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CRISPR-mediated targeted mRNA degradation in the archaeon Sulfolobus solfataricus.

Authors
  • Zebec, Ziga
  • Manica, Andrea
  • Zhang, Jing
  • White, Malcolm F
  • Schleper, Christa
Type
Published Article
Journal
Nucleic Acids Research
Publisher
Oxford University Press
Publication Date
Apr 01, 2014
Volume
42
Issue
8
Pages
5280–5288
Identifiers
DOI: 10.1093/nar/gku161
PMID: 24603867
Source
Medline
License
Unknown

Abstract

The recently discovered clustered regularly interspaced short palindromic repeat (CRISPR)-mediated virus defense represents an adaptive immune system in many bacteria and archaea. Small CRISPR RNAs cause cleavage of complementary invading nucleic acids in conjunction with an associated protein or a protein complex. Here, we show CRISPR-mediated cleavage of mRNA from an invading virus in the hyperthermophilic archaeon Sulfolobus solfataricus. More than 40% of the targeted mRNA could be cleaved, as demonstrated by quantitative polymerase chain reaction. Cleavage of the mRNA was visualized by northern analyses and cleavage sites were mapped. In vitro, the same substrates were cleaved by the purified CRISPR-associated CMR complex from Sulfolobus solfataricus. The in vivo system was also re-programmed to knock down mRNA of a selected chromosomal gene (β-galactosidase) using an artificial miniCRISPR locus. With a single complementary spacer, ∼50% reduction of the targeted mRNA and of corresponding intracellular protein activity was achieved. Our results demonstrate in vivo cleavage of mRNA in a prokaryote mediated by small RNAs (i.e. analogous to RNA interference in eukaryotes) and the re-programming of the system to silence specific genes of interest.

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