Multiplexed base-editing in Arabidopsis thaliana using RNA viral vectors
Site-directed genome editing technologies in plants have promising potential for plant gene function research and crop improvement .Targeted base editing in plants without a foreign DNA donor or double-stranded DNA cleavage would accelerate genome modification and breeding in plants. Agrobacterium-mediated techniques have been used to generate base editing through in planta transformation or embryogenic callus-based transformation through tissue culture. The generated transgenic lines expressing base-editor and sgRNA have mainly been mosaic in the first generation. To date, base-editing technologies have been difficult to achieve in the model plant Arabidopsis Thaliana. Moreover, conventional base-editing relies heavily on tissue culture and transformation. Tissue culturing is time-consuming, only eligible for certain species, and may result in unpredictable changes in the genome and epigenome. Heritable targeted mutagenesis has been achieved in plants using a RNA virus guide delivery system, but base-editing has not been reported yet. In this study, we showed that duplexed and multiplexed targeted base-editing could be rapidly generated in plants at high frequencies using Tobacco Rattle Virus delivered guide RNAs into the germline of transgenic Arabidopsis thaliana which express a cytidine deaminase base-editor. Enhanced sgRNA (esg RNA) targeting PDS3, CLA1, and CESA3 were designed with a modified scaffold that increases their stability and promotes interactions with Cas9. Mutagenesis using these esgRNAs would convert a CAG codon of PDS3 and a CGA of CLA1 to premature TAG and TGA stop codons, respectively (Figure Disruption of PDS3 function results in a photobleached phenotype due to impaired carotenoid. CLA1 is required for chloroplast development; homozygous knockout mutant plants have an albino phenotype. We were able to detect C to T mutation in all three loci in the systemic leaves in infected plants. The results suggest virus-mediated multiplexed base-editing can be performed in A. thaliana, allowing high-throughput functional analysis of genes through precise mutagenesis.