Elucidation of the molecular mechanism of Cas-endonucleases from bacteria and cyanobacteria

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CRISPR-Cas systems (CRISPR = Clustered Regulatory Interspaced Short Palindromic Repeats, Cas= CRISPR-associated gene) are used by prokaryotes to defend themselves against phages, invading nucleic acids and mobile genetic elements. These adaptive immune systems consist of a genomic CRISPR-locus, which is transcribed in the crRNA (CRISPR-RNA) and one (or a complex) effector nuclease, that cleaves the target nucleic acid in a crRNA-sequence dependent manner. The RNA-guided, sequence-dependent cleavage of double stranded DNA by the Cas9 endonuclease of Streptococcus pyogenes (SpyCas9) is currently exploited for the site-specific genome modification of various organisms and has revolutionised the field of biology. Structural biology played a crucial role to engineer and harness the SpyCas9-system as a universal genome editing tool by deciphering its molecular mechanism. About 100 structures of Cas-proteins and CRISPR-associated proteins have been determined by now. However, prokaryotes and archaea possess numerous, highly diverse Cas-systems, with to date unknown natural functions and mechanisms of action as well as a large number of such systems still remaining to be discovered. Based on bioinformatics analysis of bacterial and archaeal genomes, many novel subtypes of Cas-proteins were predicted and classified in bacteria, archaea and cyanobacteria. Furthermore, recent studies show that an active Class 1 CRISPR-Cas system generates oligoadenylate secondary messengers, resulting in the activation of another endonuclease and RNA-degradation. In addition, current data suggest that CRISPR-Cas systems are not merely prokaryotic defence mechanisms, but also play a role in DNA repair, regulation of gene expression, virulence and horizontal gene transfer. This clearly emphasis that despite the tremendous progress in the characterisation of these intriguing systems which has already been made, a lot of information on their molecular mechanisms and functions in the natural organisms is largely missing. My research group focuses on the structure-function relationship of nucleic acids and their recognition by protein factors and small molecules. In this proposed project we want to biochemically and structurally characterise Class 2 Cas-proteins from cyanobacteria and bacteria. This will lead to a detailed elucidation of the molecular mechanisms of these bacterial and cyanobacterial Cas-proteins. In order to obtain a full picture of the atomic details and their function in the natural host, we will cooperate with the groups of G. Bange, W. Hess, G. Klug and A. Marchfelder within the framework of this priority program as outlined in the present proposal. Within the funding period novel Cas- and Cas-associated proteins with particular roles in their natural organisms might be discovered by members of the priority program. Here we will be able to bring our expertise into play and to decipher the molecular mechanisms of these proteins.