Transgene-Free Genome Editing in Nicotiana benthamiana with CRISPR/Cas9 Delivered by a Rhabdovirus Vector

The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas systems have become the most widely adopted genome editing platform owing to their unprecedented simplicity, efficiency, and versatility. Typically, the genome editing enzyme is expressed in plant cells from an integrated transgene delivered by either Agrobacterium-mediated or biolistic transformation. Recently, plant virus vectors have emerged as promising tools for the in planta delivery of CRISPR/Cas reagent. Here, we provide a protocol for CRISPR/Cas9-mediated genome editing in the model tobacco plant Nicotiana benthamiana using a recombinant negative-stranded RNA rhabdovirus vector. The method is based on infection of N. benthamiana with a Sonchus yellow net virus (SYNV)-based vector that carries the Cas9 and guide RNA expression cassettes to target specific genome loci for mutagenesis. With this method, mutant plants free of foreign DNA can be obtained within 4–5 months.