CRISPR-Cas with a grain of salt

The recently discovered CRISPR-Cas system (CRISPR: clustered regularly interspaced short palindromic repeats, Cas: CRISPR-associated) is an adaptive and heritable resistance mechanism against foreign genetic elements. The CRISPR-Cas system consists of clusters of short palindromic DNA repeats which are separated by short spacers, the latter being sequences derived from the invader. In addition, a set of proteins (the Cas proteins) is involved.

We are investigating the CRISPR-Cas system in the halophilic archaeon Haloferax volcanii. H. volcanii is an archaeal model organism, which requires about 2.1 M NaCl for optimal growth and raises the intracellular salt concentration to similar values to cope with the high salt concentration in the medium. The genome is sequenced and Haloferax is one of the few archaeal organisms where genetic systems are available.

We could show that H. volcanii has three active CRISPR loci, one located on the chromosome and two located on one of the chromosomal plasmids. Next to one of the CRISPR loci the Cas proteins are encoded in one multicistronic operon including genes for Cas1-8b. According to the new nomenclature by Makarova et al. the H.volcanii CRISPR/Cas-System is of type I-B. Therefore we would expect Haloferax to use a Cascade-like complex for the processing of the pre-crRNA. To identify the composition of such a Cascade complex, we use FLAG-tagged Cas proteins and co-immuno-precipitation. In addition, we generate deletion mutants of all Cas protein genes to analyse their biological function.

Moreover, we aim to identify the PAM (protospacer adjacent motif) sequences, which might play an essntial role in the daptation and interference stage of this defence system.