Heritable Tissue-Culture-Free Gene Editing in <i>Nicotiana benthamiana</i> through Viral Delivery of SpCas9 and sgRNA

Conventional plant gene editing requires laborious tissue-culture-mediated transformation, which restricts the range of applicable plant species. In this study, we developed a heritable and tissue-culture-free gene editing method in Nicotiana benthamiana using tobacco ringspot virus (TRSV) as a vector for in planta delivery of Cas9 and single-guide RNA (sgRNA) to shoot apical meristems. Agrobacterium-mediated inoculation of the TRSV vector induced systemic and heritable gene editing in Nicotiana benthamiana PHYTOENE DESATURASE. Transient downregulation of RNA silencing enhanced gene editing efficiency, resulting in an order of magnitude increase (0.8-13.2%) in the frequency of transgenerational gene editing. While the TRSV system had a preference for certain sgRNA sequences, co-inoculation of a TRSV vector carrying only Cas9 and a tobacco rattle virus vector carrying sgRNA successfully introduced systemic mutations with all five tested sgRNAs. Extensively gene-edited lateral shoots occasionally grew from plants inoculated with the virus vectors, the transgenerational gene editing frequency of which ranged up to 100%. This virus-mediated heritable gene editing method makes plant gene editing easy, requiring only the inoculation of non-transgenic plants with a virus vector(s) to obtain gene-edited individuals.