A tomato bushy stunt virus-based vector for simultaneous editing and sensing to survey the host antiviral RNA...

A tomato bushy stunt virus (TBSV)-derived vector system was applied for the delivery of CRISPR/Cas9 gene editing materials, to facilitate rapid, transient assays of host-virus interactions involved in the RNA silencing pathway. Toward this, single guide RNAs designed to target key components of the virus-induced host RNA silencing pathway (AGO2, DCL2, HEN1) were inserted into TBSV-based GFP-expressing viral vectors TBSV-GFP (TG) and its P19 defective mutant TGΔP19. This produced rapid, efficient, and specific gene editing in planta. Targeting AGO2, DCL2, or HEN1 partially rescued the lack of GFP accumulation otherwise associated with TGΔP19. Since the rescue phenotypes are normally only observed in the presence of the P19 silencing suppressor, the results support that the DCL2, HEN1, and AGO2 proteins are involved in anti-TBSV RNA silencing. Additionally, we show that knockdown of the RNA silencing machinery increases cargo expression from a nonviral binary Cas9 vector. The TBSV-based gene editing technology described in this study can be adapted for transient heterologous expression, rapid gene function screens, and molecular interaction studies in many plant species considering the wide host range of TBSV. In summary, we demonstrate that a plant virus can be used to establish gene editing while simultaneously serving as an accumulation sensor for successful targeting of its homologous antiviral silencing machinery components.