Gene Editing of Arabidopsis thaliana RNA Dependent RNA Polymerases Using CRISPR/Cas9

Abstract

Viruses are a prolific force able to infect any form of life on the planet, including plants. Around the world, plant viruses cause considerable yield loss in many agriculture crops every year. Antiviral immunity in plants is mediated by RNA silencing and RNA decay mechanisms, which proceed as follows: once a virus inserts its genetic material into a cell, dicer‐like ribonucleases (DCLs) detect the viral RNA and fragment it into small interfering RNAs (siRNAs). These RNA fragments are then bound by Argonaute proteins and form an RNA‐induced silencing complex (RISC). The RISC binds to complementary sequences of the viral RNA and remains bound, thus deactivating or degrading the fragment by enzymatically cleaving the RNA. siRNA antiviral RNA silencing is utilized for acute or cellular antiviral defense, but it also plays a role in systemic antiviral defense. In plants the systemic silencing mechanism is mediated by enzymes called RNA‐dependent RNA polymerases (RDR) that synthesize double stranded RNA (dsRNA) from single strand RNA. These dsRNAs are then processed through the same siRNA‐silencing pathway, producing new siRNAs that can silence viral genes. The supplementary siRNAs can then be transported between cells through the plants’ vasculature and the plasmodesmata. This leads to systemic treatment and defense in the plant. Though the core genetic components of antiviral RNA silencing have been determined, additional components remain to be identified and characterized. For example, the A. thaliana genome encodes six RDR genes. It is known that RDR1 and RDR6 have major roles in antiviral RNA silencing and that RDR2 mediates biogenesis of cellular siRNAs. However, the role of the RDR3, RDR4, and RDR5 genes is not known. Though single mutants of RDR3, RDR4, and RDR5, have been constructed, double or triple mutants cannot be obtained by tDNA mutation because all three genes are linked together on the second chromosome. Our hypothesis was that RDR3 and RDR4 can be inactivated by site‐specific genome editing using the CRISPR/Cas9 technique to develop mutants lacking these genes. Further experiments show the expression of the Cas9 protein in the second generation, confirming transformation of A. thaliana proteins and RNA. We expect that creating this sextuple mutant, in which all six RDR genes are deactivated, allows the continued exploration of the role of RDRs in antiviral silencing. The RDR3 and RDR4 inactive mutants will also facilitate the necessary positive and negative control mutants needed for future analytical studies.Support or Funding InformationUndergraduate Creative Activities and Research Experience Project, funded by the PepsiQuasi Endowment and Union Bank & Trust. National Institute of Health (R01GM120108). Institute of Agriculture and Natural Resources, Agricultural Research Division, Undergraduate Student Research ProgramCRISPR single guide RNAs.A) Target Sites and sgRNAs in RDR3a an dRDR3b found using CCTOP (Stemmer et al. 2015). Small guide RNAs are the target sites minus the PAM colored in red. B) Single guide RNAs targeting RDR3a and RDR3b.Figure 1