Cross-talk and extensive rewiring of CRISPR-Cas systems with the cellular regulatory machinery in cyanobacteria

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With this proposal we aim at analyzing the CRISPR-Cas systems in cyanobacteria with a focus on their interaction with the regulatory machinery of the host. As model organism we have chosen the unicellular strain Synechocystis sp. PCC 6803. This strain carries three CRISPR-Cas systems. Two of them were classified as subtype I-D and subtype III-D. The third constitutes a III-B variant (III-Bv), characterized by a Cmr6_Cmr1 fusion, a replaced Cmr5 protein, the possible involvement of a peptidase and the lack of an associated Cas6 maturation endoribonuclease. Bioinformatic predictions suggest similar variant systems to exist also in other prokaryotes. Instead of Cas6 we found that the host-encoded endoribonuclease E was recruited as the major maturation enzyme, which raises questions about the involved molecular and evolutionary mechanisms. It directly suggests that there are additional host- and CRISPR-encoded factors that need to be identified, e.g., in loading mature crRNAs into the surveillance complexes, in performing the 3’ end maturation of crRNAs, in modulating RNase specificity as well as the type of nucleic acid targeted by the CRISPR-Cas complexes. We will address these questions using pull-down assays, the characterization of deletions for respective candidate genes in interference assays and by molecular analyses, the elucidation of an RNase E-CRISPR-repeat-RNA cocrystal structure and determination of the entire CRISPR3 complex structure. However, the possible interplay between CRISPR-associated RNases and the processes governing the maturation and stability of transcripts appears more complex than previously anticipated also with regard to the Cas6-dependent other two CRISPR-Cas systems. Therefore, we will also address the possible impact on the host transcriptome as well as redox control through the host-encoded RpaB-NblS two-component system. In both cases, the involvement of additional enzymes and the possible consequences for the post-transcriptional regulation of gene expression will be followed. The project work will be complemented by the metagenomic analysis of cyanobacterial mass developments to understand the evolving architecture of the studied types of CRISPR-Cas systems and their integration into and interaction with the cellular regulatory apparatus.