species (Koonin and Makarova, 2019). Depending on their function, Cas proteins can be primarily classified into four categories; recombinases/nucleases that aid the acquisition of spacers, ribonucleases that regulate the processing of crRNAs, scanning complexes like the crRNP complex, and nucleases that mediate the cleavage of the intended target sequences (Van Der Oost et al., 2014).

Class 1 CRISPR systems, types I and III, bear structural similarities suggesting evolution via a common ancestor (Chaudhuri et al., 2022). In addition, they employ Cas9 endonuclease to process crRNA. Type I CRISPR/Cas systems are further divided into six subtypes, types I-A, I-B, I-C, I-D, I-E, and I-F, depending on the distinct PAMs that the subunits require to regulate recognition and acquisition. The type III systems are divided into four subtypes, type III-A, III-B, III-C, and III-D, based on variation in adaptation, recognition, and interference modules of the effector protein complex. Chaudhuri et al. (2022) discussed the further classification of type I and type III into subtypes at length. Class 2 CRISPR/Cas system is divided into three types, types II, V, and VI. Out of these, the type II system is the most dissected and well-understood system so far (Koonin and Makarova, 2019; Chaudhuri et al., 2022). This system employs the Cas9 endonuclease as the effector. Type V system uses a single effector protein, Cas12. However, Cas12 has six subtypes, types V-A, V-B, V-C, V-D, V-E, and V-U, that identify distinct PAM sequences (Chaudhuri et al., 2022). Owing to obvious advantages such as smaller size, no dependency on tracr for target recognition, and asymmetric cleavage sites, Cas12 has now been actively replacing the Cas9 system for GE in many animal and plant species. Type VI systems are characterized by the presence of higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains with RNase activity (Koonin and Makarova, 2019; Chaudhuri et al., 2022). Cas13a was the first protein identified for type VI CRISPR/Cas systems (Chaudhuri et al., 2022). The evolution of type VI-B, such as Cas13b, is thought to have occurred from transmembrane systems, making them unique from type VI systems into a new subtype type VI-B (Chaudhuri et al., 2022). Type VI systems only target RNAs, thus thought to have lower instances of off-targeting and, in turn, do not harm the host cell much. The extensive diversity of the CRISPR/Cas system, as evident by their classification, reflects the evolution of the CRISPR/Cas-based defense mechanism in both archaea and bacteria. In addition, this diversity of CRISPR/Cas systems presents researchers with varied tools of GE to introduce precise changes with efficacy. Table 1 summarizes the classification of the CRISPR/Cas systems identified so far.

Class II CRISPR/Cas systems were found to be most suitable for development into a tool for genetic manipulation owing to the simplicity of their mechanism of action (Makarova et al., 2015). Type II CRISPR/Cas systems employ Cas9 protein that relies only on an RNA complex of crRNA:tracrRNA that is easy to engineer into a single guide DNA (gDNA) molecule (Jinek et al., 2012). These systems employ only two components: Cas9, a DNA endonuclease, and a customizable gRNA. A single gRNA is sufficient to direct the cleavage of the intended sequences. The gRNA molecules are customized to contain a sequence that Cas9 recognizes and a target sequence that guides the complex to the intended locus (Anders et al., 2014). To identify the intended target site, the Cas9-sgRNA complex scans the targeted DNA for a PAM site, following this 12 bases (seed region) of gRNAs proximal to PAM pair with the intended target sequence (Semenova et al., 2011). Mismatches in the seed region have been found to affect the activity of Cas9 adversely. However, mismatches in the 5’ PAM distal region are well-tolerated without affecting Cas9 nuclease activity (Liu et al., 2016). Catalytic domains of Cas9, HNH, and RuvC invariably result in a DSB in the DNA. Following this, DSB repair is initiated that is mediated either by homology direct repair (HDR) or the NHEJ pathway. The latter does not require a template for DNA repair and hence is error-prone. NHEJ is the active DNA repair mechanism in nature wherein Cas9-induced DSBs are repaired (Moore and Haber 1996). NHEJ can, therefore, lead to small insertions or deletions that could yield a host of mutations (Calvache et al., 2022; Wada et al., 2022). Such mutations are beneficial while knocking out a targeted gene using CRISPR/Cas9 systems. However, being random and unpredictable makes this mode of DNA repair unsuitable for precise editing of intended genes. To this effect, HDR is a more obvious choice of DSB repair mechanism for incorporation of desired sequences following cleavage by Cas9. In plants, GE HDR relies on a DNA template along with the gDNA and Cas9 for a successful DSB repair (Calvache et al., 2022; Wada et al., 2022). In plants, through genetic engineering, many outstanding repairs have been achieved via HDR, leading to gene replacement, DNA correction, and targeted knockouts. Figure 1 illustrates a diagrammatic representation of the adaptation to the CRISPR/Cas9 system in plants for gene editing.

Present-day agriculture faces serious threats from both abiotic and biotic stresses. Rapidly changing climate and exponentially growing world population increase the pressure of ensuring food security for both present and future generations. To mitigate agricultural losses and to aid crops in realizing their full potential, the only sustainable solution is to develop climate-resilient crops. Since its discovery in 2012 as a potential tool for genetic engineering, CRISPR/Cas9 system and its derivatives have rapidly replaced genome engineering methods in crop improvement programs across the globe. In model crops such as maize, a CRISPR/Cas9 mediated knocking and replacement in the liguleless-1 (LIG1) was reported (Svitashev et al., 2016). Similarly, in wheat, CRISPR/Cas9 GE system was employed to introduce targeted mutations in two wheat genes, TaLox2 and TaUbiL1. This study also validated the efficiency of using the CRISPR/Cas9 system in combination with microspore technology in plants for both trait improvement and discovery (Bhowmik et al., 2018). In tomato, complete expression of the susceptibility gene SlyPMR4 was knocked down to generate tomato plants with resistance against powdery mildew (Martínez et al., 2020). CRISPR/Cas9-based GE systems are now employed to improve multigenic traits such as biotic and abiotic stresses in many crops. Table 2 summarizes studies wherein CRISPR/Cas has been used successfully for trait manipulation in crop plants. Figure 2 depicts schematic representation of the domains of crop sciences wherein CRISPR/Cas platforms have largely contributed.

One of the most important applications of CRISPR/Cas9 platforms across the globe has been to engineer disease resistance in crop plants. Plant pathogens such as bacteria, viruses, nematodes, insects, and fungi are the most potent biotic stress factors that impact the yield potential of crops across the globe. Continuously evolving new strains of lethal pests make the battle against the pathogens even more complicated and daunting (Razzaq et al., 2019). Therefore, to protect and aid crops, methodologies routed in concepts of genome engineering have been successfully developed (Jaganathan et al., 2018). Peng et al. (2017) reported the development of varieties of Citrus sinensis (Wanjincheng orange) with increased resistance to Xanthomonas citri, which is responsible for the citrus canker disease in oranges. In this study, the expression of the gene, CsLOB1, which is responsible for the development of the disease, was disrupted using the CRISPR/Cas9 system. Two alleles (cslob1g and cslob1) exist for the gene CsLOB1. The promoter region of both these alleles inhibits an effector binding site (EBE) that is recognized by the main effector PthA4 of Xcc to drive the expression of cslob1 and results in the development of the disease. Five independent constructs pCas9/CsLOB1sgRNA were employed to modify the effector binding site EBE in the promoter region of CsLOB1 alleles. Homologous mutants wherein the EBE was completely disrupted were obtained, displaying no disease development following infection with Xanthomonas citri (Peng et al., 2017). In rice, an ethylene-responsive gene OsERF922 was knocked out using the CRISPR/Cas9 tool, which led to a marked reduction in the size and number of the blast lesions. This work led to the development of a rice cultivar with increased resistance against Magnaporthe oryzae (Wang et al., 2016). In another study, blight-resistant plants were produced using CRISPR/Cas9 system-mediated targeted mutagenesis of the SWEET13 gene (Zhou et al., 2015).

Management of diseases in crop plants is dominated by the frequent use of insecticides to curb yield losses. The development of crops resistant to viruses is, therefore, an efficient strategy to yield a stable yet economically viable alternative (Wang W et al., 2021). To this effect, inducing deletions and introducing point mutations in the genes using the CRISPR/Cas9 system is one of the most organic adaptations of the platform. The eukaryotic translation initiation factor genes such as eIF4E and eIF4G are an absolute requirement for the translation of RNA viruses (Shopan et al., 2020). Therefore, CRISPR/Cas9 technology has been employed in numerous plant species to engineer induced mutations in these genes. In Arabidopsis, point mutations in eIF(iso)4E gene were found to impart complete resistance against the turnip mosaic virus (Pyott et al., 2016). Likewise, in cucumber, eukaryotic translation initiation factor eIF(iso)4E was engineered using the CRISPR/Cas9 system to generate heritable homozygous point mutations that conferred resistance to the mutants against zucchini yellow mosaic virus, papaya ringspot mosaic virus-W, and vein yellowing virus (Chandrasekaran et al., 2016). In Nicotiana benthamiana, sgRNA/Cas9-mediated broad-spectrum immunity was achieved against viruses such as beet curly top virus, Tomato leaf curl Sardinia virus, Tomato yellow leaf curl virus, and Cotton leaf curl Kokhran virus (Ali et al., 2016). In rice, the CRISPR/Cas9 system was used to generate eIF4G alleles that conferred resistance against the Rice tungro spherical virus (Macovei et al., 2018). Recently, Wang et al. (2021) employed the CRISPR/Cas9 system to generate novel eIF4G alleles to yield transgenic plants displaying complete resistance to rice black-streaked dwarf virus. Engineering these mutations via the traditional backcrossing would have taken years, but using the CRISPR/Cas9 system expedited the process, and the goal was achieved in just a single generation.

The CRISPR/Cas9 system has also been used extensively over the past decade in generating climate-resistant cultivars in various crop species such as cotton, maize, rice, wheat, potato, soybean, and tomato (Khan et al., 2021; Wang et al., 2021; Rahman et al., 2022). In wheat, two regulatory genes (i.e., TaDREB3 and TaDREB2) were mutated using the CRISPR/Cas9 system, which resulted in increased drought tolerance in the mutated plants in comparison to the wild cultivars (Kim et al., 2017). In maize, the ZmARGOS8 gene that negatively regulates ethylene response was studied using the CRISPR/Cas9 system. The promoter of this gene was knocked out and replaced with maize GOS2 promoter in 5′-UTR of the target gene. The mutant plants were found to overexpress ARGOS8, which led to a stupendous increase in the yield in comparison to the wild type under drought conditions during the flowering stage without any yield penalty under irrigated environment (Shi et al., 2017). In rice, the CRISPR/Cas9 system was used to knock out gene OsRR2. The homozygous mutants obtained displayed increased tolerance to salinity stress (Zhang et al., 2019). In another study, three genes, OsPIN5b, GS3, and OsMYB30, that determine panicle length, grain size, and cold tolerance, respectively, were simultaneously edited using the CRISPR/Cas9 system (Zeng et al., 2020). T2 generations of the homozygous mutants of these genes displayed increased panicle length, enlarged grain size, and increased cold tolerance, respectively. The CRISPR/Cas9 tool has also been employed for the functional characterization of genes that regulate stress responses in plants. In Arabidopsis, three genes (CBF1, CBF2, and CBF3) have been identified to confer cold acclimatization and tolerance. However, the underlying mechanism remained undeciphered owing to the absence of any loss-of-function lines for these genes. Zhao et al. (2016) generated mutants of the cbf gene family, cbf1, cbf2, and cbf3. They generated cbf single, double, and triple mutants using the CRISPR/Cas9 platform. Interestingly, for the three genes, cbf triple mutants displayed compromised seedling development and reduced salt tolerance. However, both triple and double (cbf2cbf3) mutants displayed increased sensitivity to feeding post-cold acclimatization in comparison to the wild-type control. The cb1/cb3 double mutants displayed increased resistance, indicating that accumulation of CBF2 is more important than CBF1 and CBF3 in regulating cold acclimation-dependent freezing tolerance. The functional role of many other genes with a potential role in stress tolerance was also investigated in the model system Arabidopsis. The expression of UGT79-B2 and B3 genes was induced by abiotic stresses such as salinity, drought, and cold. Overexpression of these genes was found to increase the resistance of the transgenics. However, gene ugt79b2/b3 double mutants generated using the CRISPR/Cas9 system were found to be susceptible to abiotic stresses compared to the wild-type control. The overexpression mutants accumulated anthocyanins, but the ugt79b2/b3 double mutants that displayed lower levels of anthocyanins were also found to be more susceptible to stresses than the wild-type control plants. These findings also suggested that an array of anthocyanins impart resistance against abiotic stresses (Li et al., 2017). In rice, knockout mutants for the OsSAPK2 gene were developed for functional characterization of the gene. The mutants showed insensitivity to abscisic acid and increased 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 in mediating drought tolerance through increased stomatal closure (Lou et al., 2017). In another study, OsAnn3, a rice annexin gene, was knocked out in rice using the CRISPR/Cas9 system. The survival ratio of T1 mutant lines was found to be adversely affected, indicating that the expression of OsAnn3 was central in imparting cold tolerance in rice (Shen et al., 2017).

Drought stress in plants is governed by mitogen-activated protein kinases (MAPKs). In tomato, functional characterization of MAPKs was achieved by knocking down SlMAPK3 using the CRISPR/Cas9 system (Wang et al., 2017). The resulting slmapk3 mutants displayed severe wilting symptoms along with lower antioxidant enzymes, increased hydrogen peroxide, and increased membrane damage in comparison to the wild-type control. In another study, a multiplex CRISPR/Cas9 system was used simultaneously to edit five tomato γ-aminobutyric acid (GABA) shunt genes (CAT9, SSADH, GABA-TP1, TP2, and TP3). These genes are repressors of GABA metabolism. Hence, targeted mutagenesis of these genes led to a 19-fold increase in the accumulation of GABA in fruits and leaves (Li R et al., 2017).

The multiplex CRISPR/Cas9 system has proven to be 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 AND ERECT PANICLE (DEP1)] were edited using the multiplex CRISPR/Cas9 technique. The mutant plants displayed marked improvement in all the aforesaid traits and resulted in better and improved yields concerning tiller number and grain yield (Li et al., 2016). Similarly, multiplex editing using the CRISPR/Cas9 system of four genes, that is, GS3, Grain Widths 2, 5, and 6 (GW2, GW5, and GW6), which are negative regulators of grain weight, was investigated in rice. A remarkable improvement was observed in grain weight and size (Xu et al., 2016). The CRISPR/Cas9 system was also employed in rice to knockout three heading date genes (i.e., Hd2, Hd4, and Hd5) (Li et al., 2017). The mutants displayed early heading and higher yield under drought stress conditions. Furthermore, a CRISPR/Cas9 mediated disruption of the OsSWEET11 gene, known for grain filling and sucrose transportation in rice, led to reduced sucrose concentration and grain weight, which suggested that overexpression of these genes would be beneficial in obtaining a better grain quality (Ma et al., 2017). In wheat, GASR7 was knocked out using the CRISPR/Cas9 tool, and the resulting mutants showed increased kernel weight (Zhang et al., 2016). In tomato, the use of CRISPR/Cas9 methods has also delivered seedless tomatoes (Ueta et al., 2017). In this study, a novel sgRNA/Cas9 was employed, resulting in additional somatic mutation in SlIAA9, a key parthenocarpy gene. The mutation rate was 100%, and there were no off-target mutations. The mutants hence obtained displayed parthenocarpic fruit along with an altered leaf shape.

CRISPR/Cas9 systems have evolved over the years, and many other approaches have also been routed in this technology. As discussed earlier, CRISPR/Cas9-mediated gene editing necessarily introduces DSBs that are subsequently repaired by either NHEJ or HDR mechanisms (Kantor et al., 2020). This results in two major challenges in using CRISPR/Cas9 mechanisms. Firstly, although HDR promises insertion of only sequence-specific DNA, this pathway is synonymous with increased instances of indels and limited efficiency (Song et al., 2017). Secondly, reliance on the HDR mechanism of gene repair restricts gene editing to only dividing cells, adversely affecting the efficiency of this platform in manipulating the disease resistance in plants (Bollen et al., 2018). Many newer technologies that are primarily rooted in the CRISPR/Cas mechanism overcome some of these limitations and are more precise in achieving genome restructuring in plants. Some of these technologies are detailed in the following sections.

Under the scenario of ever-rising food demands and climate change, there is tremendous pressure on scientists and breeders to speed up the development of climate-resilient-high-yielding cultivars. The application of molecular breeding approaches has achieved great success in accelerating performance gains in various crops in the past decade. However, the need of the hour is to integrate new biotechnological methods and technologies in the existing breeding programs to further realize genetic gains. The unprecedented advances made in GE technologies have shown great potential in genetic enhancement and boosting crop production. This review highlights how newly evolved CRISPR/Cas systems have successfully brought about a paradigm shift in crop improvement programs. There has been a significant advancement in understanding the functions of gene complexes underpinning complex traits, which was extremely daunting using the existing gene discovery approaches. The efficient use of GE tools in manipulating complex traits, especially in polyploid crops, has now become feasible, especially when used in combination with the next-generation sequencing platforms.

Despite the substantial deployment of the CRISPR/Cas platform in developing crops with desired traits, studies demonstrating the translation of the laboratory-based results into the field have been anecdotal. In addition to being relevant at the genome level, the improved traits must also be realized in the field without any trade-offs or counter effects on other traits of importance. Additionally, any genome strategy developed should pose no threat to the environment and should be able to reduce the application of pesticides and fertilizers. One of the major challenges in developing cultivars by the GE route is rooted in low transformation and regeneration efficiencies. Numerous agronomically important crops such as sunflower, cotton, and many others either have long transformation protocols with low efficiencies or are outrightly recalcitrant. In addition, in crops where transformation protocols have been established, regeneration efficiencies remain low, making the application of GE strategies challenging.

Furthermore, public acceptance of GE-modified crops has not come of age yet. A common misconception about these crops adversely affecting health and the environment has led many farmers to avoid reaping benefits from growing these crop cultivars. This bias automatically trickles down to the consumers and, in turn, results in limited acceptance of these crops for public consumption. Therefore, we believe, scientists across the globe need to ensure a healthy flow of information using present-day outreach tools, including social media, to educate the consumers about the differences between transgenic approaches 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, these methods still go through governmental scrutiny and assessment in many countries. Nonetheless, in the foreseeable future, new-age GE platforms in plants are contemplated to be employed as a tool for efficiently engineering the majority of crop plants. We expect and hope that these methods can be integrated into breeding programs globally with relatively lesser regulatory procedures compared to conventional transgenic approaches. The development of these measures will need comparable attention and consistent research efforts to continually assess developed crop varieties on various climatic and genomic parameters, especially in our present-day rapidly changing climate and pest pressure.

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 recently, techniques such as epigenome editing, prime editing, and base editing have been used for gene editing. These techniques are powerful alternative strategies that have been developed for gene editing in plants. However, glaring challenges still exist that continue to impede the goals of achieving sustainable crop production. These challenges stem 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 protocols need to be primed toward generating platforms that are more precise, efficient, accurate, and, most importantly, feasible. At first, no off-target silencing should result from using these methods. Secondly, the delivery and results obtained in crop plants should not vary from species to species. In addition, the genomic changes should be traceable in future generations with precision and also remain feasible with respect to cost and labor. Lastly, at present, we need more dynamic regulatory measures in place to ease the development and use of these platforms in crop improvement programs.