Language
English
Publication Date
9-11-2026
Journal
bioRxiv
DOI
10.64898/2026.09.07.749908
PMID
42818330
PMCID
PMC13622603
PubMedCentral® Posted Date
9-11-2026
PubMedCentral® Full Text Version
Pre-Print
Abstract
Circular RNAs (circRNAs) are generated by backsplicing of eukaryotic protein-coding transcripts and can regulate microRNAs and RNA binding proteins, or serve as translation templates. Their covalently closed structure confers resistance to exonuclease-mediated degradation, extending their half-life and supporting their development as RNA therapeutics. However, existing overexpression methods often yield substantial contaminating linear RNAs, limiting their utility. Here, we systematically benchmarked plasmid-based circRNA overexpression strategies in human cells, comparing spliceosome- and ribozyme-based mechanisms across constructs incorporating widely used flanking sequences. The ribozyme-based Tornado system produced the highest circRNA yield but introduced extraneous “molecular scars” into the mature product. By contrast, spliceosome-mediated circularization using introns from the Drosophila Laccase2 gene, which contain imperfect complementary repeats, produced scarless circRNA with substantially lower linear RNA contamination. Linear RNA was further reduced by engineering the primary transcript to terminate in a non-polyadenylated end, increasing its susceptibility to exonucleases. Building on this optimized system, we developed a dual-output platform co-expressing a linear fluorescent reporter alongside a circRNA from a single promoter (CIRCUS, circRNA and upstream linear system), enabling efficient screening of circRNA-driven cellular phenotypes, including site-specific A-to-I editing of target mRNAs. Together, this toolkit provides high-purity circRNA production suitable for mechanistic studies and circRNA-based therapeutic development.
Keywords
circRNA, backsplicing, ribozyme-based circularization, RNA therapeutics, A-to-I RNA editing, Laccase2 introns, circRNA overexpression, CIRCUS, Tornado
Published Open-Access
yes
Recommended Citation
Fields, Christopher J; Fujiwara, Rina; Wright, Bradley W; et al., "Engineering Circular RNA Expression Systems To Minimize Contaminating Linear RNA Byproducts" (2026). Faculty, Staff and Students Publications. 8319.
https://digitalcommons.library.tmc.edu/baylor_docs/8319