Gene cloning in vegetable improvement programme

Introduction

Interest in the health benefits of consuming vegetables has surged due to their rich array of nutritional compounds, including vitamins, minerals, antioxidants, dietary fibre, and a diverse spectrum of phytochemicals (Septembre-Malaterre et al., 2018) [70] . While vitamins and minerals are crucial for human health, antioxidant compounds found in fruits and vegetables are known to mitigate cellular oxidative stress and lower the risk of chronic diseases such as diabetes, cancer, and cardiovascular disorders (Aune et al., 2017; Miller et al., 2017) [8,59] . Nevertheless, like all crops, vegetables are typically sensitive to biotic and abiotic stresses, making diseases, high temperatures, and limited water availability significant constraints on their productivity. These challenges are expected to intensify due to climate change (Bisbis et al., 2018) [13] . Consequently, researchers are focused on enhancing vegetable varieties by improving yield and yield stability, nutritional quality, and tolerance to biotic and abiotic stresses through both traditional breeding methods and advanced plant molecular breeding technologies (Abdallah et al., 2015) [1] . Plant breeding is a multifaceted process that involves developing new crop varieties with desirable traits and strategizing the combination of these traits to create superior varieties (Glenn et al., 2017) [29] . The initial step in breeding involves leveraging genetic variation among individuals within a plant species. For many years, this has been accomplished by crossing parental lines or employing physical or chemical mutagenesis on crop plants (Holme et al., 2019; Kleter et al., 2019) [31,42] . Since the early 20 th century, when pioneering research discovered the ability of X-rays to alter plant phenotypes in crops such as barley and maize, a variety of innovative mutagenesis breeding tools have been developed (Muller, 1927; Stadler, 1928) [60,76] . Subsequently, more advanced radiation techniques, including gamma radiation, UV light, and particle radiation, were introduced to generate novel agronomic traits and explore gene functions in crops (Shu et al., 2012) [74] .

Over time, chemical mutagens became preferred for their ease of use and higher mutation frequency compared to radiological methods. Today, ethyl methane sulfonate is the most widely used chemical mutagen, with other agents like sodium azide and methyl nitrosourea (Az-MNU) also commonly employed for mutagenesis in plants. Physical and chemical mutagenesis has been effectively utilized to induce point mutations and deletions within plant genomes, leading to the release of thousands of varieties developed through mutation breeding (Ahloowalia et al., 2004) [2] . While the potential of mutagenesis in vegetable breeding has not been fully realized, it has found extensive application in generating variation in ornamental plants (Bernardo, 2016) [10] . With the advancement of molecular technologies and DNA sequencing, researchers began investigating the functional properties of genes using T-DNA-tagged mutant pools created through random T-DNA insertional mutagenesis. This approach allows for the identification of DNA sequences flanking T-DNA, facilitating the exploration of genetic elements potentially responsible for phenotypic changes in mutants. The data gathered from analyzing T-DNA flanking sequences has been pivotal, providing new avenues for developing improved crop varieties with desirable traits using molecular breeding tools (Chaudhary et al., 2019; Kleter et al., 2019) [20,42] . In recent decades, significant advancements have been made in molecular biology techniques. The discovery of sequence-specific nucleases (SSNs) and the development of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein (Cas) system have enabled precise, programmable gene editing at the DNA level, allowing for the creation of vegetable crops with modified functions and desired traits (Abdallah et al., 2015; Nunez de Caceres Gonzalez and De la Mora Franco, 2020) [15,61] .

History of gene cloning/gene-editing technology

In recent years, the advancement of gene and genome-editing technologies has significantly enhanced the precise and efficient modification of genomes across various organisms, including vegetable crops, to improve yield and quality (Chen and Gao, 2014) [21] . Sophisticated molecular biology techniques utilizing sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas system (Chen and Gao, 2014) [21] , have empowered plant researchers to perform targeted gene and genome engineering with remarkable precision and efficiency. These methods induce double-strand breaks (DSBs) at specific DNA loci, which are subsequently repaired by the cell's endogenous DNA repair mechanisms. This repair can either involve the insertion or deletion of nucleotides through non-homologous end joining (NHEJ) or gene replacement via homologous recombination (HR), thereby causing loss-offunction or gain-of-function in target genes (Symington and Gautier, 2011; Figure 1) [79] . The various CRISPR-Cas systems, in particular, have emerged as powerful tools for gene and genome editing, leading to the creation of numerous genomeedited plants using these technologies (Gao, 2014) [27] .

Zinc-Finger Nucleases

Zinc-finger nucleases (ZFNs), the first generation of sitespecific nucleases, enabled rapid and targeted modifications of the genome. ZFNs are typically constructed by fusing zincfinger protein (ZFP) domains, which can bind to DNA sequences in a sequence-specific manner, with a nonspecific DNA cleavage domain derived from the bacterial FokI endonuclease (Figure 1; Petolino, 2015) [63] . Each ZFP, consisting of a tandem array of cysteine2 and histidine2 (Cys2-His2) domains (Miller et al., 1985) [58] , recognizes approximately three base pairs (bp) of DNA. Generally, four to six ZFPs are linked together to recognize a specific DNA sequence of 12 to 18 bp (Urnov et al., 2010) [83] . For DNA cleavage to occur, the FokI catalytic domain must dimerize, meaning ZFNs function as heterodimers to target and cut DNA. Once the zinc-finger domain identifies and binds to the target DNA, the attached FokI nuclease domain cleaves the DNA, creating double-strand breaks (DSBs) at the targeted locus (Lee et al., 2016) [43] . ZFNs have been utilized to modify specific gene sequences in various crops, including maize (Shukla et al., 2009; Ainley et al., 2013) [75,3] , soybean (Curtin et al., 2011) [24] , rice (Cantos et al., 2014; Jung et al., 2018) [18,39] , and apple (Peer et al., 2015) [62] .

Transcription Activator-Like Effector Nucleases

Transcription activator-like effector nucleases (TALENs) have emerged as an alternative to zinc-finger nucleases (ZFNs) for genome editing (Sakuma and Yamamoto, 2017) [67] . Like ZFNs, TALENs are composed of DNA-binding and nuclease domains derived from the FokI enzyme. The DNA-binding domain in TALENs, known as transcription activator-like effector (TALE) repeats, is typically made up of repetitive sequences derived from TALEs secreted by Xanthomonas bacteria, which facilitate the infection of plant species (Figure 1). Each TALE repeat is designed to recognize a single nucleotide within a genomic sequence (Joung and Sander, 2013) [38] . A single DNA-binding repeat consists of a conserved 34-amino acid sequence with a highly variable region at positions 12 and 13, referred to as the repeat variable di-residue (RVD), which determines the nucleotide-binding specificity (Richter et al., 2020) [65] . By combining specific RVDs, a TALE DNA-binding domain, linked to the FokI nuclease domain, can generate double-strand breaks (DSBs) at a desired genomic target site. Similar to ZFNs, TALEN-mediated genome editing has been successfully employed for targeted mutagenesis in various crop species, including rice (Shan et al., 2013) [71] , soybean (Haun et al., 2014) [30] and potato (Sawai et al., 2014) [68] .

Precise genome editing in plants

Targeted mutagenesis in plants primarily relies on nonhomologous end joining (NHEJ) to repair double-strand breaks (DSBs). This repair mechanism is inherently error-prone, often resulting in small deletions, insertions, or nucleotide substitutions (Atkins and Voytas, 2020) [7] . In contrast, homology-directed repair (HDR) is a less frequent pathway due to its lower editing efficiency. However, HDR allows for precise gene insertions or replacements when a suitable repair template is provided. The advent of modern mutagenesis tools, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas systems, has significantly transformed the landscape of plant molecular biology and functional genomics. These tools hold considerable promise for advancing crop development through the targeted modification of agronomic traits (Chen et al., 2019) [22] . Although ZFNs and TALENs have been instrumental in targeted mutagenesis across various plant species such as Arabidopsis, maize, Brassica, rice, barley, soybean, tobacco, tomato, wheat, potato, and sugarcane (Shelake et al., 2019) [72] , the CRISPR-Cas system has emerged as the most widely adopted genome editing tool. Its popularity is attributed to its straightforward engineering process, versatility, affordability, high efficiency, and remarkable specificity (Brandt and Barrangou, 2019) [15] . This system shows immense potential for genome editing aimed at developing improved cultivars with added value (Zhang et al., 2020c) [88] .

Basic CRISPR-Cas System

The CRISPR-Cas9 system has become a widely used platform for genome engineering, primarily due to its simplicity, versatility, and precision (Figure 2). The system's central component, Cas9 (SpCas9), originates from the type II CRISPR-Cas Class 2 system of Streptococcus pyogenes. It was the first nuclease adapted for genome editing and remains the most frequently utilized enzyme in this domain (Mali et al., 2013) [56] . The CRISPR-Cas9 system functions with the help of a singleguide RNA (sgRNA), designed to operate in the crRNA-tracrRNA complex. The sgRNA guides the Cas9 protein to specific genomic loci through complementary base pairing between the sgRNA and the target DNA sequence, located next to a protospacer adjacent motif (PAM). The Cas9 protein then cleaves both DNA strands, typically generating blunt-end cuts three base pairs upstream of the PAM site (5'-NGG; Makarova et al., 2015) [55] . To overcome the limited availability of target sites recognized by SpCas9, various Cas9 variants have been developed to recognize different PAM sequences. These include xCas9, which recognizes 5'-NG, 5'-GAA, and 5'-GAT (Hu et al., 2018) [34] ; Staphylococcus aureus Cas9 (SaCas9), which recognizes 5'-NNGRRT; Streptococcus thermophilus Cas9 (StCas9), which recognizes 5'-NNAGAAW (Cong et al., 2013) [23] ; Neisseria meningitidis Cas9 (NmCas9), which recognizes 5'-NNNNGATT (Esvelt et al., 2013) [25] ; Campylobacter jejuni Cas9 (CjCas9), which recognizes 5'-NNNVRYM (Yamada et al., 2017) [85] ; and CasX, also known as Cas12e, which recognizes 5'-TTCN (Burstein et al., 2017) [17] . Additionally, Cas13 variants, which target RNA instead of DNA, have been employed to study virus biology in plants, showcasing high interference activities against RNA viruses (Mahas et al., 2019) [54] . These innovations have significantly expanded the flexibility and applicability of CRISPR-Cas systems, enabling targeted genetic modifications across a broad spectrum of organisms, including plants, for enhanced trait development and disease resistance. Base Editing Deaminase-mediated base editing technology is an advanced genome-editing tool that enables precise point mutations in target genomic regions without inducing double-strand breaks (DSBs). This technology involves a base editor fused to a Cas9 nickase, which is directed to a specific DNA sequence by a guide RNA (gRNA). Two primary base editing tools, cytosine base editors (CBEs) and adenine base editors (ABEs), have been developed for this purpose (Shimatani et al., 2017;) [73] . Furthermore, dual base editor-mediated precise genome-editing technology has also been applied to plants. Cytosine Base Editors (CBEs) consist of a Cas9 nickase (nCas9) with a D10A mutation that inactivates one of the nucleolytic domains, RuvC, of Cas9. This nCas9 is fused with a cytidine deaminase and a uracil glycosylase inhibitor (UGI; Figure 2C). The cytidine deaminase catalyzes the deamination of cytidine in the DNA strand, converting it into uridine. When the nCas9 (D10A) protein nicks the non-target DNA strand, the U-G mismatch induces a T-A substitution in the resulting DNA strands through DNA repair and replication processes (C-to-T conversion). CBE-mediated base editing has been effectively employed in various crops such as rice, maize, wheat, and potato, showing relatively high efficiency (Shimatani et al., 2017; Zong et al., 2017) [73,92] . Adenine Base Editors (ABEs), another class of base editing technology, facilitate the conversion of A-T base pairs to G-C. Adenine base editors utilize an adenosine deaminase fused with nCas9 (D10A), which deaminates adenosine to inosine. Inosine pairs with cytidine and is interpreted as guanine by DNA polymerase during the DNA repair and replication process, leading to an A-T to G-C conversion (Gaudelli et al., 2017) [28] . ABEs have been applied in crops like Arabidopsis, wheat, rice, and rapeseed, although the efficiency is generally lower compared to the original SpCas9 or SaCas9 systems (Kang et al., 2018) [40] . However, recent development of ABE variants in mammalian cells could potentially enhance editing efficiency in plants (Richter et al., 2020) [65] .

Allows simultaneous conversion of C-G to T-A and A-T to G-C.

This method utilizes the fusion of nCas9 with both a CBE and an ABE, a system known as the Saturated Targeted Endogenous Mutagenesis Editor (STEME). This tool enables the generation of dual base substitutions (C-G to T-A and A-T to G-C) using a single guide RNA (sgRNA). The STEME system, which comprises an ABE, a CBE, nCas9 (D10A), sgRNA, and UGI, was recently used for genome editing in rice (Richter et al., 2020) [65] . STEMEs can produce a diverse array of mutations, including base substitutions and in-frame insertions and deletions (indels) with high efficiency, making dual base editing particularly useful for studying cis-elements in noncoding regions and for genome-wide screening of cis-regulatory regions.

Prime Editing

One of the primary challenges with current genome-editing tools is their technical complexity and extremely low efficiency in introducing customized sequences at specific target sites. To address this limitation, Anzalone et al. (2019) [5] developed a ground-breaking genome-editing technology called prime editing. This innovative approach enables 12 types of base conversions in target genes at locations ranging from 3 base pairs (bp) upstream to 29 bp downstream of the protospacer adjacent motif (PAM). Prime editing allows for precise insertions of up to 44 bp and deletions of up to 80 bp, all without causing double-strand breaks (DSBs) (Anzalone et al., 2019) [5] . The prime editing system is composed of a Cas9 nickase (nCas9) with an H840A mutation fused to a reverse transcriptase (RTase) and a prime editing guide RNA (pegRNA). The pegRNA directs the nCas9 (H840A) to bind to the target DNA sequence and consists of two key regions: a spacer that is complementary to the non-edited DNA strand at the 5' end of the pegRNA, and a prime binding site (PBS) located at the 3' end of the pegRNA. The PBS is crucial for recognizing the sequence of the DNA strand to be edited and includes the desired sequences to be introduced into the target site (Figure 2E). In this system, the PBS functions as a primer for the RTase linked to nCas9 (H840A). The RTase uses the pegRNA as a template, which pairs with the nCas9 (H840A)-nicked single-stranded DNA (ssDNA) strand, effectively copying the genetic information from the pegRNA into the target genome site (Anzalone et al., 2019) [5] . After reverse transcription, the system undergoes equilibration between the 3' edited DNA flap and the 5' unedited DNA flap, followed by the integration of the edited DNA into the target site via ligation and the DNA repair system (Anzalone et al., 2019) [5] . Despite its potential, the prime editing system's efficiency in plants has been limited, and its ability to achieve precise gene editing has only been verified in two food crops: rice and wheat (Lin et al., 2020) [50] . Therefore, additional studies are required to demonstrate the applicability of prime editing technology in a wider range of organisms. These studies will be crucial for advancing the use of prime editing tools and making continuous improvements to the technology, thereby expanding its utility in various fields of genetic research and crop development.

Chromosome Engineering

In plant breeding, the goal is to combine numerous favourable traits into a single breeding line. Since traits are determined by genes located on chromosomes, genes that are located close to each other on the same chromosome are often inherited together, a phenomenon known as complete linkage. However, this linkage can sometimes include unfavourable traits, making it challenging to isolate and maintain beneficial traits through traditional breeding methods. To address this issue, CRISPR-Cas9 technology offers a promising solution by enabling precise genome editing. The technology can induce double-strand breaks (DSBs) in DNA, which, when followed by homologous recombination (HR), can potentially disrupt unwanted linkages and facilitate the separation of target genes from linked unfavourable traits (Filler Hayut et al., 2017) [26] . Despite this potential, the frequency of crossover (CO) events that lead to the separation of linked genes remains relatively low, and efficient detection systems are required to identify successful HR events. Early studies demonstrated that CRISPR-Cas9-mediated DSB induction could lead to DNA fragment inversions in plants, a technique that has been used to revert large inversions and thus make the region accessible to meiotic crossovers (Zhang et al., 2017) [86] . This approach has allowed for the elimination of unwanted inversions and has facilitated the recombination of desirable traits. For instance, research has shown that the introduction of multiple DSBs, although with low efficiency, can cause inversions, and the reversal of a 1.1 Mb inversion on Arabidopsis chromosome 4 using SaCas9 has been successfully achieved (Schmidt et al., 2020) [69] . Moreover, CRISPR-Cas9 technology has enabled the creation of heritable reciprocal translocations in the mega base pair (Mbp) range between nonallelic chromosomes, a process that has significant implications for plant breeding (Beying et al., 2020) [12] . This approach could substantially enhance the introgression of genes from wild relatives into cultivated varieties by reducing linkage drag, thereby making extensive backcrossing less necessary.

Gene cloning strategies for vegetables with improved traits

Vegetables play a crucial role in human nutrition due to their rich content of essential nutrients and phytochemicals, which are vital for disease prevention and overall health maintenance. To reduce the risk of cardiovascular diseases and cancer, recent guidelines recommend consuming over 400 grams of vegetables and fruits daily (Aune et al., 2017) [8] . However, vegetable crops face numerous challenges, including susceptibility to pests, diseases caused by viruses, bacteria, and fungi, and abiotic stresses such as drought, salinity, flooding, and nutrient deficiencies (Boscaiu and Fita, 2020) [14] . To address these challenges, breeding programs aim to develop vegetable varieties that are resistant to biotic stresses, tolerant to abiotic conditions, and high yielding with enhanced nutritional content. In this context, CRISPR-Cas9 technology has emerged as a promising tool to achieve these goals (Khatodia et al., 2017) [41] . CRISPR-Cas9 allows for precise genome editing, which can be utilized to enhance resistance to pests and diseases, improve stress tolerance, and boost nutritional qualities in vegetable crops. By enabling targeted modifications in the plant genome, CRISPR-Cas9 can help create varieties that better withstand environmental challenges and provide greater health benefits to consumers (Table 1).

Tomato

Tomato (Solanum lycopersicum L.) serves as a prominent model for testing CRISPR-Cas9 approaches due to its wellcharacterized genome and significant economic value. Since the advent of CRISPR-Cas9 in tomato research, several notable advancements have been made: 1. Initial Applications: The first successful CRISPR-Cas9mediated genome editing in tomato was reported in 2014.

Researchers targeted the SIAGAMOUS-LIKE 6 (SIAGL6) gene, which is involved in leaf development, demonstrating the tool's potential in manipulating developmental pathways (Brooks et al., 2014) [16] .

Fruit Ripening:

The CRISPR-Cas9 system was further applied to the RIPENING INHIBITOR (RIN) gene, which encodes a MADS-domain transcription factor crucial for fruit ripening. Mutations in RIN resulted in incomplete fruit ripening, underscoring the gene's critical role and the potential for CRISPR-Cas9 to influence fruit quality (Ito et al., 2015) [37] .

Targeted Mutagenesis and Marker-Free Editing:

Shimatani et al. (2017) [73] employed a base-editing approach known as Target-AID (CRISPR-AID), which combines a Cas9 nickase (nCas9, D10A) with a cytidine deaminase. This method was used to create marker-free, homozygous DNA substitutions in endogenous tomato genes, DELLA (Solyc11g011260) and Ethylene Resistance 1 (ETR1; Solyc12g011330), which are involved in plant hormone signalling (Shimatani et al., 2017) [73] . 4. Stress Tolerance: Tomato plants with CRISPR-Cas9induced mutations in the Solanum lycopersicum mitogenactivated protein kinase 3 (slmapk3) gene exhibited enhanced tolerance to heat stress. These plants showed reduced wilting, less membrane damage, lower reactive oxygen species (ROS) levels, and increased antioxidant enzyme activity, highlighting the utility of CRISPR-Cas9 for improving stress resilience. [83] .

Dwarfism and Gibberellin

Cucumber

Cucumber (Cucumis sativus L.), a member of the Cucurbitaceae family, is a widely cultivated vegetable with significant economic value across temperate and tropical regions. The CRISPR-Cas9 system has been utilized to advance cucumber breeding and improve traits such as disease resistance and flowering characteristics:

• Viral Resistance: The initial application of CRISPR-Cas9 in cucumber focused on enhancing viral resistance. By targeting the eukaryotic translation initiation factor 4E (eIF4E) gene, researchers successfully conferred broadspectrum resistance to various viral infections. This approach highlights CRISPR-Cas9's potential for developing crops with improved disease resistance (Chandrasekaran et al., 2016) [19] . • Gynoecious Phenotype: In cucumber breeding, gynoecious lines are valuable due to their higher fruit production and reduced labour costs for crossing. Hu et al. (2017) [32] employed CRISPR-Cas9 to generate mutants of the WPP trp/pro/pro domain Interacting Protein1 (CsWIP1) gene, which encodes a zinc-finger transcription factor involved in flower development. The resulting Cswip1 T0 mutants displayed a gynoecious phenotype, bearing only female flowers. This result indicates that CsWIP1 plays a role in suppressing cucumber carpel development, making it a key target for manipulating flowering traits (Hu et al., 2017) [32] .

Watermelon

Watermelon (Citrullus lanatus), a member of the Cucurbitaceae family, is valued for its nutritional content, including citrulline, vitamins, and lycopene (Maoto et al., 2019) [57] . The CRISPR-Cas9 system has been instrumental in watermelon genetic modification, with several applications aimed at improving fruit characteristics and resistance to pests and diseases:

1. Albino Phenotype: The CRISPR-Cas9 system was used to target the phytoene desaturase (ClPDS) gene, which encodes an essential enzyme in carotenoid biosynthesis.

Mutations in this gene resulted in an albino phenotype, demonstrating the system's efficacy in altering pigment production (Tian et al., 2017) [81] . 2. Herbicide Resistance: Base editing technology was applied to the acetolactate synthase (ClALS) gene, achieving single-nucleotide conversions that conferred resistance to sulfonylurea herbicides. Watermelon plants with C to T mutations at Pro 190 in the ClALS gene were resistant to these herbicides while maintaining fruit and seed size and yield (Tian et al., 2018) [80] .

Disease Resistance:

The CRISPR-Cas9 system was employed to knockout the phytosulfokine1 (ClPSK1) gene, which is involved in susceptibility to Fusarium oxysporum f. sp. niveum (FON). The loss-of-function mutations in ClPSK1 resulted in increased resistance to FON, enhancing plant resilience to this pathogen (Zhang et al., 2020b) [88] . 4. Gynoecious Lines: The ClWIP1 gene, a homologue of CsWIP1 in cucumber and CmWIP1 in melon, was targeted to create gynoecious watermelon lines. ClWIP1 is involved in carpel development and its mutation led to the production of lines with a gynoecious phenotype, showing its role in floral development (Zhang et al., 2020a) [88] .

Eggplant

Eggplant (Solanum melongena L.), the fifth most produced vegetable globally with 52.3 million tons in 2017 (Alam and Salimullah, 2021) [4] , faces challenges such as enzymatic browning, which affects the quality and marketability of its fruits. Polyphenol oxidase (PPO) enzymes are primarily responsible for this browning process. In eggplant, three PPO genes-SmelPPO4, SmelPPO5, and SmelPPO6-have been identified as having high transcript levels in fruit after cutting, indicating their significant role in enzymatic browning. To address this issue, researchers have utilized CRISPR-Cas9-based mutagenesis to simultaneously knockout these three PPO genes. This approach aims to reduce or eliminate the browning of eggplant fruit flesh, thereby improving its quality and shelf life.

The application of CRISPR-Cas9 technology in this context demonstrates its potential to enhance the post-harvest characteristics of eggplants by targeting specific genes involved in undesirable traits, such as enzymatic browning.

Leafy vegetable

Leafy vegetables have benefited from CRISPR-Cas applications for trait improvement, including the development of methods for precise genome editing without the integration of foreign DNA.

In lettuce and cabbage, for example, CRISPR-Cas9 and Cpf1 (Now known as Cas12a) RNPs (Ribonucleoprotein complexes) have been directly delivered into protoplasts-a method known as a DNA-free genome-editing approach. Woo et al. (2015) [84] demonstrated successful delivery of CRISPR-Cas9 RNPs into lettuce protoplasts using PEG-mediated transfection. This technique led to the regeneration of plants with the intended mutations. More recent studies suggest that electroporation may offer improved efficiency over PEG-mediated methods for delivering RNPs into cabbage protoplasts. Electroporation is believed to have lower chemical toxicity, which may enhance its effectiveness for genome editing in leafy vegetables (Lee et al., 2020) [44] . Additionally, genome editing has been applied to modulate the translation of mRNA in leafy vegetables. Editing the upstream open reading frame (uORF) of genes involved in vitamin C biosynthesis, such as LsGGP1 and LsGGP2, has shown promising results. By disrupting the uORF, researchers increased mRNA translation efficiency, leading to elevated ascorbate content and improved tolerance to oxidative stress. This demonstrates the potential of CRISPR-Cas9 technology to enhance nutritional quality and stress resilience in leafy vegetables.

Perspectives

The debate over the regulation of new gene-editing techniques is indeed critical, especially given the potential benefits these technologies offer in crop improvement. While strict regulatory policies, like those in the EU, might limit the application of these innovations, recent reports from the European Commission suggest a cautious optimism about their potential. The advancement of more efficient and precise gene-editing systems, alongside the integration of multiple omics approaches, will be crucial in overcoming these regulatory hurdles and enhancing vegetable improvement. Identifying and targeting specific genes responsible for desirable traits, particularly in complex genetic backgrounds, will be key to maximizing the benefits of genome editing. [19] CsWIP1 KO/gynoecious phenotype Enhanced Agrobacterium-mediated transformation using vacuum infiltration Hu et al., 2017 [32] Watermelon ClPDS KO/albino phenotype Agrobacterium-mediated transformation / PEG-mediated protoplast transfection Tian et al., 2017 [81] ClALS Point mutation/herbicide resistance Agrobacterium-mediated transformation Tian et al., 2018 [80] ClPSK1 Chinese kale BaPDS1, BaPDS2 KO or KD/albino phenotype Agrobacterium-mediated transformation Sun et al., 2018 [78] BoaCRTISO KD/yellow color of Chinese kale with improved market prospects Agrobacterium-mediated transformation Sun et al., 2020 [77] Cabbage BoPDS KO/albino phenotype Agrobacterium-mediated transformation Ma et al., 2019a [51] Electro-transfection in RNP delivery to protoplast Lee et al., 2020 [44] BoPDS1, BoSRK3, BoMS1

Multisite and multiple gene KO using an array of sgRNA-tRNA/male-sterile line Agrobacterium-mediated hypocotyls transformation Ma et al., 2019b [51]

Conclusion

Advancements in genome editing technologies, such as base editing and prime editing, offer unprecedented precision in modifying genetic sequences, enabling the development of crops with improved traits. Techniques like CRISPR-Cas9 facilitate targeted modifications, enhancing resistance to pests, abiotic stresses, and boosting nutritional content across various vegetable species. Notable applications in crops like tomato, cucumber, and watermelon demonstrate the potential to improve traits and reduce undesirable characteristics. As research progresses, continued refinement of these tools and addressing regulatory challenges will be essential for unlocking their full potential in agricultural biotechnology and sustainable crop development.

References

1. Abdallah NA, Prakash CS, McHughen AG. Genome editing for crop improvement: Challenges and opportunities. GM Crops Food. 2015;6(3):183-205.
2. Ahloowalia BS, Maluszynski M, Nichterlein K. Global impact of mutation-derived varieties. Euphytica. 2004;135(2):187-204.
3. Ainley WM, Sastry-Dent L, Welter ME, Murray MG, Zeitler B, Amora R, et al. Trait stacking via targeted genome editing. Plant Biotechnol J. 2013;11(9):1126-1134.
4. Alam I, Salimullah M. Genetic engineering of eggplant (Solanum melongena L.): progress, controversy and potential. Horticulturae. 2021;7(4):78.
5. Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576(7785):149-157.
6. Arazoe T, Nishida K, Kondo A. Targeted nucleotide substitution in mammalian cells by target-AID. BioProtocol. 2017, 7(9).
7. Atkins PA, Voytas DF. Overcoming bottlenecks in plant gene editing. Curr Opin Plant Biol. 2020;54:79-84.
8. Aune D, Giovannucci E, Boffetta P, Fadnes LT, Keum N, Norat T, et al. Fruit and vegetable intake and the risk of cardiovascular disease, total cancer and all-cause mortalitya systematic review and dose-response meta-analysis of prospective studies. Int. J Epidemiol. 2017;46(3):1029-56.
9. Bernard G, Gagneul D, Alves Dos Santos H, Etienne A, Hilbert JL, Rambaud C. Efficient genome editing using CRISPR/Cas9 technology in chicory. Int J Mol Sci. 2019;20(5):1155.
10. Bernardo R. Bandwagons I, too, have known. Theor Appl Genet. 2016;129(11):2323-2232.
11. Bertier LD, Ron M, Huo H, Bradford KJ, Britt AB, Michelmore RW. High-resolution analysis of the efficiency, heritability, and editing outcomes of CRISPR/Cas9-induced modifications of NCED4 in lettuce (Lactuca sativa). G3: Genes, Genomes, Genetics. 2018;8(5):1513-1521.
12. Beying N, Schmidt C, Pacher M, Houben A, Puchta H. CRISPR-Cas9-mediated induction of heritable chromosomal translocations in Arabidopsis. Nat Plants. 2020;6(6):638-645.
13. Bisbis MB, Gruda N, Blanke M. Potential impacts of climate change on vegetable production and product quality -a review. J Clean Prod. 2018;170:1602.
14. Boscaiu M, Fita A. Physiological and molecular characterization of crop resistance to abiotic stresses. Agronomy. 2020;10(9):1308.
15. Brandt K, Barrangou R. Applications of CRISPR technologies across the food supply chain. Annu Rev Food Sci Technol. 2019;10:133-150.
16. Brooks C, Nekrasov V, Lippman ZB, Van Eck J. Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol. 2014;166(3):1292-1297.
17. Burstein D, Harrington LB, Strutt SC, Probst AJ, Anantharaman K, Thomas BC, et al. New CRISPR-Cas systems from uncultivated microbes. Nature. 2017;542(7642):237-241.
18. Cantos C, Francisco P, Trijatmiko KR, Slamet-Loedin I, Chadha-Mohanty PK. Identification of "safe harbor" loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair. Front Plant Sci. 2014;5:302.
19. Chandrasekaran J, Brumin M, Wolf D, Leibman D, Klap C, Pearlsman M, et al. Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology. Mol Plant Pathol. 2016;17(8):1140-1153.
20. Chaudhary J, Alisha A, Bhatt V, Chandanshive S, Kumar N, Mir Z, et al. Mutation breeding in tomato: advances, applicability and challenges. Plants. 2019;8(5):128.
21. Chen K, Gao C. Targeted genome modification technologies and their applications in crop improvements. Plant Cell Rep. 2014;33(4):575-583.
22. Chen K, Wang Y, Zhang R, Zhang H, Gao C. CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu Rev Plant Biol. 2019;70:667-697.
23. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819-823.
24. Curtin SJ, Zhang F, Sander JD, Haun WJ, Starker C, Baltes NJ, et al. Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases. Plant Physiol. 2011;156:466-473.
25. Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods. 2013;10:11161121.
26. Filler Hayut S, Melamed Bessudo C, Levy AA. Targeted recombination between homologous chromosomes for precise breeding in tomato. Nat Commun. 2017;8:15605.
27. Gao C. Genome editing in crops: from bench to field. Nat Sci Rev. 2014;2:13-15.
28. Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature. 2017;551:464-471.
29. Glenn K, Alsop B, Bell E, Goley M, Jenkinson J, Liu B, et al. Bringing new plant varieties to market: plant breeding and selection practices advance beneficial characteristics while minimizing unintended changes. Crop Sci. 2017;57(6):2906-2921.
30. Haun W, Coffman A, Clasen BM, Demorest ZL, Lowy A, Ray E, et al. Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family. Plant Biotechnol J. 2014;12(7):934-940.
31. Holme IB, Gregersen PL, Brinch-Pedersen H. Induced genetic variation in crop plants by random or targeted mutagenesis: convergence and differences. Front Plant Sci. 2019;10:1468.
32. Hu B, Li D, Liu X, Qi J, Gao D, Zhao S, et al. Engineering non-transgenic gynoecious cucumber using an improved transformation protocol and optimized CRISPR/Cas9 system. Mol Plant. 2017;10(12):1575-1578.
33. Hu J, Li S, Li Z, Li H, Song W, Zhao H, et al. A barley stripe mosaic virus-based guide RNA delivery system for targeted mutagenesis in wheat and maize. Mol Plant Pathol. 2019;20(11):1463-1474.
34. Hu JH, Miller SM, Geurts MH, Tang W, Chen L, Sun N, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature. 2018;556(7701):57-63.
35. Huang S, Weigel D, Beachy RN, Li J. A proposed regulatory framework for genome-edited crops. Nat Genet. 2016;48(2):109-111.
36. Ito Y, Nishizawa-Yokoi A, Endo M, Mikami M, Toki S. CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening. Biochem Biophys Res Commun. 2015;467(1):76-82.
37. Joung JK, Sander JD. TALENs: A widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol. 2013;14(1):49-55.
38. Jung Y-J, Nogoy FM, Lee S-K, Cho Y-G, Kang K-K. Application of ZFN for site-directed mutagenesis of rice SSIVa gene. Biotechnol Bioprocess Eng. 2018;23(1):108115.
39. Kang B-C, Yun J-Y, Kim S-T, Shin Y, Ryu J, Choi M, et al. Precision genome engineering through adenine base editing in plants. Nat Plants. 2018;4(5):427-431.
40. Khatodia S, Bhatotia K, Tuteja N. Development of CRISPR/Cas9 mediated virus resistance in agriculturally important crops. Bioengineered. 2017;8(3):274-279.
41. Kleter GA, Kuiper HA, Kok EJ. Gene-edited crops: towards a harmonized safety assessment. Trends Biotechnol. 2019;37(5):443-447.
42. Lee J, Chung J-H, Kim HM, Kim D-W, Kim H. Designed nucleases for targeted genome editing. Plant Biotechnol J. 2016;14(2):448-462.
43. Lee MH, Lee J, Choi SA, Kim Y-S, Koo O, Choi SH, et al. Efficient genome editing using CRISPR-Cas9 RNP delivery into cabbage protoplasts via electro-transfection. Plant Biotechnol Rep. 2020;14(6):695-702.
44. Li C, Li W, Zhou Z, Chen H, Xie C, Lin Y. A new rice breeding method: CRISPR/Cas9 system editing of the Xa13 promoter to cultivate transgene-free bacterial blightresistant rice. Plant Biotechnol J. 2020;18(2):313-315.
45. Li C, Zhang R, Meng X, Chen S, Zong Y, Lu C, et al. Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors. Nat Biotechnol. 2020;38:875-882.
46. Li C, Zong Y, Wang Y, Jin S, Zhang D, Song Q, et al. Expanded base editing in rice and wheat using a Cas9adenosine deaminase fusion. Genome Biol. 2018;19:59.
47. Li J, Manghwar H, Sun L, Wang P, Wang G, Sheng H, et al. Whole genome sequencing reveals rare off-target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9-edited cotton plants. Plant Biotechnol J. 2019;17:858-868.
48. Li T, Yang X, Yu Y, Si X, Zhai X, Zhang H, et al. Domestication of wild tomato is accelerated by genome editing. Nat Biotechnol. 2018;36:1160-1163.
49. Lin Q, Zong Y, Xue C, Wang S, Jin S, Zhu Z, et al. Prime genome editing in rice and wheat. Nat Biotechnol. 2020;38:582-585.
50. Ma C, Liu M, Li Q, Si J, Ren X, Song H. Efficient BoPDS gene editing in cabbage by the CRISPR/Cas9 system. Horticult Plant J. 2019;5:164-169.
51. Ma C, Zhu C, Zheng M, Liu M, Zhang D, Liu B, et al. CRISPR/Cas9-mediated multiple gene editing in Brassica oleracea var. capitata using the endogenous tRNAprocessing system. Horticult Res. 2019;6:20.
52. Ma X, Zhang X, Liu H, Li Z. Highly efficient DNA-free plant genome editing using virally delivered CRISPR-Cas9. Nat Plants. 2020;6:773-779.
53. Mahas A, Aman R, Mahfouz M. CRISPR-Cas13d mediates robust RNA virus interference in plants. Genome Biol. 2019;20:263.
54. Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, et al. An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol. 2015;13:722736.
55. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339:823-6. doi:10.1126/science.1232033.
56. Maoto MM, Beswa D, Jideani AIO. Watermelon as a potential fruit snack. Int J Food Prop. 2019;22:355-370.
57. Miller J, McLachlan AD, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985;4:1609-1614.
58. Miller V, Mente A, Dehghan M, Rangarajan S, Zhang X, Swaminathan S, et al. Fruit, vegetable, and legume intake, and cardiovascular disease and deaths in 18 countries (PURE): A prospective cohort study. Lancet. 2017;390:2037-49. doi:10.1016/S0140-6736(17)32253-5.
59. Muller HJ. Artificial transmutation of the gene. Science. 1927;66:84-87.
60. Peer R, Rivlin G, Golobovitch S, Lapidot M, Gal-On A, Vainstein A, et al. Targeted mutagenesis using zinc-finger nucleases in perennial fruit trees. Planta. 2015;241:941-951.
61. Petolino JF. Genome editing in plants via designed zinc finger nucleases. In Vitro Cell Dev Biol Plant. 2015;51:1-8.
62. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520:186-191.
63. Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol. 2020;38:883-91.
64. Rodríguez-Leal D, Lemmon ZH, Man J, Bartlett ME, Lippman ZB. Engineering quantitative trait variation for crop improvement by genome editing. Cell. 2017;171:470.e478-80.e478.
65. Sakuma T, Yamamoto T. Current overview of TALEN construction systems. Methods Mol Biol. 2017;1630:25-36.
66. Sawai S, Ohyama K, Yasumoto S, Seki H, Sakuma T, Yamamoto T, et al. Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal glycoalkaloids in potato. Plant Cell. 2014;26:3763-3774.
67. Schmidt C, Fransz P, Rönspies M, Dreissig S, Fuchs J, Heckmann S, et al. Changing local recombination patterns in Arabidopsis by CRISPR/Cas mediated chromosome engineering. Nat Commun. 2020;11:4418.
68. Septembre-Malaterre A, Remize F, Poucheret P. Fruits and vegetables, as a source of nutritional compounds and phytochemicals: Changes in bioactive compounds during lactic fermentation. Food Res Int. 2018;104:86-99.
69. Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol. 2013;31:686-688.
70. Shelake RM, Pramanik D, Kim JY. Exploration of plantmicrobe interactions for sustainable agriculture in CRISPR era. Microorganisms. 2019;7:269.
71. Shimatani Z, Kashojiya S, Takayama M, Terada R, Arazoe T, Ishii H, et al. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat Biotechnol. 2017;35:441-443.
72. Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE, et al. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature. 2009;459:437-441.
73. Stadler LJ. Genetic effects of X-rays in maize. Proc Natl Acad Sci U S A. 1928;14:69-75.
74. Sun B, Jiang M, Zheng H, Jian Y, Huang WL, Yuan Q, et al. Color-related chlorophyll and carotenoid concentrations of Chinese kale can be altered through CRISPR/Cas9 targeted editing of the carotenoid isomerase gene BoaCRTISO. Horticult Res. 2020;7:161.
75. Sun B, Zheng A, Jiang M, Xue S, Yuan Q, Jiang L, et al. CRISPR/Cas9-mediated mutagenesis of homologous genes in Chinese kale. Sci Rep. 2018;8:16786.
76. Symington LS, Gautier J. Double-strand break end resection and repair pathway choice. Annu Rev Genet. 2011;45:247271.
77. Tian S, Jiang L, Cui X, Zhang J, Guo S, Li M, et al. Engineering herbicide-resistant watermelon variety through CRISPR/Cas9-mediated base-editing. Plant Cell Rep. 2018;37:1353-1356.
78. Tian S, Jiang L, Gao Q, Zhang J, Zong M, Zhang H, et al. Efficient CRISPR/Cas9-based gene knockout in watermelon. Plant Cell Rep. 2017;36:1-8.
79. Tomlinson L, Yang Y, Emenecker R, Smoker M, Taylor J, Perkins S, et al. Using CRISPR/Cas9 genome editing in tomato to create a gibberellin-responsive dominant dwarf DELLA allele. Plant Biotechnol J. 2019;17:132-140.
80. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD. Genome editing with engineered zinc finger nucleases. Nat Rev Genet. 2010;11:636-646.
81. Woo JW, Kim J, Kwon SI, Corvalán C, Cho SW, Kim H, et al. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol. 2015;33:1162-64.
82. Yamada M, Watanabe Y, Gootenberg JS, Hirano H, Ran FA, Nakane T, et al. Crystal structure of the minimal Cas9 from Campylobacter jejuni reveals the molecular diversity in the CRISPR-Cas9 systems. Mol Cell. 2017;65:1109.e1103-21.e1103.
83. Zhang C, Liu C, Weng J, Cheng B, Liu F, Li X, et al. Creation of targeted inversion mutations in plants using an RNA-guided endonuclease. Crop J. 2017;5(1):83-88.
84. Zhang H, Si X, Ji X, Fan R, Liu J, Chen K, et al. Genome editing of upstream open reading frames enables translational control in plants. Nat Biotechnol. 2018;36(8):894-898.
85. Zhang J, Guo S, Ji G, Zhao H, Sun H, Ren Y, et al. A unique chromosome translocation disrupting ClWIP1 leads to gynoecy in watermelon. Plant J. 2020;101(2):265-77.
86. Zhang J, Zhang H, Botella JR, Zhu JK. Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties. J Integr Plant Biol. 2018;60(4):369-375.
87. Zhang M, Liu Q, Yang X, Xu J, Liu G, Yao X, et al. CRISPR/Cas9-mediated mutagenesis of Clpsk1 in watermelon to confer resistance to Fusarium oxysporum f.sp. niveum. Plant Cell Rep. 2020;39(5):589-595.
88. Zhang Y, Pribil M, Palmgren M, Gao C. A CRISPR way for accelerating improvement of food crops. Nat Food. 2020;1(4):200-205.
89. Zong Y, Wang Y, Li C, Zhang R, Chen K, Ran Y, et al. Precise base editing in rice, wheat and maize with a Cas9cytidine deaminase fusion. Nat Biotechnol. 2017;35(5):438-440.
90. Zsögön A, Čermák T, Naves ER, Notini MM, Edel KH, Weinl S, et al. De novo domestication of wild tomato using genome editing. Nat Biotechnol. 2018;36(12):1211-1216.