DNA-free Gene editing in Vitis vinifera L.; Knockout green fluorescent protein gene in Sultana grape by direct delivery of RNPs into protoplast

Global agricultural productivity, farm incomes, and food security will all be impacted by climate change. Grape (Vitis vinifera L.) is one of the world's most commercially significant fruit crops, and it is extensively cultivated for fruits, juice, and, most importantly, wine. Scientific evidence sharply states that climate change represents a dominant challenge for viticulture in the upcoming decades. Agriculture and farming around the world are highly depended on crops that produce food and fiber for humans, either directly or indirectly through livestock. Modern technology has improved agricultural operations over the last two centuries, complementing traditional plant breeding approaches to improve crop productivity and quality. Combining synthetic tools and traditional breeding into genomics-based breeding is a novel way to get over the limitations of traditional breeding. Genome editing offers the potential to speed up basic research and plant breeding by allowing for quick, accurate, and targeted genome editing. The revolutionary CRISPR-Cas system offers enormous potential for editing gene expression for crop improvement and food production. Genome editing is a powerful way to find and precisely locate a specific region inside a genome, then edit the targeted regions for a variety of applications. Unlike traditional transgenic technology, which randomly introduces genetic components into a genome, genomic editing operates on a specific genome sequence inside the genome. The risks of altering genomes via genome-editing technologies are considerably less than those associated with genetically modified (GM) crops since most edits typically affect a few nucleotides leading to changes similar to those found in naturally occurring populations. There is no way to tell the difference between a 'naturally occurring' mutation and a gene edit after the genomic-editing agents have separated. This DNA-free RNP delivery approach is promising for plant breeding since the resulting edited crops are likely falling outside of GMO regulation. Consequently, DNA-free genome editing is a game-changing technique that allow for faster and more precise crop development. In this study, we described a successful knockout of a green fluorescent protein (GFP) reporter gene, that is already integrated into the grape genome with a single copy, in V. vinifera "Sultana" by direct delivery of RNPs into protoplast. We demonstrated the use of this powerful new tool in targeted knockout of a gene settled in the grape genome. By following the loss of the GFP fluorescence signal, we were able to observe the cells that had endured targeted mutations as a result of CRISPR/ Cas9 activity. In addition, we presented examples of the various types of indels obtained by Cas9-mediated cleavage of the GFP gene, guided by two independent sgRNAs. The application of the CRISPR/Cas9 RNP system enables the generation of grape plants engineered by DNA-free gene editing. Eventually, we provided an optimized protocol to target important native genes in the grape plant in the future. In this study for the first time, we managed to achieve whole plants regenerated from DNA-free genome edited protoplasts. Monitoring the protoplast to whole plant developmental stages demonstrated that regenerated plantlets derived from gene-edited protoplasts exhibited a normal phenotype concerning leaf shape, color and growth habits compared to wild-type plants. Here an efficient protocol has been presented for foreign DNA-free CRISPR/Cas9 mediated gene editing in Vitis vinifera Sultana including detailed protoplast-to-plant steps.