Bioinformatic approaches to identify target specific guide-RNAs for CRISPR-Cas genome editing in Vitis vinifera L.

Grapevine (Vitis vinifera L. ssp. Sativa) is an economically important cultivated species for the production of wines, raisins, and juices. It is characterized by thousands of different varieties, the majority of which are highly susceptible to several fungal pathogens. Among them, Erysiphe necator, the causal agent of powdery mildew, can causes severe crop and quality losses if no control measures are carried out. In most conventional-managed vineyards, powdery mildew and other diseases are mainly treated by chemical fungicides, making viticulture one of the agricultural activities with the greatest chemical input. To reduce the environmental impact of grapevine cultivation many efforts are ongoing through biological or biotechnological approaches. In this context, the grapevine genetic improvement is gaining great benefit from the New Plant Breeding Technologies, which resemble traditional breeding but with shorter time and less impact on the varieties ’s genomes. In order to reduce the fungal susceptibility, we plan to produce two different knock-out mutants for two members of the MLO gene family (VvMLO6 and VvMLO7), well known for their involvement in powdery mildew susceptibility. Moreover, to further evaluate their functional effect(s), three different editing events will be performed: each gene will be switched-off independently and we will produce a third mutant carrying both the mutations. The last editing event is designed in order to evaluate the possibility of any synergistic outcome. The above mentioned purposes will be achieved by using CRISPR-Cas technology on Glera, Pinot Nero and Sangiovese varieties. Despite the ability of this technique to introduce Insertion and/or Deletion (InDel) in very specific target sequences, off target activities can occur. To minimize the latter possibility, we took advantages from different bioinformatics tools in order to: i) design guides, ii) predict on-target editing efficiency and iii) minimize unspecific editing events. First, SNPs calling analysis of the above mentioned varieties was performed using the near-homozygous Pinot PN40024 genome as reference. Then, guide RNAs were designed using CRISPRdirect tool. Guides specificity was analysed using CRISPR_P tool and, as final criteria for guide selection, absence of unspecific target on genomic DNA was evaluated with CRISPOR and RGEN. The two selected gRNAs will be implemented into expression plasmids, together with the Cas9 and used to perform Agrobacterium-mediated transformation of embryogenic calli.