Versatile toolkit for highly-efficient and scarless overexpression of circular RNAs.

Autor:	Stringer BW; College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, 5032, Australia.; South Australian Health and Medical Research Institute (SAHMRI), Adelaide, South Australia, 5000, Australia., Gabryelska M; College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, 5032, Australia., Marri S; College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, 5032, Australia., Clark L; College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, 5032, Australia.; South Australian Health and Medical Research Institute (SAHMRI), Adelaide, South Australia, 5000, Australia.; South Australian Genomics Centre (SAGC), Adelaide, South Australia, 5000, Australia., Lin H; College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, 5032, Australia., Gantley L; College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, 5032, Australia., Liu R; College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, 5032, Australia., Wilusz JE; Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology, Therapeutic Innovation Center, Baylor College of Medicine, Houston, TX, 77030, USA., Conn VM; College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, 5032, Australia., Conn SJ; College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, 5032, Australia.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2023 Nov 22. Date of Electronic Publication: 2023 Nov 22.
DOI:	10.1101/2023.11.21.568171
Abstrakt:	Circular RNAs (circRNAs) are a class of single-stranded, covalently closed RNA that contain a unique back-splice junction (bsj) sequence created by the ligation of their 5' and 3' ends via spliceosome-catalyzed back-splicing. A key step in illuminating the cellular roles of specific circRNAs is via increasing their expression. This is frequently done by transfecting cells with plasmid DNA containing cloned exons from which the circRNA is transcribed, flanked by sequences that promote back-splicing. We observed that commonly used plasmids lead to the production of circRNAs with molecular scars at the circRNA bsj. Stepwise redesign of the cloning vector corrected this problem, ensuring bona fide circRNAs are produced with their natural bsj at high efficiency. The fidelity of circRNAs produced from this new construct was validated by RNA sequencing and also functionally validated. To increase the utility of this modified resource for expressing circRNA, we developed an expanded set of vectors incorporating this design that (i) enables selection with a variety of antibiotics and fluorescent proteins, (ii) employs a range of promoters varying in promoter strength and (iii) generated a complementary set of lentiviral plasmids for difficult-to-transfect cells. These resources provide a novel and versatile toolkit for high-efficiency and scarless overexpression of circular RNAs that fulfill a critical need for the investigation of circRNA function. Competing Interests: CONFLICT OF INTEREST J.E.W. serves as a consultant for Laronde.
Databáze:	MEDLINE
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