Designer circRNA(GFP) reduces GFP-abundance in Arabidopsis protoplasts in a sequence-specific manner, independent of RNAi pathways.

We demonstrate non-immunogenic circRNA as a tool for targeted gene regulation in plants, where it acts in an isoform- and sequence-specific manner, enabling future agronomic applications. Circular RNAs (circRNAs) are single-stranded RNA molecules characterized 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 protoplasts as a model system. We show that PEG-mediated transfection with a 50-nt circRNA(GFP) containing a 30-nt GFP-antisense sequence results in a dose- and sequence-dependent reduction of GFP reporter target protein abundance. 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.