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Here, we developed a turnip mosaic virus (TuMV)\u2010based system for gRNA delivery in Cas9\u2010expressing\n                    <jats:italic>Nicotiana benthamiana<\/jats:italic>\n                    and tested whether Csy4\u2010mediated gRNA processing could improve editing efficiency. A TuMV construct carrying a gRNA targeting\n                    <jats:italic>PHYTOENE DESATURASE<\/jats:italic>\n                    (\n                    <jats:italic>NbPDS<\/jats:italic>\n                    ) 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\n                    <jats:italic>NbPDS<\/jats:italic>\n                    genes, raising editing in infiltrated leaves to 7.1%\u201313.8% for\n                    <jats:italic>NbPDSa<\/jats:italic>\n                    and 7.6%\u201323.0% for\n                    <jats:italic>NbPDSb<\/jats:italic>\n                    , whereas lower but reproducible editing was detectable in systemic leaves. The TuMV\u2010Csy4 platform also supported editing of a second endogenous target,\n                    <jats:italic>MAGNESIUM CHELATASE SUBUNIT H<\/jats:italic>\n                    (\n                    <jats:italic>NbChlH<\/jats:italic>\n                    ), and enabled multiplex editing of\n                    <jats:italic>NbPDS<\/jats:italic>\n                    and\n                    <jats:italic>NbChlH<\/jats:italic>\n                    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\n                    <jats:sup>Ile<\/jats:sup>\n                    mobility element was fused to the\n                    <jats:italic>NbPDS<\/jats:italic>\n                    gRNA. Although this construct increased somatic editing, no albino progeny were recovered after screening approximately 20,000 seedlings, demonstrating that heritable editing was not achieved under these conditions or did not result in mutations in all copies of the two\n                    <jats:italic>NbPDS<\/jats:italic>\n                    genes. Together, these results establish TuMV as a platform for Cas9\u2010based gRNA delivery and show that Csy4\u2010mediated processing improves editing efficiency, supports multiplex targeting, and demonstrates the feasibility of potyvirus\u2010based genome editing systems in plants.\n                  <\/jats:p>","DOI":"10.1002\/pld3.70191","type":"journal-article","created":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T03:42:03Z","timestamp":1788838923000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Turnip Mosaic Virus\u2010Based gRNA Delivery System for Plant Genome Editing"],"prefix":"10.1002","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-4013-941X","authenticated-orcid":false,"given":"Ekkachai","family":"Khwanbua","sequence":"first","affiliation":[{"name":"Department of Plant Pathology, Entomology and Microbiology Iowa State University  Ames Iowa USA"},{"name":"Department of Biology University of Washington  Seattle Washington USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3765-975X","authenticated-orcid":false,"given":"Ryan R.","family":"Lappe","sequence":"additional","affiliation":[{"name":"Department of Plant Pathology, Entomology and Microbiology Iowa State University  Ames Iowa USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-7590-9662","authenticated-orcid":false,"given":"Austin A.","family":"Bierl","sequence":"additional","affiliation":[{"name":"Department of Plant Pathology, Entomology and Microbiology Iowa State University  Ames Iowa USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3542-3188","authenticated-orcid":false,"given":"Steven A.","family":"Whitham","sequence":"additional","affiliation":[{"name":"Department of Plant Pathology, Entomology and Microbiology Iowa State University  Ames Iowa 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