Circular RNA (circRNA) has gained significant attention in RNA therapeutics due to its enhanced stability and protein-coding potential. In this study, we present two in vitro RNA circularization techniques, namely Permuted Intron-Exon through Trans-splicing (PIET) and Complete self-splicing Intron for RNA Circularization (CIRC). PIET leverages the second step of group I intron splicing, offering an alternative circularization strategy. CIRC utilizes the natural, intact forms of group I and group II introns, eliminating the need for intron engineering. Compared to Permuted Intron-Exon (PIE), CIRC exhibits enhanced RNA circularization efficiency and speed under mild conditions. Using CIRC, we successfully circula... More
Circular RNA (circRNA) has gained significant attention in RNA therapeutics due to its enhanced stability and protein-coding potential. In this study, we present two in vitro RNA circularization techniques, namely Permuted Intron-Exon through Trans-splicing (PIET) and Complete self-splicing Intron for RNA Circularization (CIRC). PIET leverages the second step of group I intron splicing, offering an alternative circularization strategy. CIRC utilizes the natural, intact forms of group I and group II introns, eliminating the need for intron engineering. Compared to Permuted Intron-Exon (PIE), CIRC exhibits enhanced RNA circularization efficiency and speed under mild conditions. Using CIRC, we successfully circularize large RNA constructs encoding full-length dystrophin, a protein whose deficiency is linked to Duchenne muscular dystrophy (DMD), thus overcoming size limitations typically associated with circRNA platforms. Notably, CIRC enables the production of scarless circRNA and circRNA with minimal immunogenicity. Additionally, CIRC supports streamlined circRNA purification using ribonuclease R (RNase R) or oligo(dT)-based methods. These advancements significantly expand the potential of the circRNA platform for both research and therapeutic applications.
