Characterization of a type I-B CRISPR-Cas system of Clostridium thermocellum

CRISPR-Cas systems are adaptive immune systems, found in bacteria and archaea that provide inheritable resistance against mobile genetic elements, e.g. viruses and plasmids. CRISPR-Cas systems comprise one or more CRISPR loci that contain virus-derived DNA sequences (spacers) that are interspaced by identical repeat sequences (repeats), and a set of cas genes. The degradation of nucleic acid targets is mediated by ribonucleoprotein (RNP) complexes, formed by Cas proteins, that are guided by small CRISPR RNA molecules (crRNAs). A Cas protein classification has been established which reflects the diversification of CRISPR systems during the co-evolution of phages and their respective hosts. In this study, the type I-B CRISPR-Cas system of the thermophilic bacterium Clostridium thermocellum was investigated. CRISPR loci are transcribed into precursor-crRNAs and individual crRNAs are produced by Cas6 endonucleases. C. thermocellum contains two Cas6 proteins and the recombinant enzymes exclusively process their respective precursor transcripts in vitro. RNA-Seq analyses confirmed crRNA maturation and highlighted crRNA abundance differences in vivo. These analyses identified internal promotion of precursor-crRNA transcription and reverse crRNA transcripts (anti-crRNAs). Anti-crRNAs display a distinct processing pattern and the abundance of the complementary crRNA is often reduced in vivo. Cleavage assays with crRNAs and double-stranded crRNA/anti-crRNA hybrids identified RNase III to be capable of anti-crRNA processing. RNase III cleavage is mediated by recognition motifs within the repeat RNA duplexes. In type I-B systems, CRISPR interference is mediated by a dsDNA targeting crRNP complex, termed Cascade, which consists of the Cas proteins Cas3, Cas5, Cas6, Cas7 and the subtype-specific Cas8b subunit. All five recombinant Cascade subunits were produced in Escherichia coli. Cascade assembly studies revealed that a stable core-complex is formed by Cas5, Cas7, Cas8b and crRNA in vitro. Cas3 does not assemble with the complex. Cas6 is a temporarily associated subunit. Mass-spectrometric analyses confirmed protein interactions between Cas5, Cas7 and Cas8b and determined an uneven complex stoichiometry of 1:1:6:2.5 for Cas5:Cas6:Cas7:Cas8b. The large subunit Cas8b forms an additional small C-terminal protein fragment that is also observed in C. thermocellum cell extracts and assembles with the complex. This thesis provides details for the in vitro assembly of individual Cas proteins into type I-B Cascade. Furthermore, RNA-Seq analyses of the CRISPR arrays highlight the impact of individual spacer and repeat sequences on the functionality of CRISPR-Cas systems