Transgene-free Genome Editing in Grapevine

CRISPR/Cas9 genome editing technology has revolutionized plant breeding by offering precise and rapid modifications. Traditional breeding methods are often slow and imprecise, whereas CRISPR/Cas9 allows for targeted genetic improvements. Previously, direct delivery of Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes to grapevine (<i>Vitis vinifera</i>) protoplasts has been demonstrated, but successful regeneration of edited protoplasts into whole plants has not been achieved. Here, we describe an efficient protocol for obtaining transgene/DNA-free edited grapevine plants by transfecting protoplasts isolated from embryogenic callus and subsequently regenerating them. The regenerated edited plants were comparable in morphology and growth habit to wild-type controls. This protocol provides a highly efficient method for DNA-free genome editing in grapevine, addressing regulatory concerns and potentially facilitating the genetic improvement of grapevine and other woody crop plants. Key features • Protoplasts are one of the most commonly used systems for the application of new breeding technologies, including DNA-free genome editing. • Protoplasts are a highly accessible platform by CRISPR-Cas9 ribonucleoparticles through chemical or physical transfection. • CRISPR-Cas9 ribonucleoparticles avoid the use of both <i>Agrobacterium tumefaciens</i> and plasmids; no stable integration of exogenous DNA occurs. • The genetic background of DNA-free edited plants regenerated from protoplasts remains unchanged and identical to the original plant.