RNA-dependent RNA targeting by CRISPR-Cas9
Abstract
Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.
Data availability
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MS2 CRISPR screen in E. coliPublicly available at NCBI SRA under the BioProject accession number PRJNA413805.
Article and author information
Author details
Funding
Howard Hughes Medical Institute
- Rachel M Torrez
- Jennifer A Doudna
National Science Foundation (MCB-1244557)
- Steven C Strutt
- Jennifer A Doudna
Paul Allen Frontiers Science Program
- Jennifer A Doudna
Laboratory Directed Research and Development (U.S. Department of Energy's National Nuclear Security Administration under contract DE-NA0003525)
- Oscar A Negrete
German Academic Exchange Program
- Emine Kaya
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Rodolphe Barrangou, North Carolina State University, United States
Version history
- Received: October 12, 2017
- Accepted: January 3, 2018
- Accepted Manuscript published: January 5, 2018 (version 1)
- Version of Record published: February 2, 2018 (version 2)
Copyright
© 2018, Strutt et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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