Inducing circular RNA formation using the CRISPR endoribonuclease Csy4

  1. Aravind Asokan2,4,5,7
  1. 1Curriculum in Genetics and Molecular Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
  2. 2Gene Therapy Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
  3. 3Department of Microbiology and Immunology,
  4. 4Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
  5. 5Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
  6. 6IBGS, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
  7. 7Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
  1. Corresponding author: aravind{at}med.unc.edu

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

  • Received April 6, 2016.
  • Accepted January 31, 2017.

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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