1.4.1. Production of Non-GM Plants Using CRISPR-Cas9 Gene Editing

Most fruit crops have heterozygous genotypes, hybrid incompatibility, and long growth periods. Some have complex triploid or polyploid genomes [90]. During the last 20 years, innovative breeding technology employing genetic engineering has provided a favourable way to accelerate crop improvement for such species [91]. For example, the introduction of foreign DNA fragments when creating transgenic lines may block the function of endogenous genes and affect the expression of adjacent genes. In contrast to transgenic approaches, CRISPR/Cas9 technology is able to generate nontransgenic plants. Because CRISPR/Cas9 expression cassettes and their target sites are located at different positions of the genome, segregation and removal of the CRISPR/Cas9 cassettes is possible via subsequent selfing or crossing; however, this is not feasible in most fruit crops, because of their complex, highly heterozygous, and polyploid genomes, and because they are usually vegetatively propagated.

Fruit trees have a long juvenile stage and take several years to reach the reproductive stage. In such cases, the CRISPR/Cas9 components can be transiently expressed in the nucleus and function for a short time to induce precise mutations. This means that transgene-free edited plants can be generated, since the CRISPR/Cas9 expression cassette is not integrated into the genome. Currently, the overall efficiency of the transient system for the production of T-DNA-free edited apple lines is very low (0.4%) [92]; thus, the next step is to improve editing efficiency and to make this system suitable for other crops.

Preassembled CRISPR-Cas-sgRNA ribonucleoproteins (RNPs) can be delivered into plant cells and used for genome editing without the integration of foreign DNA because of degradation by endogenous proteases [93]. The protoplast transformation technique has been used to transform grape, apple, and lettuce with purified Cas9 RNPs. Sequencing analysis of transformed cells revealed mutagenesis efficiencies of 0.1 to 6.9% in grapevine and apple; however, due to the poor regeneration ability of protoplasts, no plants were regenerated [93,94].

Two methods have been used to obtain transgene-free plants with mutations via CRISPR/Cas9 gene editing. The first is based on the site-specific recombinase flippase (Flp) [95], which recognises 34 bp-long flippase recognition target site (FRT) sequences. The Flp/FRT system has been extensively used to remove undesired transgenic components in transgenic apple [96,97]. The second removal method relies on the Cas9 enzyme cleavage mechanism. Two additional synthetic target sites, referred to as cleavage target sites, were added next to the left border (LB) and right border (RB) sites of the CRISPR/Cas9 vector. When plants are transformed using CRISPR/Cas9, the Cas9 cleavage activity not only edits the endogenous target site, but also removes T-DNA by inserting two additional cleavage target sites, thereby resulting in T-DNA-free plants [98].
