Engineering circular RNA expression systems to minimize contaminating linear RNA byproducts

Circular RNAs (circRNAs) are a fascinating class of non-coding RNA molecules that arise from the backsplicing of eukaryotic protein-coding transcripts. They possess unique properties that make them resistant to degradation by exonucleases, thereby extending their half-life and paving the way for their potential use in therapeutics. However, a major challenge in the overexpression of circRNAs is the generation of significant quantities of linear RNA byproducts, which can hinder their practical utility.

In this study, researchers sought to optimize the methods for producing circRNAs to minimize the unwanted linear RNA byproducts. They evaluated spliceosome- and ribozyme-based mechanisms in human cells, employing different constructs with commonly used flanking sequences. While the ribozyme-based Tornado system emerged as the most effective in producing high levels of circRNA, it unfortunately also introduced extraneous molecular scars into the mature product.

Conversely, spliceosome-mediated circularization using introns derived from the Drosophila Laccase2 gene proved to be a game-changer. These introns contain imperfect complementary repeats, which allowed for the creation of scarless circRNA with considerably lower contamination of linear RNA. To further enhance the purity of the linear RNA, the researchers engineered the primary transcript to terminate in a non-polyadenylated end, making it more susceptible to exonucleases and reducing its abundance.

Building upon this optimized production system, the scientists developed a dual-output platform that facilitated the simultaneous expression of a linear fluorescent reporter alongside a circRNA from a single promoter. This innovative approach, dubbed CIRCUS (circRNA and upstream linear system), enabled efficient screening of circRNA-driven cellular phenotypes. One such phenotype of interest was site-specific A-to-I editing of target mRNAs, which could potentially be harnessed for therapeutic applications.

In conclusion, this research provides a comprehensive toolkit for high-purity production of circRNAs, making them more suitable for mechanistic studies and the development of circRNA-based therapeutics. By addressing the challenge of contaminating linear RNA byproducts, the researchers have paved the way for more efficient and reliable use of these intriguing RNA molecules in biomedical research and potential therapeutic interventions.