Khwanbua, E.; Lappe, R. R.; Bierl, A. A.; Whitham, S.

Plant virus-based gRNA delivery systems offer a rapid alternative to stable transformation for CRISPR-mediated genome editing, but potyvirus-based platforms in Cas9-expressing plants are still underexplored. Here, we developed a turnip mosaic virus (TuMV)-based system for gRNA delivery in Cas9-expressing Nicotiana benthamiana and tested whether Csy4-mediated gRNA processing could improve editing efficiency. A TuMV construct carrying a gRNA targeting PHYTOENE DESATURASE (NbPDS) induced detectable editing in both infiltrated and systemic tissues, although editing frequencies were low. Incorporation of the bacterial endoribonuclease Csy4 increased editing efficiencies in the two NbPDS genes, raising editing in infiltrated leaves to 7.1-13.8% for NbPDSa and 7.6-23.0% for NbPDSb, while lower but reproducible editing was detectable in systemic leaves. The TuMV-Csy4 platform also supported editing of a second endogenous target, MAGNESIUM CHELATASE SUBUNIT H (NbChlH), and enabled multiplex editing of NbPDS and NbChlH regardless of guide order. Editing efficiencies were consistently higher in infiltrated leaves than in systemic leaves, and no visible photobleaching or chlorosis was observed in systemic tissues despite confirmed molecular editing. To assess the potential for heritable editing, a tRNA<S>Ile</S> mobility element was fused to the NbPDS gRNA. Although this construct increased somatic editing, no albino progeny were recovered after screening approximately 20,000 seedlings, indicating that heritable editing was not achieved under these conditions. Together, these results establish TuMV as a platform for Cas9-based gRNA delivery and show that Csy4-mediated processing improves editing efficiency, supports multiplex targeting, and demonstrates the feasibility of potyvirus-based genome editing systems in plants.