Potato virus X Vector-Mediated DNA-Free genome editing in plants.

Ariga, Hirotaka; Toki, Seiichi; Ishibashi, Kazuhiro

Genome editing technology is important for plant science and crop breeding. Genome-edited plants prepared using general CRISPR-Cas9 methods usually contain foreign DNA, which is problematic for production of genome-edited transgene-free plants for vegetative propagation or highly heterozygous hybrid cultivars. Here, we describe a method for highly efficient targeted mutagenesis in Nicotiana benthamiana through expression of Cas9 and single guide (sg)RNA using a potato virus X (PVX) vector. Following Agrobacterium-mediated introduction of virus vector cDNA, >60% of shoots regenerated without antibiotic selection carried targeted mutations, while ≤18% of shoots contained T-DNA. The PVX vector was also used to express a base-editor consisting of modified Cas9 fused with cytidine deaminase to introduce targeted nucleotide substitution in regenerated shoots. We also report exogenous DNA-free genome editing by mechanical inoculation of virions comprising the PVX vector expressing Cas9. This simple and efficient virus-vector-mediated delivery of CRISPR-Cas9 could facilitate transgene-free gene editing in plants.