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Elife. 2018 Jan 5;7. pii: e32724. doi: 10.7554/eLife.32724.

RNA-dependent RNA targeting by CRISPR-Cas9.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, United States.
2
Sandia National Laboratories, Biotechnology and Bioengineering Department, Livermore, United States.
3
Howard Hughes Medical Institute, Maryland, United States.
4
Department of Chemistry, University of California, Berkeley, United States.
5
Innovative Genomics Institute, University of California, Berkeley, United States.
6
MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, 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.

KEYWORDS:

CRISPR/Cas9; E. coli; RNA; biochemistry; programmable

PMID:
29303478
PMCID:
PMC5796797
DOI:
10.7554/eLife.32724
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

SS is listed on a patent application (No. 62598888) related to this work, RT, EK No competing interests declared, ON Oscar A Negrete: is listed on a patent application (No. 62598888) related to this work, JD 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

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