Genome editing technology is a plant breeding innovation that allows rapid targeted modification of plant genome, similar to natural mutations, to improve crop traits such as yield and disease tolerance. Objective of this project is to apply CRISPR/Cas9 gene-editing to produce genome-edited non-transgenic (devoid of foreign genes) grapevine. We proposed to edit the susceptibility genes for powdery mildew (PM) in grapevine named ‘mildew locus O’ genes (MLO) through a combination of plasmid- and ribonucleoprotein (RNP)-delivered CRISPR/Cas9 into the intact embryonic cells to produce DNA-free PM-resistant grapevines. We adopted this two-phase gene-editing approach rather than RNP-delivery alone to overcome the large-scale screening logistics to identify mutated plants in the absence of selection marker genes.
* At the end of the 2nd quarter of Year 2, the conventional Agrobacterium-mediated transformation of microvine to generate mlo mutants has been concluded. Single, double and quadruple mutants of MLOs 3, 4, 13, and 17, involved in grapevine PM-susceptibility, are expected from the first phase gene-editing experiments. The CRISPR-Cas9 plasmid used for MLO gene editing was prepared with an alternative strategy which is explained in the following main text of the report. Gene edited mlo mutants are expected in 3-4 months after which characterization and identification of the specific MLO genes involved in grapevine PM-resistance and assessment of the level of resistance will be performed. As part of the second phase, removal of the transgene T-DNA cassette, integrated into the plant genome, by employing Cas9 RNP delivery has been proposed to generate transgene-free mlo mutant microvines. For that purpose, we plan to use Cell Penetrating Peptides (CPP) fused to Cas9 to facilitate the entry of the RNP into the embryogenic cells. Experiments to optimize the RNP delivery using CPP have continued during the 2nd year. After confirming the efficacy of the candidate CPP for cell penetration and internalization using fluorescent dye, we proceeded to test the delivery of CPP-conjugated Cas9 RNPsinto embryogenic cells. For these trials, GFP-expressing embryogenic callus has been used and the gene-editing rate of the GFP target gene has been assessed. Lower than expected editing rate was observed in these preliminary experiments and experiments to improve the editing rate and to optimize the methods to regenerate gene-edited embryos have been continuing in the year 2. When the MLO gene-edited microvines from first phase are ready, the RNP-delivery methods that are being optimized during the 2nd year will be employed to get the final transgene-free mlo mutant microvines.