Inducing circular RNA formation using the CRISPR endoribonuclease Csy4

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


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.

MeSH terms

  • Bacterial Proteins / metabolism*
  • CRISPR-Associated Proteins / metabolism*
  • Endoribonucleases / metabolism*
  • HEK293 Cells
  • Humans
  • RNA / metabolism*
  • RNA Splicing
  • RNA, Circular


  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • RNA, Circular
  • RNA
  • Csy4 endoribonuclease, Pseudomonas aeruginosa
  • Endoribonucleases