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Article|26 Oct 2022|OPEN
DNA-free genome editing in grapevine using CRISPR/Cas9 ribonucleoprotein complexes followed by protoplast regeneration 
Samaneh Najafi1 , Edoardo Bertini1 , Erica D’Incà1 , Marianna Fasoli1 and Sara Zenoni,1 ,
1Department of Biotechnology, University of Verona, 37134 Verona, Italy
*Corresponding author. E-mail:

Horticulture Research 10,
Article number: uhac240 (2023)
Views: 425

Received: 13 Jul 2022
Accepted: 17 Oct 2022
Published online: 26 Oct 2022


CRISPR/Cas9 genome editing technology can overcome many limitations of traditional breeding, offering enormous potential for crop improvement and food production. Although the direct delivery of Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes to grapevine (Vitis vinifera) protoplasts has been shown before, the regeneration of edited protoplasts into whole plants has not been reported. Here, we describe an efficient approach to obtain transgene-free edited grapevine plants by the transfection and subsequent regeneration of protoplasts isolated from embryogenic callus. As proof of concept, a single-copy green fluorescent protein reporter gene (GFP) in the grapevine cultivar Thompson Seedless was targeted and knocked out by the direct delivery of RNPs to protoplasts. CRISPR/Cas9 activity, guided by two independent sgRNAs, was confirmed by the loss of GFP fluorescence. The regeneration of GFP protoplasts into whole plants was monitored throughout development, confirming that the edited grapevine plants were comparable in morphology and growth habit to wild-type controls. We report the first highly efficient protocol for DNA-free genome editing in grapevine by the direct delivery of preassembled Cas9-sgRNA RNP complexes into protoplasts, helping to address the regulatory concerns related to genetically modified plants. This technology could encourage the application of genome editing for the genetic improvement of grapevine and other woody crop plants.