Target-specific sequence recognition by Cas9 is led by guide RNAs (gRNAs) ( van der Oost et al., 2014 Jiang and Doudna, 2017), a short synthetic RNA fragment composed of a scaffold sequence necessary for Cas-binding, and a user-defined spacer of ∼20 nucleotides based on the genomic target to be modified ( Sternberg et al., 2014). It allows the direct generation of target-specific sequence modifications in the genome, opening DNA repair pathways via donor-dependent homology-directed recombination or error-prone non-homologous end joining, activated after the Cas9-induced DNA double-stranded break (DSB). The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is currently one of the most powerful GenEd techniques available. These insights enable precision breeding using GenEd tools ( Capriotti et al., 2020 Paul et al., 2021). Complete sequencing of the grapevine genome ( Jaillon et al., 2007) has identified novel genes, analyzed structural gene variants, discovered new single nucleotide polymorphisms, and clarified regulatory regions. The latter involves gene editing (GenEd) techniques actively under development ( Zhu et al., 2020). In this regard, strategies for genetically improving grapevine rely on both conventional and precision breeding. Genomic studies and technological advances in plant genetic engineering provide a path for developing new varieties compatible with today’s market and production challenges ( Gomès et al., 2021). Therefore, solutions with a trend toward a sustainable and agrochemical-free agriculture and production chain are needed. Containing the growth of pathogens and the progress of plant disease depend on chemicals, with application regimes that even include preventive treatments. On a productive scale, the management of fungal diseases represents a major challenge in viticulture. Grape ( Vitis vinifera L.) is a perennial fruit crop with regionally high economic activity due to its multiproduct nature ( Moriondo et al., 2011). These results point to the use of geminivirus-based replicons for gene editing in grapevine and other relevant fruit species. Assays have shown that a transgene-free VviDEL1 double-cut edited line exhibits over 90% reduction in symptoms triggered by powdery mildew infection. After approximately 18 months, the edited grapevine plants were preliminary evaluated regarding its resistance to Erysiphe necator and Botrytis cinerea. These experiments allowed for the establishment of greenhouse individuals exhibiting a double-cut edited status for all targeted genes under different allele-editing conditions. The editing functionality of gRNA pairs in pGMV-U was evaluated by grapevine leaf agroinfiltration assays, thus enabling longer-term embryo transformations. 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). 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. 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. 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. 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. 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 woody nature of grapevine ( Vitis vinifera L.) has hindered the development of efficient gene editing strategies to improve this species. 2Phytopathology Laboratory, La Platina Research Station, National Institute of Agriculture Research, Santiago, Chile.1Biotechnology Laboratory, La Platina Research Station, National Institute of Agriculture Research, Santiago, Chile.Felipe Olivares 1†, Rodrigo Loyola 1†, Blanca Olmedo 1, María de los Ángeles Miccono 1, Carlos Aguirre 1, Ricardo Vergara 1, Danae Riquelme 2, Gabriela Madrid 1, Philippe Plantat 1, Roxana Mora 1, Daniel Espinoza 1 and Humberto Prieto 1*
0 kommentar(er)
