Developing an Efficient DNA-free, Non-transgenic Genome Editing Methodology in Grapevine

Genome editing is a plant breeding innovation that allows rapid targeted modification of plant genome, like natural mutations, to improve traits such as yield and disease tolerance. Loss-of-function mutations in one or more of ‘Mildew resistance Locus O’ (MLO) genes impart protection to plants from powdery mildew fungi and confer durable broad-spectrum resistance. This mlo-based resistance, detected initially as a natural mutation in barley, has been successfully employed for nearly four decades. Recently, mlo-based resistance was found to be engineered or identified, from natural mutants, in many plant species of economic relevance. Genome editing in grapevine is currently performed through conventional Agrobacterium[1]mediated plasmid delivery, which integrates foreign genes into the genome and is labeled GMO plants. A more recent CRISPR RiboNucleoProtein (RNP) delivery in protoplasts is a DNA-free approach but makes it difficult to regenerate plantlets and identify the gene-edited mutants. This project aims to establish an alternative gene-editing approach for the grapevine model to generate powdery mildew-resistant mutant vines free of foreign genes. We proposed to edit the MLO susceptibility genes through a combination of plasmid- and RNP-delivered CRISPR/Cas9 in microvine to produce DNA-free powdery mildew-resistant plants. The project is currently in the 3rd quarter of the final year, and the first phase of generating mlo mutants through conventional Agrobacterium-mediated gene editing has been concluded. MLOs 3, 4, 13, and 17 in grapevine have been identified as the closest orthologs of MLO genes responsible for powdery mildew resistance in other species. We obtained single mutants of candidate MLOs and the combinations of double and quadruple mlo mutants. The mutant combinations will help identify the appropriate MLO genes for mildew resistance in grapevine. Currently, the genome-edited mlo mutant embryos are at various stages of plantlet regeneration. Preliminary genotyping analyses of the transformants showed mutations in the predicted areas of the MLO genes targeted by the CRISPR system. So far, we have identified over 200 potential mlo mutants. The next phase of the project is the removal of the transgene cassette, integrated into the genome of mlo mutants by employing CRISPR RNP delivery to obtain transgene-free mlo vines. Towards this goal, we concluded the experimental work. We established 1) the methodology to purify the modified Cas9 protein, 2) the RNP complexations and in vitro cleavage tests, 3) the excision of the transgene cassette using in vitro RNPs, 4) the use Cell-Penetrating Peptides (CPP) to facilitate the entry of the CRISPR RNP into regenerable embryogenic cells, and 5) the optimization of the CPP-RNP delivery conditions. The editing of the target genes through the CPP-conjugated RNP delivery into the embryogenic cells was also tested on GFP-expressing embryogenic cell lines. We expect the regenerating mlo-edited plantlets to be ready for genotyping and powdery mildew resistance assays in 3 months, after which the second phase of the project (in vivo excision of the transgenic cassette) will begin to obtain transgene-free powdery mildew resistant vines.