Grape and apple fruit crop plants are a major source of fiber, nutrients, and antioxidants, all essential for a healthy diet. These crops play a key role in the economy of many developed and developing countries and considerable efforts are being made to improve commercial traits using both conventional breeding and genetic engineering. However, growing social distrust in genetically modified (GM) crops in many countries has resulted in the adoption of very stringent and costly regulations disciplining the authorization of these crops, with the result that they have become very difficult or impossible to commercialize successfully (Sprink et al., 2016). Although genetic transformation of grape and apple crops has been used for the past two decades to enhance primarily biotic and abiotic tolerance, there are only a few examples of field evaluation or commercialization of transgenic plants worldwide (Kanchiswamy et al., 2015). A transition is needed toward more efficient and productive use of available genome sequences to meet growing demands for sustainable and safe intensification of food production. Here we explore the possibility of adopting next-generation plasmid-independent CRISPR/Cas9 genome editing approaches to develop improved grape and apple varieties that will probably avoid current GM regulations, and thus broaden the utility of this technology, with greater global acceptance levels. US Department of Agriculture (USDA) does not impose any GMO regulations to the plants with targeted mutagenesis generated by self-repair mechanisms, if they are free from Agrobacterium or any transgenic or foreign genetic materials; consequently, we assume there is high probability that CRISPR/Cas9 RNPs could be exempted from current GMO regulations (Waltz, 2012; Ledford, 2013; Jones, 2015). Nevertheless, the EU is uncertain to approve them and has yet to provide information on whether targeted mutation made by CRISPR/Cas9 or CRISPR/Cas9 RNPs fall outside regulatory criteria (Waltz, 2012; Jones, 2015).

CRISPR/Cas9 editing tools are efficiently exploited for gene mutation, repression, activation and epigenome editing in several model and crop plants, such as Arabidopsis, tobacco, rice, sorghum, maize, wheat, poplar, tomato, soya bean, petunia, citrus and recently grape and apple (Nishitani et al., 2016; Ren et al., 2016; Song et al., 2016). Meanwhile CRISPR/Cas9 RNPs DNA-free genome editing tools are successfully demonstrated in Arabidopsis, tobacco, lettuce, rice, petunia, and recently in wheat (Woo et al., 2015; Subburaj et al., 2016; Zhang et al., 2016).

To date, CRISPR/Cas9 RNPs editing tools have not been applied to genetic modification of grape and apple crops. Here, we demonstrate adoption of next-generation CRISPR/Cas9 RNPs technology for these fruit species to establish an efficient DNA-free method for the site-directed mutagenesis system. In the grapevine, PM (Gadoury et al., 2012; Pessina et al., 2016) is caused by the destructive fungal pathogen Erysiphe necator, an obligate biotroph infecting all green tissues and berries, resulting in drastic losses in yield and berry quality. Currently PM can be effectively controlled by frequent applications of fungicides in the field. However, the rapid emergence of new fungal strains and the hazardous effect of fungicides on the environment, combined with additional costs to growers (which can reach up to 20% of total production costs), demand the development of sustainable alternative strategies. Recently, it has been demonstrated that RNAi-mediated silencing of the susceptible gene (S-gene) MLO-7 significantly increases resistance to PM in the grapevine (Pessina et al., 2016). Here we targeted MLO-7 for mutagenesis in order to increase resistance to PM in commercially important cultivar such as Chardonnay.

The enterobacterial phytopathogen Erwinia amylovora causes fire blight, an invasive disease that threatens the apple and a wide range of commercial and ornamental Rosaceae host plants. Although, many studies have identified candidate genes as suitable targets for increasing fire blight resistance via genetic engineering, currently no resistant cultivars are commercially available, due to the social and regulatory hurdles associated with GMO plants (Singh et al., 2006). Here we selected important fruit producing apple cultivar Golden delicious to target DIPM-1, DIPM-2, and DIPM-4, in order to increase resistance to fire blight disease (Meng et al., 2006). DIPM sequence structures are closely aligned with leucine-rich repeat receptor-like kinase receptors from several organisms, and furthermore DIPMs show direct physical interaction with the disease-specific (dsp) gene of Erwinia amylovora, which may act as a susceptible factor. Mutagenesis of DIPM-1, 2, and 4 could provide apple cultivars resistant to fire blight disease.

We successfully show direct delivery of CRISPR/Cas9 RNPs to the grape and apple protoplast and efficient mutation of the targeted candidate genes. Targeted gene mutation, such as indel (insertion or deletion), was observed in 2 out of 2 specific sites of MLO-7 in grapevine cultivars and single specific sites of each DIPM-1, 2, and 4 in apple cultivars.

Plant protoplasts constitute a dynamic and versatile system for CRISPR/cas9 genome editing in plants and has been widely adopted in several crop species for functional analysis of the traits concerned, cellular localization, and studies of multiple signaling cascades (Shan et al., 2013; Xie and Yang, 2013; Zhao et al., 2016). CRISPR/Cas9 or other genome editing tools mediated protoplast transfection system has been successfully adopted in Arabidopsis, rice, wheat, maize, tobacco, lettuce, and petunia (Jiang et al., 2013; Li et al., 2013; Shan et al., 2013; Wang et al., 2014; Gao et al., 2015; Woo et al., 2015; Subburaj et al., 2016), however, a similar system has not been developed for the grapevine and apple. In this study, we isolated protoplast from embryogenic calli and leaves of grapevine and apple cultivar in order to standardize an efficient protocol for the transient expression system of CRISPR/Cas9 RNPs. Protoplast isolation, transfection and transient gene expression system in grape and apple has been little explored and most of the available methods have not been updated for two decades (Doughty and Power, 1988; Patat-Ochatt et al., 1993; Patat-Ochatt, 1994; Reustle et al., 1995; Zhu et al., 1997; Saito and Suzuki, 1999; Fontes et al., 2010). Protoplast viability, yield, and efficient transfection depend on various factors, such as the concentration of cell wall digestion enzymes, buffer conditions, the osmotic status of protoplasts, the incubation period, and the type of explants used for protoplast isolation. In the current study, all these variables were updated and optimized in order to achieve a better yield. This is the first report of successful demonstration of CRISPR/Cas9 RNPs mediated protoplast transformation in grapevine and apple cultivars. Our method for transient expression of genome editing tools in the protoplast to target the gene of interest with specificity and higher efficiency should help grapevine and apple scientists to analyze the traits concerned in the host plant within a day or two. Furthermore, future work on regeneration of genome edited protoplast will provide an opportunity to develop DNA-free genome edited grapevine and apple fruit crop plants. One such example is regeneration of apple plants from meristem derived callus protoplast (Saito and Suzuki, 1999).

CRISPR/Cas9 is easy to prepare, scalable and affordable compared to ZFNs and TALENs. However, the broader application of plasmid mediated CRISPR/Cas9 to life sciences, biotechnology and medicine is limited by off-target effects, unwanted integration of plasmid vectors into the genome and possible GMO regulations (Hendel et al., 2015; Zhang et al., 2015). In order to overcome these limitations, we delivered CRISPR/Cas9 RNPs rather than plasmids directly into the protoplast cells and showed that RNPs enable efficient genome editing, while avoiding unwanted integration of plasmid DNA in the host genome, similar to other recent studies done in human, animal and plant cells (Figure 3; Kim et al., 2014; Lee et al., 2014; Liu et al., 2015; Woo et al., 2015; Subburaj et al., 2016).

Recently several groups have demonstrated that CRISPR/Cas9 can induce unwanted mutations in off-target sites, differing from on-target sites by up to 5 nt, leading to questions about their specificity (Cradick et al., 2013; Fu et al., 2013; Hsu et al., 2013; Tan et al., 2015). We and others have proposed various strategies to improve the on-target specificity of CRISPR/Cas9 (Koo et al., 2015; Kanchiswamy et al., 2016). Synthesis of unique sgRNA (Cho et al., 2014; Fu et al., 2014; Sung et al., 2014), Cas9 nickases and web based computer programs to identify unique target sites (Mali et al., 2013; Ran et al., 2013; Bae et al., 2014) and CRISPR/Cas9 RNPs (Woo et al., 2015). In order to facilitate the highest site-specific mutation frequency in grape and apple protoplast, we titrated the ratio of Cas9 and sgRNAs, in a similar way to our previous studies (Hsu et al., 2013; Woo et al., 2015). In this study, we employed three different Cas9: sgRNA ratios, i.e., 3:1, 1:1, and 1:3, for protoplast PEG mediated transformation in the grape and the apple. We determined that the 3:1 ratio for MLO-7 in the grapevine, the 3:1 ratio for DIPM 1, the 1:1 and 3:1 ratio for DIPM 2 and the 1:1 ratio for DIPM 4 in the apple resulted in highest mutation frequency. Here, we showed the critical advantage over plasmid mediated genome editing delivery by titrating the Cas9:sgRNA ratio to achieve maximum mutation frequency (Liu et al., 2015; Kanchiswamy et al., 2016).

This study demonstrated direct delivery of CRISPR/Cas9 RNPs to grape and apple protoplasts and site-directed mutation of the grape gene locus MLO-7 and the apple gene loci DIPM-1, 2, and 4.

We demonstrated efficient targeted mutagenesis in the grapevine gene locus MLO-7 and the apple gene loci DIPM-1, 2, and 4, using direct delivery of CRISPR RNPs. Although the mutation efficiency was found to vary with the targeted gene locus and the ratio of Cas9 and sgRNA, mutation patterns and frequency assays showed CRISPR RNPs to be an effective strategy for targeted mutagenesis of gene loci in grape and apple protoplasts. This method has already shown improved features compared to plasmid-mediated genome editing in humans, animals and plants, such as higher efficiency, significantly reduced off-target effects and more rapid editing activity after delivery (Liu et al., 2015; Woo et al., 2015; Kanchiswamy, 2016; Kanchiswamy et al., 2016). Furthermore, in plants, the new varieties obtained with this approach may be deregulated from current GMO legislations, as the Cas9 protein-guide RNA complexes will rapidly decompose in regenerating cell cultures. Further studies are now required to optimize plant regeneration from CRISPR RNPs transformed protoplast to explore the applications of this technology at field level.

CN, O-JK, and M-HJ performed and designed experiments. RV, SK, J-SK, RiV, and MM provided materials, funding, plan for the experiment. CN wrote manuscript with the cooperation and support of all co-authors.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.