ZFNs are proteins that have been engineered with a zinc finger domain located at the N-terminal and an endonuclease domain situated at the C-terminal end, crucial for specific DNA sequence recognition and cleavage (Kim et al., 1996; Porteus and Carroll, 2005). This former domain enables the precise recognition of the designated DNA bases, and the FokI restriction enzyme’s endonuclease domain ensures cleavage of specific DNA sequences (Bitinaite et al., 1998). Heterodimerization of FokI RE is essential for ZFN functionality, requiring two ZFNs to dimerize and bind both DNA strands. In ZFN, a consecutive series of three to six zinc fingers is arranged, with each zinc finger recognizing approximately 3 base pairs of DNA, and directs the nuclease to cut a specific genomic site (Wolfe et al., 2000). ZFNs find broad application in tailored genome engineering across diverse organisms (Urnov et al., 2010). Although initially applied for gene knockout in mice, ZFNs have limited applications in agriculture for crop improvement, mainly in crops like Arabidopsis, tobacco, and maize (Petolino, 2015). However, their off-target binding and the challenging and time-consuming nature of ZFN molecule design make them inefficient and less cost-effective for specific mutations (Pattanayak et al., 2011).

Zinc finger nucleases (ZFN) held the position as the predominant programmable site-specific nuclease until the emergence of the DNA sequence binding effector protein, i.e., transcriptional activator-like effector (TALE), derived from Xanthomonas, a bacterium known for its pathogenicity in plants (Boch et al., 2009; Moscou and Bogdanove, 2009). TALE is primarily responsible for regulating disease susceptibility associated (S) genes in rice. It is composed of a transcriptional activation domain (AD) at the C-terminal and a nuclear localization signal signature (NLS) for transcriptional regulation, along with a crucial tandem repeats acting as a DNA sequence binding domain (DBD), and a translocation signal sequence at the N-terminal (Jankele and Svoboda, 2014). The DBD contains a short amino acid (33–35 long) repetitive stretch, with peptides at the 12th and 13th positions, termed repeat-variable di-residues (RVDs), exhibits significant variability and is accountable for the specific detection of nucleotide bases (Mak et al., 2013). Utilizing the sequence-specific binding characteristic of the DBD has resulted in the creation of a novel gene editing (GE) technique known as transcription activator-like effector nuclease (TALEN).

Like ZFN, TALENs are tailored by merging the DBD of TALE with the FokI, a restriction enzyme that identifies asymmetric DNA sequences and cuts them outside of their detection sequence (Joung and Sander, 2013). However, TALEN design is simpler than ZFN, as TALE repeat sequences exhibit precision in targeting individual sites within a genome. Unlike ZFN, constructing a long array of DBD does not necessitate the multimerization of the repeat sequence, making TALEN engineering easier and less time-consuming (Gaj et al., 2013). RVDs in TALE repeat regions aid in recognizing specificity for various binding targets, allowing flexibility in designing TALENs for a broader range of potential target sites compared to ZFNs’ targeting sites. TALENs have found extensive application in genome editing across various plant species. Additionally, TALEs binding with gene activators and receptors, beyond nucleases, creates effective engineered transcriptional controllers for preferred gene expression level. In spite of TALENs’ benefits in high target specificity and little off-target effects than ZFNs exhibit, the intricate repetitive pattern within the DBD part of the TALE protein limits their application in editing multiple genomes with target specificity, and protein modification remains challenging (Chattopadhyay et al., 2022). To address these issues, the utilization of programmable RNA-directed DNA endonucleases for genome editing has become increasingly popular.

CRISPR, short for clustered regularly interspaced short palindromic repeats, denotes a distinct nucleotide sequence discovered within prokaryotic genomes. It structurally consists of CRISPR repeat-spacer arrays. This structure forms a locus containing conserved protein-coding genes associated with CRISPR (Cas). These genes are intricately involved in prokaryotic acquired defense mechanisms against bacteriophage invasions, akin to eukaryotic RNA interference (RNAi), thus providing genetic memory from previous encounters (Makarova et al., 2006; Bhushan et al., 2021). CRISPR utilizes small CRISPR RNA (crRNA) molecules synthesized from inherited genetic long-term memory as guide RNAs to cleave the viral genome (Barrangou et al., 2007; Brouns et al., 2008). This customizable feature of CRISPR has led to the development of the popular RNA-directed DNA endonuclease-centered genome editing technique known as the CRISPR/Cas system. CRISPR/Cas-centred genome editing relies on RNA-DNA sequence-pairing to precisely manipulate host DNA bases. It has emerged as a novel approach for genome manipulation across various species, including plants (Mushtaq et al., 2021). This technique offers robustness, simplicity, cost-effectiveness, ease of application, and high versatility compared to ZFN and TALEN (Ahmad et al., 2018). Its accuracy remains high even in multiplex genome editing, allowing for the simultaneous targeting of multiple genes (McCarty et al., 2020). Demonstrated in model plant systems (such as tobacco and arabidopsis) and diverse crops (including tomato, rice, maize, soybean, wheat, and potato), along with woody plant systems (like poplar, apple), CRISPR/Cas facilitates resilient trait improvements, addressing challenges related to both quantity and quality enhancement, and alleviating abiotic and biotic stresses (Arora and Narula, 2017; Jaganathan et al., 2018; Montecillo et al., 2020). The CRISPR/Cas technology induces dsDNA breaks at specific genome sites using a dual guide RNA system crafted to match target sequences. Watson–Crick base pairing enables binding with one strand of genomic DNA, facilitating cleavage of dsDNA by Cas9 endonucleases (Jiang and Doudna, 2017). Cellular repair mechanisms, comprising NHEJ or HDR, then mend the double-stranded breaks, resulting in genomic modifications like mutations through deletion and insertion (Shin et al., 2017). The application of these technologies for enhancing resistance to biotic stress in plants represents a promising avenue for improving rice crops.

Modifying pathogen targets involves functional knockout of host susceptibility (S) genes, as demonstrated in rice, cucurbits, and tomato with eIF4E and eIF(iso)4E knockout for improved immunity to plant viruses (Chandrasekaran et al., 2016; Macovei et al., 2018; Moury et al., 2020; Yoon et al., 2020). However, complete disruption of pleiotropic genes may adversely affect plant health and harvest. Alternatively, crafting cis-regulatory elements in S gene promoters offers broad-spectrum defense that is derived by the mechanism of effector binding elements (EBEs) removal from the S genes in the rice crop (Oliva et al., 2019). Modification of S gene coding sequences through precise genome or base editing, using CRISPR/Cas9 and base editors, introduces single-base changes to create single nucleotide polymorphisms in pathogen effector binding sites, thus reducing fitness costs. Additionally, altering amino acids in surface receptor proteins, such as AtBAK1 and RIN4, can prevent evasion of secreted pathogen effectors without completely abolishing their natural function (Rodriguez et al., 2016; Li et al., 2019).

Regulating the host immune response entails knocking out negative regulators’ transcription factors (TFs), as illustrated by the deletion of OsERF922 in rice, which reduces abscisic acid accumulation and enhances resistance to blast pathogens (Wang et al., 2016). Similarly, modifying central regulators like NPR1, NPR3, etc., can lower infection levels; for instance, CRISPR/Cas-directed alteration in OsNPR3 enhances the resistance mechanism in cocoa against Phytophthora tropicalis (Fister et al., 2018). Utilizing genome editing approaches to reduce the expression of non-coding RNAs (siRNAs and miRNAs) has been demonstrated, such as CRISPR/Cas9 editing of OsMIR408 and OsMIR528 in rice (Zhou et al., 2017). Alternatively, modifying plant defense through metabolic pathway engineering using genome editing approaches has been confirmed in plants like tomato and Opium poppy (Alagoz et al., 2016; Li R. et al., 2018). Genome editing tools are effectively employed for in planta intervention, managing the growth of pathogen infection in the plants. Primarily validated against DNA and RNA viruses (Beet curly top virus, Tomato yellow leaf curl virus, Cucumber mosaic virus) through CRISPR/Cas protein expression (Cao et al., 2020; Varanda et al., 2021), this strategy has now been extended to disrupt bacterial genomes using CRISPR nucleases, confirming a new resistance process in plants (Lee et al., 2021).

Lastly, it should also be feasible to alter specific amino acids within a plant’s focused protein that are crucial for detection and cutting by pathogen effector molecules. For instance, effector proteases from a bacterium Pseudomonas syringae, such as AvrPphB chopping the signaling constituents PBL- 1, 2, 6, PBS1, and BIK1, or HopB1 slicing the co-receptor BAK1, could be targeted (Zhang et al., 2010; Li L. et al., 2016). A mere change of one amino acid within the cleavage motif disrupts the cleavage functionality mediated by AvrPphB. Nonetheless, a dual alteration in BIK1 protease at the positions G230A and D231A revealed a central-negative effect on flg22-triggered defense related signaling mechanism, suggesting the functional significance of the altered residues in the protease (Zhang et al., 2010). Hence, it is crucial that the change in amino acid does not entirely eliminate the inherent working mechanism of the targeted protein. Conversely, it is also plausible to increase the disease reaction through nucleotide alterations, which is reported for a resistance gene in wheat plant (Stirnweis et al., 2014). Dual amino acids substitution within the NBS domain increased the defense induced hypersensitive response (HR), thus enhancing protection towards powdery mildew disease. RIN4 serves as a focused molecule for four bacterial effectors, at minimum, two of them trigger hyperphosphorylation of RIN4, reducing the RIN4-ROC1 binding interface (Rodriguez et al., 2016). Some hyperphosphorylated amino acids could be interchanged to attenuate effector-elicited vulnerability. For instance, the 141th serine phosphorylation of RIN4 triggered by FLS2 activation is vital for the immune suppression, while the phosphorylation of threonine at position 166 is induced by another effector molecule AvrB (Chung et al., 2014). Moreover, the AvrB also inhibited pathogen-triggered callose deposition in wild type plants, whereas this immune suppression was not observed in mutant plants expressing RIN4 (T166A), which cannot be phosphorylated. This illustrates that numerous factors must be considered, but generally, substituting amino acid residues in the effector targets should make plants more immune. In this regard, precise nucleotide excision by ABEs or CBEs will emerge as a powerful technique, allowing researchers to swap particular amino acid residues, thereby disrupting interactions between effectors and their targets.

Rice yield suffers significant losses due to various viruses, with rice tungro disease posing a particularly severe threat in Southeast Asia (Bunawan et al., 2014). Managing this disease through conventional methods is challenging due to simultaneous infections by two distinct viruses, tungro bacilliform, and tungro spherical. The scarcity of suitable resistance sources hampers the utilization of resistant genotypes, making genetic engineering an attractive alternative. Extensive research highlights the importance of certain host factors, such as eIF4G and eIF(iso)4G, in the progression of plant diseases caused by viruses. RNA viruses exploit such host factors during infection for protein translation from transcript and to control host replication mechanism for their multiplication (Hwang et al., 2013). Consequently, these host factors have been targeted and mutated in various plants to obtain resistance against different RNA viruses (Chandrasekaran et al., 2016; Pyott et al., 2016). Similarly, eIF4G also assists tungro spherical virus during infection, and a single nucleotide polymorphism in the rice host factor eIF4G (in 9th exon), affecting the conserved amino acids Y1059V, 1060V 1061, contributes to the resistant characteristics (Lee et al., 2010). Moreover, mutations in the NL region of the eIF4G factor are also associated with viral resistance in rice (Macovei et al., 2018). Similarly, another mutation in the coding region of the eIF4G confers resistance in rice against a different virus (black-streaked dwarf virus) using CRISPR/Cas9 system (Table 1). However, this alteration does not confer resistance in rice against stripe virus (Wang et al., 2021). The black-streaked dwarf virus is a rice-infecting plant RNA virus. Plant RNA viruses display significant diversity, with their genetic compositions evolving rapidly. Consequently, it is imperative to engineer a CRISPR/Cas system capable of conferring robust and broad-spectrum immunity against these RNA viruses. The effective targeting of viral RNA genomes in model plants has paved the way for the application of this potent technology in enhancing viral resistance in crop plants (Aman et al., 2018a; Aman et al., 2018b). Zhan et al. (2019) achieved noteworthy success in using the CRISPR/Cas13 system to effectively hinder the replication of potato virus Y (PVY) in potato plants. Likewise, Zhang T. et al. (2019) employed CRISPR/Cas13 to develop transgenic rice varieties resistant to southern rice black-streaked dwarf virus and rice stripe mosaic virus. Genome editing thus emerges as the most suitable tool for rapidly generating plant virus-resistant genotypes.

Bacterial leaf blight disease poses a formidable challenge to rice cultivation, primarily due to the extensive variability of the X. oryzae pv oryzae (Xoo) pathogen, which complicates management efforts (Chattopadhyay et al., 2017). Host resistance remains the most effective strategy to combat this challenge, with the identification of 44 resistant (R) genes to date (Kumar et al., 2020). Notably, approximately 35% of these R genes exhibit recessive alleles (Zhang and Wang, 2013; Hu et al., 2017). However, ironically, certain recessive R alleles like xa- 1, 4, 21, alongside dominant R alleles like Xa- 5, 13, serve as susceptibility factors. They facilitate disease progression by supporting the Xoo pathogen (Hu et al., 2017). This is achieved through the pathogen’s utilization of host machinery via its TALE effectors, to stimulate the expression of susceptibility genes (S genes), among which the OsSWEET family plays a crucial role by providing sucrose/sugar to the pathogen (Streubel et al., 2013; Gupta et al., 2021). Specific recognition of effector binding element sequences within the promoter region of OsSWEET genes by pathogen effectors (TALEs) initiates disease onset (Oliva et al., 2019). Therefore, modifying the OsSWEET promoter sequences emerges as a pivotal strategy for establishing comprehensive resistance to Xoo. Mainly, genome editing using TALENs focused on modifying rice S genes. Specifically, OsSWEET 13 and 14 were targeted (Table 1). The engineering primarily involved the promoter sequence, which contained the AvrXa7 effector binding element (EBE) in the OsSWEET14. This modification led to boosted resistance against Xoo strains that secrete AvrXa7 (Li et al., 2012). Likewise, enhancing the disease immunity in rice variety Nipponbare or Kitaake against Xoo strains bearing the Tal5(F) or AvrXa7 effector was achieved by editing the promoter of OsSWEET13, which contained EBEs for TalC, Tal5, and AvrXa7 effectors. However, this modification did not confer resistance against TalC effector (Blanvillain-Baufumé et al., 2017). Subsequently, the rapid advancement in CRISPR/Cas-mediated genome editing facilitated the precise sequence editing of S gene promoters (OsSWEET 11 and 14) to boost host plant immunity (Jiang et al., 2013). Therefore, CRISPR/Cas9 technology was repeatedly employed to target cis-elements within the promoters of S genes, aiming to safeguard rice against a range of Xoo strains (Zhou et al., 2015; Kim et al., 2019; Oliva et al., 2019; Xu et al., 2019; Zafar et al., 2020; Duy et al., 2021). Additionally, TALEN and CRISPR technologies were utilized to elucidate the function of new genes such as Xa23-Ni and Xa10-Ni to Xoo strains (Wang et al., 2017). Similarly, bacterial leaf streak (BLS) disease, caused by another X. oryzae pathotype oryzicola (Xoc), poses a serious threat to the rice crop. It employs different effectors to control host S genes and facilitate subsequent disease development. For instance, Tal7 is a Xoc effector that suppresses AvrXa7-Xa7-mediated defense by activating the Os09g29100 gene. Engineering the effector Tal7 interacting region (EBEtal7) of the Os09g29100 promoter via TALEN enhances resistance to BLS pathogens (Xoc strain RS105) by suppressing AvrXa7-Xa7-mediated defense (Cai et al., 2017). Editing of promoters in additional S genes aimed to boost resistance in rice. This was shown through the disruption of the EBETal5d/Tal2g segment within the promoter of the OsSULTR3;6 gene. This alteration conferred increased immunity in the rice line IRBB10 against Xoc strains containing either the Tal5d or Tal2g effectors (Xu et al., 2019). Furthermore, efforts to attain comprehensive protection from both Xoc and Xoo pathogens via CRISPR/Cas9 GET have shown promising results (Jiang et al., 2013), with mutant lines displaying broad-spectrum resistance without compromising normal agronomic traits, including yield (Kim et al., 2019). These findings highlight the promise of cutting-edge GETs for investigating and strengthening rice resistance towards different pathogenic bacteria.

Rice faces significant challenges from various fungal pathogens, leading to considerable yield losses (Singh et al., 2016). Among these, rice blast presents a severe menace, accountable for roughly 30% of global crop losses. The hemibiotrophic characteristic of the blast causing fungus (M. oryzae) exacerbates its extensive and catastrophic impact, resulting in the frequent occurrence of new variant races and speedy disintegration of host resistance (R) genes (Ning et al., 2020). Consequently, depending entirely on the implementation of newly discovered R genes, which alter the pathogen population structure, becomes increasingly challenging. Understandably, there is a growing demand for alternative options. Various strategies have been employed to confer blast disease protection in rice, with gene editing emerging as the really advanced approach for increasing protection towards diverse rice fungal pathogens. Primarily, rice genes such as OsMPK5, encoding stress-sensitive mitogen-activated protein kinase, were aimed to establish genome editing strategy in rice using RNA-guided editing approach. Employing the CRISPR/Cas9 platform, three guide RNAs were crafted to induce mutations in OsMPK5, leading to comprehensive disease protection in the rice variety Nipponbare (Table 1). Mutated protoplasts exhibited a mutation frequency ranging from 3% to 8% (Xie and Yang, 2013). Subsequently, different gene editing tools were used to knockout various disease susceptibility factors to achieve broad-spectrum defense. For instance, the negative controller of defense mechanisms in rice, OsERF922TF, was targeted for knockout using CRISPR/Cas9 platform (Wang et al., 2016). Binary vector carriers delivering the Cas9/sgRNA cartridge induced mutations and insertions-deletions (InDels) in Kuiku131 rice plants, yielding a 42% mutation rate in the transgenic T0 generation. This targeted gene knockout reduced abscisic acid accumulation, enhancing defense mechanisms without affecting other agronomically beneficial traits, demonstrating the precision of gene editing. Employing Cas9/multi-target sgRNA vector constructs, genome editing targeted multiple base positions in the OsERF922 sequence, increasing mutation efficiency and improving defense against the blast pathogen (Wang et al., 2016). Changes made in the OsSEC3A sequence, encoding a subunit for the exocyst protein complex, using the CRISPR/Cas9 platform targeted multiple sites within the gene, resulting in upregulation of defense-related genes and increased resistance to the blast fungus. However, it also caused stunted growth and lesion-like phenotypes (Ma et al., 2018). A similar method induced mutations in the resistance Pi21 gene, enhancing rice immunity against blast disease without affecting key agronomic traits (Nawaz et al., 2020). A BSR-D1 gene responsible for susceptibility factor was also selected for mutation through CRISPR/Cas9 platform to stimulate rice immunity against blast disease. The modification of the cis-regulatory element through single base mismatch led to enhanced interaction of MYBS1 TF, resulting in the suppression of peroxidase expression and subsequent augmentation of H₂O₂ production (Li W. et al., 2020). Likewise, the functional significance of a newly discovered gene, Perox3, involved in peroxidase synthesis, in blast disease resistance was investigated by knocking it out using CRISPR/Cas9 platform in rice (Zhu et al., 2020).

In addition to blast disease, rice sheath blight poses significant challenge in rice production. In order to develop a wide-ranging defense mechanism against this disease, successful mutation of the OsPFT1 gene in variety ASD16 was achieved via CRISPR/Cas9 platform, with the functional evaluation of the mutated lines pending determination (Shah et al., 2019). Besides targeting host genes, a range of fungal pathogen genes were also addressed to elucidate the roles of gene in the pathogenicity. For example, the removal of two USTA and UvSLT2 genes of the false smut fungal pathogen (U. virens), respectively encode Ustiloxin and MAP kinase, resulted in two significant outcomes: hindrance in Ustiloxin formation and raised sensitivity to cell wall disruption. These changes consequently led to diminished fungal pathogen virulence to rice (Liang et al., 2018). Similarly, disrupting the blast fungal scytalone dehydratase gene using CRISPR/Cas9 affected melanin synthesis and appressoria development (Yamato et al., 2019), thereby reducing rice infection. However, achieving a consistent expression system through the application of Cas9 plasmids in fungal species poses challenges. Hence, a new genome-editing methodology was established. It involved the temporary use of refined CRISPR/Cas9 ribonucleoprotein, pre-added with guide RNA, for specific genetic modification in rice blast pathogen (Foster et al., 2018). This approach, devoid of plasmids, can also be leveraged for the genetic manipulation in various fungi. Consequently, the precise alteration of host genes using gene knock-out or knock-in approaches along with base editing strategies, without causing off-target effects, remains an ongoing imperative (Arazoe, 2021). Thus, continuous enhancement of GET is crucial for rapid and adaptable genome modification of host rice plants and its numerous pathogens.

Numerous studies have aided understanding about how to apply genome editing techniques to generate novel alleles. In rice research, for instance, CRISPR/Cas has been instrumental in creating novel alleles linked to increased yields and resistance towards diseases such as rice leaf blight and rice blast (Zhou et al., 2018; Zhou et al., 2022). Zhou et al. (2018) identified and characterized a rice mutant, bsr-k1, which provides a high disease resistance towards both pathogens Xoo and M. oryzae. The deactivation of the Bsr-k1 gene results in the accumulation of OsPAL1-7 mRNA transcripts in the mutant, leading to improved rice yield with enhanced resistance to leaf blight and blast diseases (Zhou et al., 2018). Furthermore, Zhou et al. (2022) introduced mutations in specific genes, namely, Pi21, Bsr-d1, and ERF922, within the rice lines, longke638S and TGMS (male sterile line). The resulting mutated lines displayed increased resistance towards rice blast in contrast to the wild type, demonstrating the potential for multi-gene editing in disease resistance. This genetic manipulation technique facilitates targeted alterations in the DNA sequence, potentially resulting in changes in the traits or characteristics of rice varieties.

Moreover, Macovei et al. (2018) identified novel alleles for resistance to rice tungro disease (RTD) by mutating the eIF4G factor gene of IR64 rice variety, susceptible to tungro visus, using CRISPR/Cas9 editing technology. The identified eIF4G alleles with mutations close to specific residues conferred resistance to RTD, offering potential targets for breeding resistant varieties. These studies underscore the successful creation of novel alleles in rice through genome editing techniques, highlighting the versatility and effectiveness of CRISPR/Cas in improving rice biotic tolerance traits. Continued research in this domain holds promise for addressing global challenges in food security and sustainable rice production.

Furthermore, the disruption of OsCPR5.1 in a rice cultivar Kitaake conferred a high level of resistance to rice yellow mottle virus (RYMV) isolate BF1 without any yield penalty (Arra et al., 2023). This study suggests that introducing novel mutations into rice cultivars using CRISPR/Cas technology can be exploited to obtain resistance towards rice yellow mottle virus, potentially facilitating accelerated breeding applications to develop resistance in elite cultivars. Likewise, in rice, an allelic variant of the SF3B1 splicing factor demonstrated resistance to the herbicide herboxidiene, acts as a splicing inhibitor (Butt et al., 2019). Recently, targeted evolution of OsACC and OsALS within rice germplasm through a dual Base Editor (BE) discovered new allelic variations that provide herbicide tolerance (Li H. et al., 2020; Kuang et al., 2020). Likewise, a novel mutant wx allele of the rice waxy gene was created using a BE, altering the content of amylose in the mutant plants for the development of soft rice (Xu et al., 2020). Though establishing a platform for selecting desired traits with allelic evolution poses challenges, applying a parallel approach for generating in vivo allele in various crop plants presents an intriguing avenue for exploration.

In addition, functional genomics approaches based on the CRISPR platform open up the prospect of revisiting neglected under-utilized genetic materials to rediscover alleles that were lost through domestication process. Throughout domestication, several crop species shed certain genes deemed unnecessary for survival but potentially governing key agronomic attributes. A myriad of various alleles controlling phenotypic traits were consolidated in domesticated crops through traditional breeding methods aimed at enhancing the fundamental attributes, such as growth, yield, tolerance to abiotic and biotic stress problems, etc. (Jacob et al., 2018). The non-domesticated wild members of cultivated crops acted as gene pool reservoirs and were utilized for hybridization of desired traits in breeding programs. However, a major drawback of traditional breeding programs is the lengthy time needed and the loss of associated genes due to the genetic hitchhiking effect, decreases heterozygosity (Smith and Haigh, 2008). The advent of CRISPR technology has spurred interest in the domestication of non-domesticated plants through introduction of domestication genes, a process referred to de novo domestication (DND), which offers a novel approach to adapting plants for cultivation (Osterberg et al., 2017; Fernie and Yan, 2019). Technological advancements based on CRISPR have demonstrated prospective in the DND of wild relative plants harboring a desired trait without sacrificing genome diversity. For example, Zsogon et al. (2018) achieved successful domestication of wild tomatoes, improving nutritional qualities and yield quantity by employing CRISPR approaches to target various genes including those related to fruit shape, fruit weight, lycopene beta cyclase, and self-pruning. In a parallel manner, the speedy domestication of wild strawberry tomato (Physalis pruinosa) was achieved by editing the CLV1 (receptor kinase) and SP5G (associated with flower growth and development) genes using CRISPR/Cas9, leading to enhanced yield in the domesticated plants (Lemmon et al., 2018). Additionally, with the application of CRISPR/Cas9, desirable traits were introduced into wild tomatoes by aiming the SlWUS, SlCLV3, SP5G, and SP genes (Li T. et al., 2018). In a recent investigation, the CRISPR approach was utilized to investigate salt resilience in rice and tomato, uncovering SlHAK20 as a vital factor controlling K+/Na+ homeostasis and contributing to the diminished salt resistance observed in domesticated plants (Wang et al., 2020). This SlHAK20 represents the initially recognized gene involved in the domestication process linked to decreased tolerance to salt stress, offering valuable molecular insights for enhancing other crops. Furthermore, in rice, Abdullah et al. (2022) discussed the prospective and potential of the DND of wild species to enhance global rice production. They emphasized the advantageous traits found in a wild rice (Oryza australiensis), accessible genomic data, along with the capability of genome editing, to uncover and comprehend the roles of novel beneficial alleles. The wild rice domestication through the DND approach holds a lots promise in the crop improvement (Li T. et al., 2018). This process requires the advancement in well-annotated reference genome sequence, efficient transformation method, and the genetic manipulation of various core genes essential for domestication. These genes govern traits like panicle architecture, awn length, shattering attribute, and nutritional quality benefits, aiming to enhance various features.

Applications of genome editing in creating novel alleles have revolutionized research in genetics and crop improvements. The GETs, especially the CRISPR/Cas platform, enable precise modifications of DNA sequences, offering unprecedented control over the genetic makeup of organisms. Genome editing allows scientists to introduce or modify specific useful phenotypes in crops, such as enhanced nutritional value, high tolerance against biotic stresses, and improved yield quality. Creating novel alleles for stress tolerance is crucial in the face of climate change. Genome editing has been used to develop crops with enhanced tolerance to drought, salinity, and other environmental stressors, contributing to more resilient and sustainable agriculture. Genome editing allows researchers to create targeted mutations in specific genes, enabling the study of gene function. By creating novel alleles associated with gene knockout or overexpression, scientists can elucidate the role of genes in various biological processes. Novel alleles generated through genome editing can be used to engineer metabolic pathways in microorganisms for compatible host-pathogen interaction processes, biofuel generation, pharmaceuticals, and other valuable compounds. In conclusion, the applications of genome editing in creating novel alleles extend across diverse fields, promising advancements in agriculture, medicine, and basic research. Ongoing research and technological developments in genome editing techniques continue to broaden the scope of possibilities for creating precise and tailored genetic modifications.

One of the most contentious criticisms directed at CRISPR technology revolves around the potential risk of inadvertently mutating non-target genes within the manipulated organism, which could result in unintended biological repercussions within its ecosystem. These random mutations might activate undesirable genes, including those associated with disease susceptibility. Furthermore, gene editing procedures could induce translocations of chromosomal segments, leading to genome instability. To mitigate the risks of off-target effects associated with Cas9, numerous strategies have been devised, primarily focusing on optimizing the design of sgRNA. Studies have demonstrated that the utilization of truncated sgRNA can diminish undesired mutagenesis at certain off-target sites while maintaining high efficiency in on-target genome editing (Ding et al., 2016). Additionally, Muller et al. (2016) introduced a Streptococcus thermophilus Cas9 variant featuring a longer PAM sequence, resulting in reduced off-target activity. Another promising avenue for precision genome engineering involves the nmeCas9 system, which offers a safer alternative, albeit with slightly lower efficiency compared to spCas9 (Lee et al., 2016). Paired Cas9 nickases represent another approach, generating two single-strand nicks occurring on DNA strands, thus exhibiting high specificity and efficacy without off-target activity (Cho et al., 2014). Regulating Cas9 off-target activity is also achievable by selecting appropriate sgRNA sequences and optimizing experimental conditions (Doench et al., 2016).

The transfer of CRISPR/Cas cargoes presents a significant difficult task in the application of plant GET, particularly in monocotyledons, where gene gun and agrobacterium-directed delivery effectiveness heavily depend on the recipient genotype. Notably, certain elite rice cultivars pose difficulties in transformation due to inherent limitations in their culture and regeneration capabilities (Zeng et al., 2020). Furthermore, the inevitable incorporation of T-DNA plasmid fragment followed by plant regrowth and development processes is often practically intricate and labor-intensive. Hence, the development of delivery methods that circumvent the need for tissue culture is desirable, extending their applicability in different plant systems. Nanotechnology derived materials like carbon nanotubes (CNTs) and ultrafine particles or nanoparticles (NPs) offer a promising avenue for the CRISPR/Cas system, facilitating the diffusion of desired DNA into plants passing cell wall barriers without any external assistance (Tao et al., 2021). In 2017, a novel approach termed pollen magnetofection (PMF) utilized magnetic NPs as carriers of genetic material to introduce foreign genes into the pollen of multiple model crops, resulting in approximately 1% generation of transgenic plants upon pollination with magnetofected pollen (Zhao et al., 2017). However, concerns regarding the reproducibility of pollen magnetofection have been raised by some researchers (Wei et al., 2021). If the contents of CRISPR/Cas could be effectively delivered to reproductive cells and reliably expressed through the PMF method, it could offer a streamlined method for creating heritable genetic modifications in transgenic seeds, eliminating the necessity of tissue culturing step (Molla et al., 2016). Additionally, owing to the integration and disease free properties of nano genetic transportation approaches, gene-edited crops mediated by nanomaterials may potentially avoid classification as genetically modified organisms (GMOs; Xu et al., 2017).

GETs, particularly CRISPR/Cas, have revolutionized crop improvement, offering powerful tools for targeted genetic modifications. However, the ethical considerations surrounding the application of genome editing in agriculture are paramount. Balancing the potential benefits with ethical concerns is crucial for responsible and sustainable use. Some key ethical considerations, along with relevant references, include environmental impact, social justice and access, transparency and communication, consumer acceptance and labelling, unintended consequences and long-term effects, dual-use and bioweapon concerns, and respect for biodiversity and traditional farming practices.

The unintended environmental consequences of genome-edited crops, such as gene flow to wild relatives or the potential disruption of ecosystems, raise ethical concerns. Evaluating and minimizing the environmental impact of genome-edited crops through rigorous risk assessments and containment measures is essential (Pauwels et al., 2014). Ensuring equitable access to GETs and their benefits, especially for smallholder farmers in developing countries, raises concerns about social justice. Addressing issues of access and distribution to avoid creating disparities and ensuring that benefits are shared globally is crucial for ethical genome editing applications in agriculture (Hilbeck et al., 2020). Maintaining transparency in research, development, and deployment of genome-edited crops, along with effective communication with stakeholders, is essential for ethical practice. Establishing open communication channels and involving the public in decision-making processes can build trust and address concerns related to transparency (Kuzma et al., 2016).

Ethical concerns arise regarding consumer acceptance and the labeling of genome-edited products, as some consumers may have reservations about genetically modified organisms (GMOs). Implementing clear labeling practices, coupled with effective public engagement and education, can empower consumers to make informed choices and address ethical concerns (Kjeldaas et al., 2023). The potential for unintended consequences and long-term effects of genome editing, including off-target mutations and ecological impacts, necessitates careful ethical consideration (Waltz, 2016). The dual-use nature of GET raises ethical concerns about its potential misuse for bioweapon development or harmful purposes. Implementing responsible governance frameworks, international cooperation, and strict regulations can help prevent the misuse of GETs (Ienca and Vayena, 2018). The potential impact of genome-edited crops on biodiversity and traditional farming practices requires careful ethical consideration to avoid unintended consequences. Respecting and preserving biodiversity, as well as engaging with local communities to understand their perspectives and needs, is crucial for ethically sound genome editing practices (Ishii and Beriain, 2019). Ethical concerns regarding GETs utilization in crop development demand a multidisciplinary approach, involving scientists, policymakers, ethicists, and the public. Engaging open dialogue, transparent practices, and adherence to ethical principles are crucial for responsibly navigating the complex landscape of genome editing in agriculture.

Genome editing with designed nucleases emerges as an extremely precise and effective method for enhancing crops, offering the possibility to swiftly create new beneficial traits. These techniques stimulate precise double-strand nicks in the genome, initiating DNA repair pathways that can result in base insertions, deletions via NHEJ, or precise gene swapping and transgene inclusions by HDR systems. The phenotypic variations produced by these techniques often mimic those arising through natural processes or conventional mutagenesis, blurring the lines with exiting definitions of genetically modified or engineered organisms under prevailing regulatory frameworks. The ambiguity surrounding the regulatory reputation of genome editing approaches poses a significant hurdle to their widespread adoption for economically valuable crop qualities. Regulatory emphasis on the methodology employed, instead of the characteristics of the resulting phenotype, has led to confusion among regulators, developers, and the public, fostering uncertainty regarding the utilization of genome manipulation methods in crop development programs. The regulatory landscape concerning genome-edited crops differs globally, contributing to uncertainties in their classification and acceptance (Wolt et al., 2016). Establishing clear and globally harmonized regulations for genome-edited crops, while addressing safety concerns and promoting public acceptance, is imperative for their integration into agricultural practices.

Several challenges are associated with GET, including genetic mosaicism, complex trait regulation, and epigenetic changes. Genetic mosaicism, characterized by the coexistence of edited and unedited cells within a tissue, can complicate the phenotypic analysis of edited plants. Careful screening and selection of plants with desired edits, coupled with advancements in editing efficiency, can help mitigate the impact of genetic mosaicism (Fauser et al., 2014). Resistance to certain plant diseases or pests often involves complex genetic pathways, posing challenges in achieving desired traits through single-gene modification. Employing multiplex genome editing strategies to target multiple genes involved in resistance pathways can enhance the effectiveness of genome editing for complex traits (Staskawicz et al., 1995). Genome editing may induce unintended epigenetic changes, potentially affecting gene expression and overall phenotype. Further research into understanding and minimizing epigenetic alterations, alongside improved design and delivery of editing tools, is necessary (Niederhuth et al., 2016). Addressing these challenges and limitations will require ongoing research, technological advancements, and collaborative efforts among scientists, policymakers, and the public to completely harness the potential of genome editing for developing resistant crops and ensuring global food security.