Elissa Rhuby

TRV-AmCyan: A tool for robust visualization of viral movement and Cas9 editing in Nicotiana benthamiana

Tobacco rattle virus (TRV) vectors are used in the Voytas Lab for delivery of gene editing reagents to plants in the absence of tissue culture. Upon infection of transgenic Nicotiana benthamiana plants expressing Cas9, engineered TRV vectors move systemically through the plant vasculature, expressing sgRNAs that target endogenous sequences, such as phytoene desaturase (PDS). Previous research has shown that addition of the mobile RNA sequence Flowering Locus T (FT) is necessary for obtaining heritable edits at a high frequency in N. benthamiana (Ellison et al., 2020); however, this has yet to be tested in other plant species. Translation of these findings has been difficult due to the lack of robust tools to measure infection frequency and track viral movement, leaving researchers with using labor intensive methods of RNA extraction and RT-PCR on random leaf samples, which can result in false negatives. Recent unpublished work in the Voytas Lab has shown that including a fluorescent reporter, AmCyan, on TRV is a reliable method to visualize viral movement in real-time. This project has focused on correlating AmCyan signal and Cas9 editing. If AmCyan signal can be correlated with editing in somatic cells, it could be used to quickly determine tissues that contain potential edits for screening, reducing laborious random sampling. Additionally, we investigated the importance of including FT to engineered TRV vectors and observed whether the increased cargo from including AmCyan would impact somatic editing frequency. Results obtained from this study will be an important foundation for efficient translation of TRV-mediated editing in different plant species.