Efficient CRISPR-Mediated Post-Transcriptional Gene Silencing in a Hyperthermophilic Archaeon Using Multiplexed crRNA Expression

Autor(en)
Ziga Zebec , Isabelle Anna Zink , Melina Kerou , Christa Schleper
Abstrakt

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.

Organisation(en)
Department für Ökogenomik und Systembiologie
Journal
G3: Genes, Genomes, Genetics
Band
6
Seiten
3161-3168
Anzahl der Seiten
8
ISSN
2160-1836
DOI
https://doi.org/10.1534/g3.116.032482
Publikationsdatum
10-2016
Publikationsstatus
Veröffentlicht
Peer-reviewed
Ja
ÖFOS 2012
106022 Mikrobiologie
Link zum Portal
https://ucris.univie.ac.at/portal/de/publications/efficient-crisprmediated-posttranscriptional-gene-silencing-in-a-hyperthermophilic-archaeon-using-multiplexed-crrna-expression(8c43eda1-2154-4251-b623-03574b3b9a95).html