Designer antisense circRNAGFPreduces GFP abundance in Arabidopsis protoplasts in a sequence-specific manner, independent of RNAi pathways

Circular RNAs (circRNAs) are single-stranded RNA molecules characterised by their covalently closed structure and are emerging as key regulators of cellular processes in mammals, including gene expression, protein function and immune responses. Recent evidence suggests that circRNAs also play significant roles in plants, influencing development, nutrition, biotic stress resistance, and abiotic stress tolerance. However, the potential of circRNAs to modulate target protein abundance in plants remains largely unexplored. In this study, we investigated the potential of designer circRNAs to modulate target protein abundance in plants using Arabidopsis as a model system. We demonstrate that treatment with a 50 nt circRNA GFP , containing a 30 nt GFP antisense sequence stretch, results in reduced GFP reporter target protein abundance in a dose- and sequence-dependent manner. Notably, a single-stranded open isoform of circRNA GFP had little effect on protein abundance, indicating the importance of the closed circular structure. Additionally, circRNA GFP also reduced GFP abundance in Arabidopsis mutants defective in RNA interference (RNAi), suggesting that circRNA activity is independent of the RNAi pathway. We also show that circRNA, unlike dsRNA, does not induce pattern-triggered immunity (PTI) in plants. Findings of this proof-of-principle study together are crucial first steps in understanding the potential of circRNAs as versatile tools for modulating gene expression and offer exciting prospects for their application in agronomy, particularly for enhancing crop traits through metabolic pathway manipulation. Highlights We demonstrate the potential of non-immunogenic circRNA as a tool for targeted gene regulation in plants, where circRNA acts in an isoform- and sequence-specific manner, paving the way for future agronomic applications.