Date of this Version
Leuschen, B. Mutational Inactivation of Arabidopsis RNA-dependent RNA polymerases 3 and 4 by gene editing. 2020. Undergraduate Honors Thesis. University of Nebraska-Lincoln.
The replication and movement of RNA viruses has become a major target for understanding antiviral silencing pathways. Critical components of this pathway include dicer like endonucleases, small RNAs (sRNAs), argonaute proteins, and RNA-dependent RNA polymerases (RDRs). While it is known that RDRs are mediations involved in silencing amplification, recent studies of Arabidopsis thaliana have focused on specifying the roles of each of the six known RDRs in the genome. Three of these polymerases, RDR1, RDR2, and RDR6, have been shown to be active during antiviral response while RDR3, RDR4, and RDR5 (also known as 3a-c) triple knockouts have been shown to function in the antiviral defense pathway. RDR3, RDR4, and RDR5 are linked on Arabidopsis chromosome two, which has proven difficult to create mutants lacking multiple RDRs through conventional crossing. In this study, we aimed to use the CRISPR/Cas9 system to induce mutations in RDR3 and RDR4 for further analysis of the specified roles of each RDR. This method involved locating possible regions for targeting, creating the CRISPR/Cas9 vector, transformation using the floral dipping technique, and DNA/Protein analysis but held very low efficiency. As a result, a new vector has been constructed with higher efficiency to target the RDR regions of interest.