Diagram of TG and TGΔP19 TBSV-based expression vectors and the Cas9 binary expression vector. A) The native TBSV coat protein (CP) was replaced with GFP, directly followed by a gRNA designed to target a gene of interest. The transcription initiation sites of two subgenomic RNAs are indicated by right-angled arrows. Expression was driven by the cauliflower mosaic virus (CaMV) 35S promotor (gray arrow). TG retains a functional P19 suppressor protein (solid blue). B) TGΔP19 contains a P19 coding sequence with a premature stop codon, preventing translation of a functional VSR protein product (broken blue). MP, movement protein. C) The pHcoCas9 binary expression vector utilizes human codon-optimized Cas9 (HcoCas9) and is expressed under a CaMV 35S double promotor. Tobacco etch virus (TEV) 5′ and 3′ UTR; 3X FLAG (black); nuclear localization signal (NLS, light purple) is present on either side of the HcoCas9 coding region. Term, terminator of nos (nopaline synthase) gene.