CRISPR-Cas functions beyond defence in haloarchaea

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Since its discovery as prokaryotic immune system more than ten years ago, more and more data show that the CRISPR-Cas system or its components have additional cellular roles beyond the defence function. These additional functions encompass regulation of processes like virulence, group behaviour, stress tolerance and gene expression as well as roles in DNA repair and impacts on ecology and evolution. In the previous funding period we investigated CRISPR-Cas functions beyond defence in the halophilic archaeon Haloferax volcanii. We could show that CRISPR-Cas is involved in the maturation and function of the small RNA s479 that regulates expression of an endogenous gene. Furthermore, we collected data that suggest involvement of Cas1 in oxidative stress repair. We also initiated the analysis of a recently identified CRISPR-Cas variant, the HRAMP (haloarchaeal repeat associated mysterious protein) system. In addition, we investigated self-targeting induced genome cleavage revealing that DNA fragments are generated from the entire genome and are incorporated as new spacers into the CRISPR arrays.In the next funding period we want to further investigate the potential role of CRISPR-Cas in endogenous gene regulation. Copurification experiments with Cascade proteins will show whether mRNAs and crRNA-like sRNAs are bound and reveal whether Cascade together with crRNAs and sRNAs is active in endogenous gene regulation. We will study the molecular mechanism of the interaction of s479 with Cascade and its target mRNA in detail.We also want to continue our studies on the interaction between the CRISPR-Cas system and the cellular repair pathways. To this end we will investigate the function of Cas1 in DNA repair in more detail and identify direct interaction partners. In addition, we will examine how self-targeting induced cleavages are repaired and if CRISPR-Cas components are involved in the repair reactions. The involvement of DNA repair and recombination enzymes in interference and adaptation reactions will also be analysed.As a third goal we will investigate the non-canonical CRISPR-Cas variant HRAMP. We want to determine whether the HRAMP proteins form a complex and whether this complex binds DNA or RNA. Furthermore we want to identify the targets of this system.In the frame of this project we want to identify novel functions of haloarchaeal CRISPR-Cas systems. (1) We want to determine additional functions of the Cas1, Cas2 and Cas4 proteins. (2) We will investigate orphan CRISPR-cas genes. Several haloarchaeal organism contain only CRISPR genes or an incomplete cas gene set. We will analyse whether these genes are still active and what kind of function they have. (3) We want to unravel the regulation of endogenous genes by the CRISPR-Cas system. CRISPR loci can contain spacers matching the own genome. We want to determine whether these spacers can regulate the expression of the endogenous genes. (4) Biofilm formation in bacteria has been reported to be regulated by CRISPR-Cas. We will investigate the regulation of biofilm formation by CRISPR-Cas in haloarchaea.