Format

Send to

Choose Destination
Nucleic Acids Res. 2014 Apr;42(8):5280-8. doi: 10.1093/nar/gku161. Epub 2014 Mar 6.

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

Author information

1
Department of Ecogenomics and Systems Biology, University of Vienna, Archaea Biology and Ecogenomics Division, Althanstr. 14, 1090 Vienna, Austria and Biomedical Sciences Research Complex, School of Biology St Andrews University, St Andrews KY16 9ST, UK.

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.

PMID:
24603867
PMCID:
PMC4005642
DOI:
10.1093/nar/gku161
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Silverchair Information Systems Icon for PubMed Central
Loading ...
Support Center