Virus-induced systemic and heritable gene editing in pepper (Capsicum annuum L.)

Genome editing using the CRISPR/Cas system enables rapid and efficient plant breeding by directly introducing desired traits into elite lines within a short time frame. However, challenges associated with conventional Agrobacterium tumefaciens-mediated transformation and regeneration have limited gene editing in pepper (Capsicum annuum L.). In this study, we applied and optimized a virus-induced gene editing (VIGE) system to overcome these limitations. We inoculated transgenic pepper seedlings already expressing Cas9 with vectors based on tobacco rattle virus 2 (TRV2) expressing single guide RNAs (sgRNAs) targeting Phytoene desaturase (PDS); shoots regenerated from inoculated cotyledons displayed photobleaching phenotypes. To promote sgRNA mobility and maintain its integrity, we modified the pTRV2-sgRNA vector by incorporating a self-cleaving hammerhead ribozyme (HH) sequence to produce an intact sgRNA fused to part of the mobile RNA of FLOWERING LOCUS T. Additionally, we tested alternative mobile elements, such as tRNA^Ile and tRNA^Met. Furthermore, we cultivated plants at the low temperature of 20°C following TRV inoculation to increase TRV persistence and spread. These optimizations, including vector modifications and cultivation conditions, resulted in a systemic editing efficiency of 36.3%, as evidenced by systemic leaves showing photobleaching phenotypes. We determined that 8.5% of progeny from plants inoculated with the pTRV-HH-CaPDS-sgRNA-FT construct were mutated at the CaPDS locus. In addition, we used our VIGE system to successfully edit FASCICULATE, producing mutants whose inflorescences showed a fasciculate phenotype. Direct inoculation with a TRV-based vector expressing a mobile sgRNA to bypass tissue culture, therefore, offers an effective tool for molecular studies and breeding in pepper.