Developing Viruses for Gene Editing to Study Virus-Specific Molecular Interactions in Nicotiana Species

Abstract

The CRISPR/Cas9 gene editing platform is capable of inducing double-stranded breaks in a targeted genomic region using a sequence specific single guide RNA (sgRNA) and programmable Cas9 endonuclease. Plant viral vectors offer flexible and efficient methods to deliver these materials. In this study, Tobacco mosaic virus (TMV) and Tomato bushy stunt virus (TBSV) have been established as biotechnological tools for this purpose. I explored the adaptability of using viral vectors to deliver gene editing materials in multiple diverse Nicotiana species as well as to target components of the plant RNA silencing pathway. First, I evaluated a TMV-based system to target the highly conserved phytoene desaturase 3 gene (PDS3) in multiple Nicotiana species as a transient screening tool. Second, I effectively and efficiently delivered components of the CRISPR/Cas9 system to induce gene editing to affect the expression of a specific component of the RNA silencing pathway, HUA Enhancer 1 (HEN1). This served as proof-of-concept that it is possible to used virus-mediated gene editing to study the influence of a specific RNA silencing modification of viral infection.