Application of the OsRPS5 promoter for CRISPR/Cas9-mediated genome editing in rice

Abstract Constitutive promoters such as CaMV 35S and ubiquitin are commonly utilized in crop genome editing. However, their ectopic overexpression patterns may lead to off-target effects. To address this limitation, tissue-specific or developmentally regulated promoters offer promising alternatives. The RIBOSOMAL PROTEIN S5A (RPS5A) promoter has demonstrated superior editing efficiency compared to the 35S and ubiquitin promoters in dicotyledonous species, yet its potential application in monocots remains unexplored. In this study, we identified and functionally characterized the Oryza sativa RPS5 (OsRPS5) promoters and evaluated their utility in CRISPR/Cas9-mediated genome editing. The activities of the OsRPS5 promoters were assessed through GFP reporter expression in rice protoplasts, and their genome editing capability was validated by targeting two endogenous genes, OsPDS and OsBADH2. Genome editing driven by the OsRPS5 promoter targeting OsPDS resulted in albino phenotypes in approximately 50% of the transgenic lines, with insertion/deletion mutations confirmed through sequencing analysis. Notably, the genome editing efficiency driven by the OsRPS5 promoter was comparable to that of the widely used constitutive promoters in monocots. These findings suggest the OsRPS5 promoter as a potentially more precise and efficient alternative to constitutive promoters for genome editing applications in monocot crops.