Regulatable CRISPR-mediated RNA silencing in the hyperthermophilic archaeon Sulfolobus solfataricus

Erika Wimmer, Isabelle Anna Zink, Christa Schleper

Sulfolobusspecies thrive in hostile environments characterized by low pH, high temperature as well as a large number of viruses. Their hyperthermophilic life style and extensive CRISPR system account for the special interest in studying Sulfolobus. S. solfataricusdoes not recombine DNA into its genome, hence a genetic tool for targeted gene silencing was developed recently in our lab. By introducing an artificial CRISPR locus on a vector, the cell’s endogenous CRISPR system is harnessed to transcriptionally silence and study the function of essential genes. In order to enable controlled expression of the artificial CRISPR locus in S. solfataricus, we designed an inducible promoter system based on the maltose operon of S. acidocaldarius. To achieve low basal transcription of the artificial CRISPR locus but highly increased transcription levels upon induction, the gene encoding for the transcriptional regulator of the operon (MalR) was introduced into the same vector. The inducibility of this promoter system has previously been assessed in reporter gene-based assays. Upon induction of the promoter by adding maltose to the medium, a 6-to 8-fold increase was measured on transcript level after 180 minutes. Based on this observation, we further investigated the applicability of the expression platform for protein production and gene silencing experiments in S. solfataricus.

Department für Ökogenomik und Systembiologie
ÖFOS 2012
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