Schematic diagram illustrating the experimental workflow and methods. By monitoring GFP accumulation as a function of viral replication and spread, observational results could be visualized within 10 days after introduction of the TBSV-based gene editing vectors via agroinfiltration. The same leaf tissue samples were collected for downstream analysis, including amplification of the targeted region of interest to assess the levels of successful gene editing. In our study, gRNAs were designed to target a region that includes a single cut site for a restriction enzyme (RE). Successful targeted editing results in disruption of the cut site, and when resolved on an agarose gel, an additional RE-resistant band can be easily quantified or cloned for sequencing. Overall, a functional genetic screen can be performed yielding rapid results to study virus–host interactions.