Inducing circular RNA formation using the CRISPR endoribonuclease Csy4

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...

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Bibliographic Details
Published in:RNA (Cambridge) 2017-05, Vol.23 (5), p.619-627
Main Authors: Borchardt, Erin K, Meganck, Rita M, Vincent, Heather A, Ball, Christopher B, Ramos, Silvia B V, Moorman, Nathaniel J, Marzluff, William F, Asokan, Aravind
Format: Article
Language:English
Subjects:
Bacterial Proteins - metabolism
CRISPR-Associated Proteins - metabolism
Endoribonucleases - metabolism
HEK293 Cells
Humans
RNA - metabolism
RNA Splicing
RNA, Circular
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Summary: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.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.056838.116