The CRISPR/Cas system in Archaea: Molecular characterization of two conserved Cas complexes

Clusters of regularly interspaced short palindromic repeats (CRISPR) are present in nearly all archaeal and in 50% of all bacterial genomes. A CRISPR consists of short repeat sequences (24 to 48 nt) that are separated by unique spacer sequences which often match viral or plasmid sequences¹. A set of conserved cas (CRISPR-associated) genes is usually found next to a CRISPR cluster and thought to mediate (i) the processing of CRISPR transcripts into small RNAs (crRNAs) and (ii) the hybridisation of spacer and the homologous phage DNA resulting in antiviral defence². The detailed molecular mechanisms of Cas and Csa (specific for the cas subtype Apern) proteins in Crenarchaeota remained mostly unknown. Here, we present detailed analyses of the seven CRISPR clusters (TTX_1-7) and ten relevant cas genes of the hyperthermophilic Crenarchaeote Thermoproteus tenax. The cas genes located between two CRISPR cluster showed two typical operon-like structures. These genes were individually cloned, heterologously expressed in Escherichia coli, the entire protein complexes CasA1 (Cas4, Cas1/2, Csa1) and CasA2 (Csa5, Csa2, Cas5a, Cas3, Cas3HD, Csa4) were purified by refolding from inclusion bodies and functionally analysed. CasA1 showed ribonuclease activity for crRNA, whereas CasA2 had a strong crRNA-binding activity.