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A TMV-based viral vector for delivering gene editing tools

Kelvin Chiong: Texas A&M University

<div>Plant viruses can be engineered and utilized as overexpression tools for transient gene modification studies and large-scale production of high value proteins. An efficient example is the agroinfiltratable TMV-based overexpression vector (TRBO), a coat protein gene deletion mutant of TMV. The CRISPR/Cas9 gene editing system involves two main components, a single guide RNA (sgRNA) and a Cas9 endonuclease which, as a complex, creates double-stranded breaks in complementary DNA sequences. 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 <em>Nicotiana benthamiana</em>. Here, we utilized a TRBO vector to transiently express high levels of Cas9, and another for simultaneous delivery of a sgRNA which targets the <em>mgfp5</em> gene (gGFP) in the <em>N. benthamiana </em>GFP-expressing line 16c. The engineered Cas9-expressing TRBO vector (TRBO-Cas9) was able to express a biologically functional protein, despite the large insert size (~4.2 kb). Co-delivery of TRBO-Cas9 and TRBO-G-3’gGFP along with P19, an RNA silencing suppressor protein of <em>Tomato bushy stunt virus </em>(TBSV), resulted in high levels of Cas9 protein and efficient gene editing <em>in planta</em>. Additionally, we combined both Cas9 and the gGFP in a single delivery vector that resulted in gene editing events. Our system demonstrates the potential of virus vectors to rapidly create non-transgenic knockout plants for plant functional genomics and proteomics.</div>