Characterization of the CRISPR-Cas type I-B DNA interference complex from Clostridiumthermocellum

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Archaea and bacteria can contain adaptive CRISPR-Cas immune systems. These diverse systems utilize small CRISPR RNAs bound to a Cas protein complex to detect viral attacks and to degrade the foreign DNA. We propose to investigate the type I-B DNA interference complex from Clostridium thermocellum. We have established the recombinant production of all five subunits (Cas3, Cas5, Cas6, Cas7, Cas8b) of this ribonucleoprotein complex (termed Cascade) and the in vitro generation of CRISPR RNA transcripts. We noted that the subtype-specific protein Cas8b is produced in two fragments and that this fragmentation pattern is also conserved in other Cas8b proteins. The Cas8b cleavage site was mapped. We will apply mutational studies of the protein and nucleic acid components of the available recombinant complex to investigate the role(s) of the individual Cas subunits in Cascade functionality. We aim to unravel the mechanism(s) and the specificity of (i) DNA target selection and (ii) CRISPR RNAs loading into Cascade. One focus will be placed on the unknown role of the subtype-specific large subunit Cas8b in these processes. We propose that the observed conserved fragmentation of this protein creates the elusive small subunit found in interference complexes of different CRISPR-Cas subtypes. We will investigate the mechanism of Cas8b fragmentation and will follow preliminary results that point at a self-cleavage reaction. Cas8b mutants that prevent internal cleavage as well as protein fragments that represent potential small and large subunits will be assembled into the DNA interference complex. In vitro DNA binding and cleavage assays will be used to deduce the roles of the small and the large Cascade subunits in DNA target recognition. These studies should enable us to compare substrate selectivity of DNA interference complexes between type I-B and type I-A which was previously reconstituted in our laboratory from the archaeon Thermoproteus tenax. We plan to collaborate with members of the DFG Forschergruppe FOR1680 to (i) map the interaction sites of CRISPR RNAs and C. thermocellum Cas proteins via mass-spectrometry and to (ii) study the functionality of small and large subunits of DNA interference complexes in vivo. The comparison of different DNA interference mechanisms should shed light on (i) the molecular bases for the diversification of CRISPR-Cas systems during evolution and (ii) the degree of flexibility in DNA target selection which is e.g. needed for CRISPR-Cas-mediated genome engineering.