*1.3. CRISPR-Cas9 in Fruit and Vegetable Crop Improvement*

In 2014, CRSPR-Cas9 was used to create the first needle-leaf mutant in tomato, by knocking out *Argonaute 7* [33]. Many studies have since been published on its possible applications in protecting plants against biotic and abiotic stresses, and improving fruit quality, plant architecture, and shelf life [34]. Currently, the system is in the research stage for many fruits and vegetables crops, such as cabbage, mustard, tomato, and watermelon.

Most gene-editing studies have evaluated mutation efficiency in terms of the number of albino plants obtained after mutation of the endogenous phytoene desaturase (*PDS)* gene. The disruption of *PDS* impairs the production of chlorophyll and carotenoid, generating an easily identifiable albinism phenotype in plants. However, the products of gene editing obtained in this way have no economic value [35–37]. Because of its high economic value and the availability of Agrobacterium-mediated transformation, tomato has become a model crop for testing CRISPR-Cas9 applications (Figure 2).

**Figure 2.** CRISPR-Cas9 mediated genome editing. (I) Selection of the desired genomic DNA target, and recognition of protospacer adjacent motif (PAM) sequences before 20 bp sequences. Design of the sgRNA using online bioinformatics tools. (II) Cloning of designed sgRNAs, and binary vector construction using promoters. (III) The delivery of CRISPR-Cas editing reagents into plant cells. The vector can be transferred into the plant via *Agrobacterium tumefaciens*, nanoparticles, biolistic bombardment, or polyethylene glycol (PEG). Alternatively, plant RNA viruses have been used to induce heritable genome editing. When the cassette harbouring the sgRNA, RNA mobile element, and tobacco rattle virus (TRV) is transformed into the Cas9 expressing plants, the systemic spread of sgRNA will introduce heritable genome editing. (IV) Plant transformation and development of transgenic plants. (V) Genotyping of transgenic plants. (VI) Transgene-free plants with the desired mutation are obtained.

## 1.3.1. Improvement of Biotic Stress Resistance

Two strategies have been used to improve plant resistance to viruses: (1) designing sgRNAs and targeting the virus genome; or (2), modifying the fruit crop genes in the antiviral pathway. The binding of virus genome linked protein (VPg) to the plant protein 'eukaryotic translation initiation factor 4E' (eIF4E) is key in Y virus infection of plants. Mutation of a key site of eIF4E can affect the virus–plant interaction, and mediate plant resistance to this virus [38]. In cucumbers, using CRISPR-Cas to target the N and C ends of eIF4E-produced nontransgenic homozygous plants in the T3 generation; these showed immunity to cucumber vein yellow virus and pumpkin mosaic virus, and resistance to papaya ring spot mosaic virus (PRSV-W) [39].

CRISPR-Cas9 can generate mutations in the coding and noncoding regions of geminivirus, effectively reducing its pathogenicity. In *Nicotiana benthamiana*, sgRNA-Cas9 constructs target beet severe curly top virus (a geminivirus), inhibiting its accumulation in leaves [40]. Geminivirus noncoding-region mutations are believed to reduce or even inhibit its replication ability. Compared with coding-region mutations, noncoding-region mutations generate fewer viral variants [41].

Fungi cause many diseases, potentially causing severe losses in crop yield and quality. For instance, downy and powdery mildews cause serious economic losses in tomato [42]. *Arabidopsis thaliana DMR6* (down mildew resistant) is a member of the 2-oxoglutarate oxygenase Fe(II)-dependent superfamily and is involved in salicylic acid homeostasis. Overexpression of *DMR6* in plants can reduce susceptibility to downy mildew [43]. The *DMR6* mutation obtained using CRISPR-Cas9 to knock out the homologous genes in tomato showed resistance to *Pseudomonas syringae*, *Phytophthora* and *Xanthomonas* spp. [44]. *Mlo1* (Mildew resistant locus 1) encodes a membrane-associated protein and is a powdery mildew disease-sensitivity gene. In tomato, *Mlo1* mutants obtained via gene editing exhibited resistance to the powdery mildew *Oidium neolycopersici*. Further, a mutant free of mlo1 T-DNA was obtained by selfing T0 generation plants [45].

The fungal pathogen *Fusarium oxysporum* can cause *Fusarium* wilt disease in fruit and vegetable crops [46]. In tomatoes, *Solyc08g075770*-knockout via CRISPR-Cas9 resulted in sensitivity to *Fusarium* wilt disease [47]. In watermelons, the knockout of *Clpsk1*, encoding the Phytosulfokine (PSK) precursor, confers enhanced resistance to *Fusarium oxysporum* f.sp.*niveum* (FON) [48]. *Botrytis cinerea*, an airborne plant pathogen that infects fruit and vegetable crops, causes great economic losses. Its initial symptoms are not obvious, and the lack of effective pesticides makes its prevention and control difficult. Pathogens can be effectively controlled in crops by the use of genetic resources that convey heritable resistance. In tomatoes, mutations in *MAPK3* (mitogen-activated protein kinase 3) produced using CRISPR-Cas9 induce resistance to *Botrytis cinerea* [49].

The bacterial pathogen *Pseudomonas syringae* causes leaf spot diseases in crops, severely impacting the yield and sensory qualities of fruits and vegetables. In *Arabidopsis thaliana*, CRISPR-Cas9 was used to mutate the C-terminal jasmonate domain (JAZ2Δjas) of *JAZ2* (jasmonate ZIM domain protein 2), causing expression of JAZ2 repressors; these repressors confer resistance to *Pseudomonas syringae* [50].

## 1.3.2. Abiotic Stress Resistance Improvement

With climate change, crop production is exposed to increased potential risks of abiotic stress. Although traditional breeding can to some extent ensure stable crop production, the application of new technologies to rapidly obtain new crop germplasm resources capable of responding to abiotic stress is essential for accelerating the cultivation of new varieties [51]. The emergence of CRISPR-Cas9 gene editing has shortened the time required to create new varieties. Brassinazole-resistant 1 gene (*BZR1*) participates in various brassinosteroid (BR) mediated development processes. The CRISPR mediated mutation in *BZR1* impaired the induction of *RESPIRATORY BURST OXIDASE HOMOLOG1*(*RBOH1*) and the production of H2O2. Exogenous H2O2 recovered the heat tolerance in tomato bzr1 mutant [52]. Further, new cold- and drought-tolerant germplasms can be created using gene-editing, for instance, of *CBF1* (C-repeat binding factor 1), which regulates cold tolerance in plants, and *MAPK3*, which participates in the drought stress response to protect plant cell membranes from peroxidative damage in tomatoes [53,54].
