High levels of ADAR overexpression induce abundant and stochastic off-target RNA editing in rice protoplasts

Adenosine deaminase acting on RNA (ADAR) mediates adenosine-to-inosine (A-to-I) conversions, with its deaminase domain (ADARdd) acting as a core component of targeted RNA editing. However, the off-target effects of ADARdd remain poorly characterized in plants. Here, we demonstrate that high-level overexpression of ADARdd induces widespread off-target editing in rice protoplasts, an effect not observed in stable transgenic lines. From six biological replicates of ADARdd-overexpressing protoplasts, we identified 2794 off-target editing sites (OFTEs). These OFTEs exhibited stochastic distribution across mRNAs and low editing efficiency (20–40 %), contrasting sharply with the high-confidence editing sites in OsDRB1-ADARdd stable lines. We further identify risky endogenous promoters and establish an empirical expression threshold for ADARdd to minimize off-target editing. Our findings highlight that controlled ADARdd expression is essential for reliable RNA editing in plants, providing valuable guidance for enhancing the precision of plant RNA editing experiments.