Abstract: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐related nuclease 9 (Cas9) system enables precise, simple editing of genes in many animals and plants. However, this system has not been applied to rose (Rosa hybrida) due to the genomic complexity and lack of an efficient transformation technology for this plant. Here, we established a platform for screening single‐guide RNAs (sgRNAs) with high editing efficiency for CRISPR/Cas9‐mediated gene editing in rose using suspension cells. We used the Arabidopsis thaliana U6‐29 promoter, which showed high activity for driving sgRNA expression, to modify the CRISPR/Cas9 system. We used our highly efficient optimized CRISPR/Cas9 system to successfully edit RhEIN2, encoding an indispensable component of the ethylene signaling pathway, resulting in ethylene insensitivity in rose. Our optimized CRISPR/Cas9 system provides a powerful toolbox for functional genomics, molecular breeding, and synthetic biology in rose.

Key words: CRISPR/CAS9, gene editing, RhEIN2, rose