Tobacco Mosaic Virus as a Gene Editing Platform

Plant viruses can be engineered and utilized as transient tools for foreign gene overexpression studies and large-scale production of high value proteins. Tobacco mosaic virus (TMV) has been widely used for these purposes. One such example is the efficient agroinfiltratable TMV-based overexpression (TRBO) vector, a coat protein deletion mutant of TMV. The CRISPR/Cas9 gene editing system involves two main components, a single guide RNA (sgRNA) and a Cas9 endonuclease that, as a complex, creates double-stranded breaks to a complementary DNA sequence. Previously, our group used the TRBO vector to express both a green fluorescent protein (GFP) and a biologically active sgRNA (TRBO-G-3′gGFP) in Nicotiana benthamiana. Here, TRBO-based delivery tools were further developed as gene-editing platforms and as a transient approach to study plant gene expression and function. First, two separate TRBO vectors were utilized to transiently express high levels of Cas9, and for delivery of a sgRNA which targets the mgfp5 gene (gGFP) in the N. benthamiana GFP-expressing line 16c. The engineered Cas9-expressing TRBO vector (TRBO-HcoCas9) was able to express a biologically functional protein, despite the large insert size (~4.2 kb). The addition of P19, an RNA silencing suppressor protein of Tomato bushy stunt virus, to the co-delivery of TRBO-HcoCas9 and TRBO-G-3'gGFP resulted in higher levels of Cas9 protein and increased indel (insertions and deletions) percentages in planta. Additionally, Cas9 protein levels were improved by the RNA silencing suppressor activity of the TMV P126 replicase complex. This study also revealed an age-related resistance phenomenon in plants influencing the performance of P19 in the RNA silencing machinery, whereby P19 works more efficiently in older plants. Lastly, I combined both Cas9 and the gGFP in a single delivery vector that resulted in gene editing events. My system demonstrates the potential of virus vectors to rapidly create non-transgenic knockout plants for functional plant genomics and proteomics