RNA-dependent RNA targeting by CRISPR-Cas9

  1. Steven C Strutt
  2. Rachel M Torrez
  3. Emine Kaya
  4. Oscar A Negrete
  5. Jennifer A Doudna  Is a corresponding author
  1. University of California, Berkeley, United States
  2. Sandia National Laboratories, United States

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

The following data sets were generated
    1. Steven Strutt
    2. Jennifer Doudna
    (2017) MS2 CRISPR screen in E. coli
    Publicly available at NCBI SRA under the BioProject accession number PRJNA413805.

Article and author information

Author details

  1. 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.
  2. Rachel M Torrez

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  3. Emine Kaya

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  4. 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.
  5. 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.
    ORCID icon "This ORCID iD identifies the author of this article:" 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

  1. Rodolphe Barrangou, North Carolina State University, United States

Publication history

  1. Received: October 12, 2017
  2. Accepted: January 3, 2018
  3. Accepted Manuscript published: January 5, 2018 (version 1)
  4. 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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  1. Steven C Strutt
  2. Rachel M Torrez
  3. Emine Kaya
  4. Oscar A Negrete
  5. Jennifer A Doudna
(2018)
RNA-dependent RNA targeting by CRISPR-Cas9
eLife 7:e32724.
https://doi.org/10.7554/eLife.32724

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