RNA-dependent RNA targeting by CRISPR-Cas9

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Cite as: eLife 2018;7:e32724 doi: 10.7554/eLife.32724

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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.

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Author details

Steven C Strutt

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

Competing interests
Steven C Strutt, is listed on a patent application (No. 62598888) related to this work.
Rachel M Torrez

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

Competing interests
No competing interests declared.
Emine Kaya

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

Competing interests
No competing interests declared.
Oscar A Negrete

Biotechnology and Bioengineering Department, Sandia National Laboratories, Livermore, United States

Competing interests
Oscar A Negrete, is listed on a patent application (No. 62598888) related to this work.
Jennifer A Doudna

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

For correspondence
doudna@berkeley.edu

Competing interests
Jennifer A Doudna, is a co-founder of to Caribou Biosciences, Intellia Therapeutics, and Editas Medicine and a scientific advisor to Caribou, Intellia, eFFECTOR Therapeutics and Driver. JAD is listed on a patent application (No. 62598888) related to this work.

0000-0001-9161-999X

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, Reviewing Editor, North Carolina State University, United States

Publication 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

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.