{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T21:19:09Z","timestamp":1792790349157,"version":"4.5.0"},"posted":{"date-parts":[[2026,9,11]]},"group-title":"Molecular Biology","reference-count":70,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T00:00:00Z","timestamp":1789084800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Institutes of Health","award":["R35-GM119735"],"award-info":[{"award-number":["R35-GM119735"]}]},{"name":"National Institutes of Health","award":["CA125123"],"award-info":[{"award-number":["CA125123"]}]},{"name":"National Institutes of Health","award":["OD036336"],"award-info":[{"award-number":["OD036336"]}]},{"name":"National Institutes of Health","award":["OD038251"],"award-info":[{"award-number":["OD038251"]}]},{"name":"Cancer Prevention and Research Institute of Texas","award":["RR210031"],"award-info":[{"award-number":["RR210031"]}],"id":[{"id":"https:\/\/ror.org\/0003xa228","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Cancer Prevention and Research Institute of Texas","award":["RP240432"],"award-info":[{"award-number":["RP240432"]}],"id":[{"id":"https:\/\/ror.org\/0003xa228","id-type":"ROR","asserted-by":"crossref"}]},{"name":"National Health and Medical Research Council","award":["GNT1198014"],"award-info":[{"award-number":["GNT1198014"]}],"id":[{"id":"https:\/\/ror.org\/011kf5r70","id-type":"ROR","asserted-by":"crossref"}]},{"name":"National Health and Medical Research Council","award":["GNT2042423"],"award-info":[{"award-number":["GNT2042423"]}],"id":[{"id":"https:\/\/ror.org\/011kf5r70","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,11]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  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 \u201cmolecular scars\u201d into the mature product. By contrast, spliceosome-mediated circularization using introns from the\n                  <jats:italic>Drosophila<\/jats:italic>\n                  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,\n                  <jats:underline>circ<\/jats:underline>\n                  RNA and\n                  <jats:underline>u<\/jats:underline>\n                  pstream linear\n                  <jats:underline>s<\/jats:underline>\n                  ystem), 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.\n                <\/jats:p>","DOI":"10.64898\/2026.09.07.749908","type":"posted-content","created":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T17:55:21Z","timestamp":1789149321000},"source":"Crossref","is-referenced-by-count":0,"title":["Engineering circular RNA expression systems to minimize contaminating linear RNA byproducts"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3151-5870","authenticated-orcid":false,"given":"Christopher J.","family":"Fields","sequence":"first","affiliation":[{"name":"Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology, Therapeutic Innovation Center, Baylor College of Medicine, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0076-0029","authenticated-orcid":false,"given":"Rina","family":"Fujiwara","sequence":"additional","affiliation":[{"name":"Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology, Therapeutic Innovation Center, Baylor College of Medicine, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6233-831X","authenticated-orcid":false,"given":"Bradley W.","family":"Wright","sequence":"additional","affiliation":[{"name":"Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology, Therapeutic Innovation Center, Baylor College of Medicine, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4552-9736","authenticated-orcid":false,"given":"Brett W.","family":"Stringer","sequence":"additional","affiliation":[{"name":"College of Medicine and Public Health, Flinders Health and Medical Research Institute (FHMRI), Flinders University, Bedford Park, South Australia, 5042, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1376-4515","authenticated-orcid":false,"given":"Simon J.","family":"Conn","sequence":"additional","affiliation":[{"name":"College of Medicine and Public Health, Flinders Health and Medical Research Institute (FHMRI), Flinders University, Bedford Park, South Australia, 5042, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6929-8755","authenticated-orcid":false,"given":"Jeremy E.","family":"Wilusz","sequence":"additional","affiliation":[{"name":"Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology, Therapeutic Innovation Center, Baylor College of Medicine, Houston, TX 77030, 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