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RNA. 2017 May;23(5):619-627. doi: 10.1261/rna.056838.116. Epub 2017 Feb 21.

Inducing circular RNA formation using the CRISPR endoribonuclease Csy4.

Author information

1
Curriculum in Genetics and Molecular Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
2
Gene Therapy Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
3
Department of Microbiology and Immunology.
4
Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
5
Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
6
IBGS, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
7
Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

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.

KEYWORDS:

CRISPR; Csy4; circular RNA; gene regulation; splicing regulation

PMID:
28223408
PMCID:
PMC5393173
DOI:
10.1261/rna.056838.116
[Indexed for MEDLINE]
Free PMC Article

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