Developing an efficient DNA-free, non-transgenic Genome editing methodology in grapevine

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 bacterial selection markers.

Since the start of project in July 2019, the CRISPR plasmid to be used in the first phase of MLO gene editing is being built. To deliver CRISPR-Cas9 RNP in the second phase, we planned to use cell penetrating peptides (CPP) fused to Cas9 to facilitate Cas9 entry into the embryogenic cells. Experiments to optimize the methods for RNP delivery using CPP and to identify the optimum stage of embryogenic callus, efficiency of the CPP to penetrate the cells, and the treatment methods have been undertaken. The CPP has been synthesized and the efficiency of cell penetration and internalization were assessed using fluorescent dye attached to the CPP. Based on the detection of the dye in majority of the cells in the treated callus we confirmed that Cas9 protein can be delivered into embryogenic cells using this CPP. Methods for the bacterial expression and purification Cas9 protein and its conjugation with CPP have been optimized. Cleavage activity of the CPP-conjugated Cas9 protein is assessed through in vitro cleavage experiments using the guide RNA targeting GFP gene. In the optimization process, GFP-expressing embryogenic callus has been used to treat with the RNP in order to assess the efficiency and accuracy of RNP-mediated gene-editing. Data from these experiments will be used to streamline the process of MLO gene editing in microvine.