A simplified RUBY reporter-enabled hairy-root system for rapid evaluation of genome editing and gene function in...

The simplified RUBY represents an efficient reporter for monitoring plant transformation and, when combined with hairy-root system in grapevine, can facilitate the application of novel CRISPR technologies and gene functional study. Monitoring successful transformation events is essential for plant transformation and genome editing. The development and application of the RUBY reporter enable effective selection of transformation events based solely on distinct red pigmentation. Here, we report that a simplified version of RUBY (siRUBY), lacking the glucosyltransferase gene, also functions effectively as a visual selection marker for plant transformation. Furthermore, the siRUBY-assisted hairy-root system was established as a rapid and efficient platform for evaluating activity of adenine base editor (ABE) in grapevine. Targeted A-to-G base editing was achieved using ABE8e, with an average efficiency of approximately 14%. Moreover, this platform is also suitable for functional investigation of genes of interest. Knockout of the MYB4a gene promoted lignin deposition, likely through upregulating key lignin biosynthesis genes while repressing transcription of downstream flavonoid biosynthesis genes. Collectively, these results demonstrate that siRUBY coupled with grapevine hairy roots provides a robust platform for rapid and efficient genome editing in grapevine.