title: Inducing circular RNA formation using the CRISPR endoribonuclease Csy4.
abstract: Circular RNAs (circRNAs) are highly stable, covalently closed RNAs that are regulated in a spatiotemporal manner and whose functions are largely unknown. These molecules have the potential to be incorporated into engineered systems with broad technological implications. Here we describe a switch for inducing back-splicing of an engineered circRNA that relies on the CRISPR endoribonuclease, Csy4, as an activator of circularization. The endoribonuclease activity and 3' end-stabilizing properties of Csy4 are particularly suited for this task. Coexpression of Csy4 and the circRNA switch allows for the removal of downstream competitive splice sites and stabilization of the 5' cleavage product. This subsequently results in back-splicing of the 5' cleavage product into a circRNA that can translate a reporter protein from an internal ribosomal entry site (IRES). Our platform outlines a straightforward approach toward regulating splicing and could find potential applications in synthetic biology as well as in studying the properties of different circRNAs.
authors.affiliation: Curriculum in Genetics and Molecular Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.|Gene Therapy Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: Erin K
authors.initials: EK
authors.lastname: Borchardt
authors.affiliation: Curriculum in Genetics and Molecular Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.|Gene Therapy Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: Rita M
authors.initials: RM
authors.lastname: Meganck
authors.affiliation: Department of Microbiology and Immunology.
authors.forename: Heather A
authors.initials: HA
authors.lastname: Vincent
authors.affiliation: Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Ball
authors.affiliation: Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: Silvia B V
authors.initials: SBV
authors.lastname: Ramos
authors.affiliation: Department of Microbiology and Immunology.|Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: Nathaniel J
authors.initials: NJ
authors.lastname: Moorman
authors.affiliation: Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.|IBGS, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: William F
authors.initials: WF
authors.lastname: Marzluff
authors.affiliation: Gene Therapy Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.|Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.|Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.|Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: Aravind
authors.initials: A
authors.lastname: Asokan
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D012313:RNA / Q000378:metabolism*; D012326:RNA Splicing; D000079962:RNA, Circular
chemical_list: D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D000079962:RNA, Circular; D012313:RNA; C552242:Csy4 endoribonuclease, Pseudomonas aeruginosa; D004722:Endoribonucleases
keywords: CRISPR; Csy4; circular RNA; gene regulation; splicing regulation