Submitted to: Nature Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/4/2024
Publication Date: 1/20/2024
Citation: Yang, Y., Wheatley, M.S., Meakem, V.M., Galarneau, E.R., Gutierrez, B.L., Zhong, G. 2024. Editing VvDXS1 for creation of muscat flavor in Vitis vinifera cv. Scarlet Royal. Nature Communications. https://doi.org/10.1111/pbi.14290.
DOI: https://doi.org/10.1111/pbi.14290

Interpretive Summary: A cutting-edge technology, recently invented and named as 'Prime Editing', has extensively been applied to many plant and animal species for making precise changes of genes. However, successful application of this technology has not been reported in grapevine or other woody species. In this report, we describe the success in using the technology to modify a grape aroma gene, VvDXS1, for producing high-level monoterpenes conferring muscat flavor in the table grape Vitis vinifera cv. Scarlet Royal. Scarlet Royal is a red seedless table grape widely grown in California, USA and it has large, well-colored, attractive berries, with firm, meaty flesh and a sweet and neutral flavor. Adding muscat flavor to this neutral-flavor cultivar could make it even more appealing to consumers. We successfully developed a dozen vines with the target gene largely modified. These vines produced much higher levels of monoterpenes in leaves than the control, similar to what was found in leaf samples between field-grown muscat and non-muscat grapes. This work represents the first time to successfully apply the 'Prime Editing' technology to grapevine and woody species.

Technical Abstract: Muscat flavor represents a group of unique aromatic attributes found in some wine and table grape varieties. Biochemically, grape berries with muscat flavor produce high levels of monoterpenes. Monoterpene biosynthesis is mainly through the DOXP/MEP pathway and VvDXS1 encodes the first enzyme in this plastidial pathway of terpene biosynthesis in grapevine. A single point mutation resulting in the substitution of a lysine with an asparagine at position 284 in the VvDXS1 protein has previously been identified as the major cause for producing muscat flavor in grapes. In this study, the same substitution in the VvDXS1 gene was successfully created through prime editing in the table grape Vitis vinifera cv. Scarlet Royal which has no muscat flavor. The targeted point mutation was detected in most of the transgenic vines with varying editing efficiencies. Interestingly, no unintended mutations were detected in the edited alleles either by PCR Sanger sequencing or amplicon sequencing. More than a dozen edited vines were identified with an editing efficiency of more than 50%, indicating that these vines were likely derived from single cells in which one allele was edited. These vines had much higher levels of monoterpenes in leaves than the control, similar to what was found in leaf samples between field-grown muscat and non-muscat grapes.