title: Efficient CRISPR-Mediated Post-Transcriptional Gene Silencing in a Hyperthermophilic Archaeon Using Multiplexed crRNA Expression.
abstract: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-mediated RNA degradation is catalyzed by a type III system in the hyperthermophilic archaeon Sulfolobus solfataricus Earlier work demonstrated that the system can be engineered to target specifically mRNA of an endogenous host reporter gene, namely the β-galactosidase in S. solfataricus Here, we investigated the effect of single and multiple spacers targeting the mRNA of a second reporter gene, α-amylase, at the same, and at different, locations respectively, using a minimal CRISPR (miniCR) locus supplied on a viral shuttle vector. The use of increasing numbers of spacers reduced mRNA levels at progressively higher levels, with three crRNAs (CRISPR RNAs) leading to ∼ 70-80% reduction, and five spacers resulting in an α-amylase gene knockdown of > 90% measured on both mRNA and protein activity levels. Our results indicate that this technology can be used to increase or modulate gene knockdown for efficient post-transcriptional gene silencing in hyperthermophilic archaea, and potentially also in other organisms.
authors: Division of Archaea Biology and Ecogenomics, Department of Ecogenomics and Systems Biology, University of Vienna, 1090, Austria.
authors: Ziga
authors: Z
authors: Zebec
authors: Division of Archaea Biology and Ecogenomics, Department of Ecogenomics and Systems Biology, University of Vienna, 1090, Austria.
authors: Isabelle Anna
authors: IA
authors: Zink
authors: Division of Archaea Biology and Ecogenomics, Department of Ecogenomics and Systems Biology, University of Vienna, 1090, Austria.
authors: Melina
authors: M
authors: Kerou
authors: Division of Archaea Biology and Ecogenomics, Department of Ecogenomics and Systems Biology, University of Vienna, 1090, Austria christa.schleper@univie.ac.at.
authors: Christa
authors: 0000-0002-1918-2735
authors: C
authors: Schleper
mesh_terms: D001105:Archaea / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D015870:Gene Expression; D056426:Genetic Loci; D034622:RNA Interference; D019642:RNA, Archaeal / Q000235:genetics*; D048229:Sulfolobus solfataricus / Q000235:genetics; D000516:alpha-Amylases / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D019642:RNA, Archaeal; D000516:alpha-Amylases; D001616:beta-Galactosidase
keywords: Archaea; CRISPR; RNA; Sulfolobus; gene silencing; hyperthermophile