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 () 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 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.