Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system (original) (raw)

1. Nolan F. Sheppard1,
2. Nancy Ramia2,
3. Trace Deighan1,
4. Hong Li2,
5. Rebecca M. Terns1 and
6. Michael P. Terns1,3,4
7. 1Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA;
8. 2Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32313, USA;
9. 3Department of Genetics, Florida State University, Tallahassee, Florida 32313, USA;
10. 4Department of Microbiology, University of Georgia, Athens, Georgia 30602, USA
11. Corresponding authors: rterns{at}bmb.uga.edu, mterns{at}uga.edu

Abstract

CRISPR–Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM.

• CRISPR–Cas
• Cmr
• PAM
• RNAi
• DNA nuclease

Footnotes

• Supplemental material is available for this article.

• Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.272153.115.

• Received September 29, 2015.

• Accepted December 3, 2015.

• © 2016 Elmore et al.; Published by Cold Spring Harbor Laboratory Press

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