Characterization of two conserved complexes of the archaeal CRISPR/Cas immune system

Clusters of regularly interspaced short palindromic repeats (CRISPR) of Bacteria and Archaea consist of short repeat sequences (24 to 48 nt) that are separated by unique spacer sequences, which often match viral or plasmid sequences. Numerous conserved cas (CRISPR-associated) genes are usually located next to a CRISPR cluster and define the antiviral CRISPR/Cas immune system¹. The immunity is thought to be mediated by (i) the acquisition of foreign protospacers, (ii) the processing of CRISPR transcripts into small RNAs (crRNA) and (iii) the interference of spacer and the homologous phage DNA resulting in antiviral defence².

The detailed molecular functions and interactions of involved Cas and Csa proteins (specific for the cas subtype Apern) in Archaea remained mostly unknown. The conserved cas genes of the hyperthermophilic Crenarchaeote Thermoproteus tenax are arranged in two operon-like structures. To facilitate functional analyses, the cas genes were individually cloned and heterologously expressed in Escherichia coli. The entire protein complexes CasA1 (Cas4, Cas1/2, Csa1) and CasA2 (Csa5, Cas7, Cas5a, Cas3, Cas3´, Cas8a2) were purified by refolding from inclusion bodies and protein interactions were analysed by gel filtration chromatography. CasA2 showed DNA and crRNA-binding, substantiating its role in interference. CasA1 is proposed to be involved in the integration of new spacers.