Application of a Transient Viral Vector for Efficient Genome Editing in Plants

CRISPR-based gene editing systems have been adapted as rapid transient screening devices in many model systems, but a completely transient genetic screening tool has not been developed for plants. An Agrobacterium launched Tobacco mosaic virus coat protein (CP) deletion mutant viral vector, TRBO, was evaluated for its ability to deliver effective single guide RNAs (sgRNAs) in planta. A Cas9 expression vector was developed to be delivered through agroinfiltration along with TRBO-sgRNA. The GFP expressing 16c Nicotiana benthamiana plants were used as a host to measure indel percentages of a sgRNA target that was designed within GFP and was flanked by either two, one or no ribozymes. Furthermore, a replicase mutant was tested to ensure mutagenesis was based on TRBO-dependent replication and transcription. Observed indel rates were highest for the replication-competent sgRNA construct carrying no ribozymes, producing transient indel rates of nearly 70% with the majority of mutations occurring 3 days post-inoculation (dpi), and peaking between 6 and 7 dpi. Reduced levels of GFP protein expression and decreased green fluorescence in imaging at 12 dpi coincide with high indel percentages. Additionally, endogenous N. benthamiana paralogs Argonaute 1-H and Argonaute 1-L were targeted using a singular sgRNA, which effectively created indels within both genes. The TRBO-sgRNA system was then tested for its ability to deliver multiple sgRNAs. Surprisingly similar indel efficiencies were reported for the delivery of two sgRNAs expressed through the same viral CP subgenomic promoter, without a separating linker sequence, as a single sgRNA. This demonstrates, to the best of our knowledge, the highest and quickest plant somatic cell mutagenesis reported. Delivery of sgRNAs using the TRBO system has the ability to reduce the time and cost of producing non-transgenic gene edited plants while also offering a transiently delivered gene knockout screening method for functional genetic studies as a more precise alternative screening tool compared to viral induced gene silencing screens. Possibly the most exciting and promising capabilities for Cas9/TRBO-sgRNA, as demonstrated by the ability to deliver multiple guides, is its potential use for plant metabolic engineering processes.