**2. Future Challenges in the Application of CRISPR-Cas Gene Editing**

CRISPR-Cas9 genome editing has been introduced and used to obtain abundant germplasm resources with genetic variation, thanks to the results of whole genome sequencing and functional genomics studies in fruit and vegetable crops. Nonetheless, its future application faces two major challenges. The first is the accurate selection of key genes for targeted mutations and the corresponding types of mutations. Important agronomic traits are often complex quantitative traits and editing a single gene does not produce phenotypic changes. Therefore, efficient CRISPR-Cas-mediated target site-specific insertion and chromosome recombination methods can be used to accumulate mutant alleles [132].

The use of gene-editing technology to inhibit the expression of specific genes in plants reduces their adaptability. Therefore, precise genome editing requires efficient and specific regulation of gene functions. Mutations in the exons of genes can change the function of proteins, and mutations in the exon-intron splice sites can result in different alternative splicing variants. Cis-regulatory elements (CREs) are noncoding DNA sequences that often serve to regulate gene transcription. In CREs, single-nucleotide mutations, insertions, deletions, inversions, and epigenetic variations in gene regulatory regions are closely associated with crop domestication [133]. The CRISPR-Cas system has been used to create mutations in the regulatory regions of promoters, and to generate a number of alleles with variable phenotypes, which serve as excellent genetic resources in breeding programs. Currently, the gene editing of CREs in fruit and vegetable crops is still in its preliminary stages. Future studies are aimed at associating changes in expression produced by different mutant CREs with the corresponding phenotypes and obtaining abundant breeding resources through gene editing of CREs. Target induction of DSBs using CRISPR-Cas results not only in small mutations, such as base substitutions, insertions, and deletions, but also large rearrangements of the genome, including large deletions, chromosomal translocations, and inversion, which is an efficient way to completely delete undesired genes, such as those encoding allergens, in fruit and vegetable crops [134–136]. Although such chromosomal rearrangements occur at a lower frequency than with conventional targeted mutagenesis, a deficiency of Ku70, which is involved in the NHEJ pathway, increases the frequency of inversion and translocation (Figure 3) [135].

The second challenge in the application of gene-editing technology is transforming the CRISPR-Cas gene-editing system into plant cells and obtaining regenerated plants. Genetic mutation vector systems mediated by conventional Cas9 or Cas variants, and precise geneediting systems mediated by CBE, ABE, and prime editing, have been successfully applied

in model crops such as rice, corn, tobacco, and tomatoes, via *Agrobacterium*, gene gun, polyethylene glycol (PEG), and electroporation-based methods, providing a foundation for application in other crops [137]. However, developing a universal and efficient genetic transformation and regeneration system for fruit and vegetable crops is more difficult. In particular, the genetic transformation efficiency of CRISPR-Cas cassettes, and the ability of transformed tissues to regenerate plants, have become limiting factors. To improve explant regeneration ability after transformation, and meristem induction activity, the plant morphogenesis regulatory genes *Bbm* (baby boom) and *Wus2* (Wuschel2) can be overexpressed at the same time as CRISPR-Cas expression cassette transformation [138]. Using plant RNA and DNA viruses as vectors to transform plant cells via CRISPR-Cas can provide sufficient sgRNAs and shorten and simplify the process of genetic transformation and regeneration, making this method suitable for in situ transformation, and facilitating the production of gene-edited plants without transgenic elements [139].

**Figure 3.** Genome-editing toolbox for trait improvement. CRISPR-Cas9, Cas9 variants, and base and primer editing enable precise gene modification and predictable rearrangement of genomes and chromosomes.

> **Author Contributions:** This review was primarily conducted by L.W., Z.W., D.H., M.T., J.R., Y.S., N.Z. and H.Z., who were the contributors for the collection of the materials and revision of the manuscript. All authors have read and agreed to the published version of the manuscript.

> **Funding:** This review was funded by the Natural Science Foundation of Hubei Province (2020CFB845), Innovation Research Foundation of Wuhan Academy of Agricultural Sciences (QNCX202110).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** We appreciate internal review by Mao Donghai in Key Laboratory of Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences.

**Conflicts of Interest:** The authors declare no conflict of interest.
