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The woody nature of grapevine ( Vitis vinifera  L.) has hindered the development of efficient gene editing strategies to improve this species. The lack of highly efficient gene transfer techniques, which, furthermore, are applied in multicellular explants such as somatic embryos, are additional technical handicaps to gene editing in the vine. The inclusion of geminivirus-based replicons in regular T-DNA vectors can enhance the expression of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) elements, thus enabling the use of these multicellular explants as starting materials. In this study, we used  Bean yellow dwarf virus  (BeYDV)-derived replicon vectors to express the key components of CRISPR/Cas9 system  in vivo  and evaluate their editing capability in individuals derived from  Agrobacterium -mediated gene transfer experiments of ‘Thompson Seedless’ somatic embryos. Preliminary assays using a BeYDV-derived vector for  green fluorescent protein  reporter gene expression demonstrated marker visualization in embryos for up to 33 days post-infiltration. A universal BeYDV-based vector (pGMV-U) was assembled to produce all CRISPR/Cas9 components with up to four independent guide RNA (gRNA) expression cassettes. With a focus on fungal tolerance, we used gRNA pairs to address considerably large deletions of putative grape susceptibility genes, including  AUXIN INDUCED IN ROOT CULTURE 12  ( VviAIR12 ),  SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER 4  ( VviSWEET4 ),  LESION INITIATION 2  ( VviLIN2 ), and  DIMERIZATION PARTNER-E2F-LIKE 1  ( VviDEL1 ). The editing functionality of gRNA pairs in pGMV-U was evaluated by grapevine leaf agroinfiltration assays, thus enabling longer-term embryo transformations. These experiments allowed for the establishment of greenhouse individuals exhibiting a double-cut edited status for all targeted genes under different allele-editing conditions. After approximately 18 months, the edited grapevine plants were preliminary evaluated regarding its resistance to  Erysiphe necator  and  Botrytis cinerea . Assays have shown that a transgene-free  VviDEL1  double-cut edited line exhibits over 90% reduction in symptoms triggered by powdery mildew infection. These results point to the use of geminivirus-based replicons for gene editing in grapevine and other relevant fruit species.

frontiers in plant science

CRISPR/Cas9 Targeted Editing of Genes Associated With Fungal Susceptibility in Vitis vinifera L. cv. Thompson Seedless Using Geminivirus-Derived Replicons