Probing the Role of RDR and DCL proteins in Virus Induced Gene Editing in Tomato Plants

Genome editing is an important tool for biological research and crop improvement. Tissue culture is the traditional method for genome editing but requires large time investments, extensive experimental procedures, and can have unintended effects on the genome. Virus induced gene editing (VIGE) uses viral vectors to create heritable edits by delivering single-guide RNA to the meristem via direct delivery. Tobacco Rattle Virus (TRV) has successfully created heritable edits in dicotyledonous plants, but many species exclude viruses from the meristem as part of protection. RNA-dependent RNA polymerase (RDR) and Dicer-like (DCL) genes encode key proteins involved in viral protection in plants. This project seeks to evaluate how they affect VIGE efficiency by creating loss of function mutant tomato plant lines using CRISPR Cas9 and tissue and conducting TRV mediated VIGE on these plants using phytoene desaturase (PDS) as a reporter gene. The loss of function mutants were crossed with high Cas9 expressing, high editing efficiency wildtype plants and then VIGE was conducted on the crossed plants. PDS photobleaching symptoms began showing 11 days post editing after which a western blot was performed to evaluate the Cas9 expression in the crossed plants. Positive sequencing results indicate successful creation of loss of function mutants. Western blot results indicate high Cas9 expression in the crossed plants. This work can lay the foundation for future work on developing protocols for more efficient VIGE work and act as progress towards the overall goal of overcoming the barriers of tissue culture-based genome editing.

I have had the pleasure of working in the Van Eck as part of the BTI/ CROPPS Research Experience for Undergraduates (REU). My project involved evaluating the role of RNA-dependent RNA polymerase (RDR) and Dicer-like (DCL) genes in the efficiency of Virus Induced Gene Editing (VIGE). My mentor, Dr. Fan Xia, has helped me gain an in-depth understanding of CRISPR-Cas9 genome editing methods, the genotyping process, experimental design and problem-solving from a scientific mindset. The Van Eck lab is a collaborative and uplifting environment, and it has been an honor work with these individuals these past 10 weeks.