CRISPR-mediated targeted mRNA degradation in the archaeon Sulfolobus solfataricus

Autor(en)

Ziga Zebec, Andrea Manica, J. Zhang, M.F. White, Christa Schleper

Abstrakt

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.

Organisation(en)

Externe Organisation(en)

University of St. Andrews

Journal

Nucleic Acids Research

Band

42

Seiten

5280-5288

Anzahl der Seiten

9

ISSN

0305-1048

DOI

https://doi.org/10.1093/nar/gku161

Publikationsdatum

03-2014

Peer-reviewed

Ja

ÖFOS 2012

106022 Mikrobiologie

Link zum Portal

https://ucrisportal.univie.ac.at/de/publications/35de0f81-0c3e-4441-a171-950fab2533c2