Targeted modification of plant genomes is a powerful strategy for investigating and engineering cellular systems, paving the way for the discovery and development of important, novel agricultural traits. Cas9, an RNA-guided DNA endonuclease from the type II adaptive immune CRISPR system of the prokaryote Streptococcus pyogenes, has gained widespread popularity as a genome-editing tool for use in a wide array of cells and organisms, including model and crop plants. Effective genome engineering requires the delivery of the Cas9 protein and guide RNAs into target cells. However, in planta genome modification faces many hurdles, including the difficulty in efficiently delivering genome engineering reagents to the desired tissues. We recently developed a Tobacco rattle virus (TRV)-mediated genome engineering system for Nicotiana benthamiana. Using this platform, genome engineering reagents can be delivered into all plant parts in a simple, efficient manner, facilitating the recovery of progeny plants with the desired genomic modifications, thus bypassing the need for transformation and tissue culture. This platform expands the utility of the CRISPR/Cas9 system for in planta, targeted genome modification. Here, we provide a detailed protocol explaining the methodologies used to develop and implement TRV-mediated genome engineering in N. benthamiana. The protocol described here can be extended to any other plant species susceptible to systemic infection by TRV. However, this approach is not limited to vectors derived from TRV, as other RNA viruses could be used to develop similar delivery platforms.
Keywords: CRISPR/Cas9; Genome editing; Genome engineering; Nicotiana benthamiana; RNA viruses; TRV; Targeted modification.