CRISPR (Clustered Regularly Interspaced Palindromic Repeats)-Cas (CRISPR associated) is a robust immune system present in prokaryotes used to combat viruses, plasmids and other mobile genetic elements. The pathway to immunity occurs in three robust stages known as adaptation, crRNA biogenesis, and defense. CRISPR-Cas systems are very diverse and are currently organized into six overarching types containing close to thirty subtypes. The focus of this dissertation is the defense stage of subtype CRISPR-Cas systems III-A (Csm) and III-B (Cmr). Specifically, the requirements for successful anti-plasmid immunity and the roles of Csm6 and Csx1 in Type-III defense, was investigated. Several different technical approaches were utilized to study these systems, drawing upon elements of biochemistry, molecular biology, genetics, microbiology, and bioinformatics. Characterization of three Csm6 homologs from Lactococcus lactis, Streptococcus thermophilus, and Staphylococcus epidermidis, was performed using the model organism Escherichia coli. Csm6 was found to be a metal-independent single-stranded RNA-specific endoribonuclease. Cleavage activity of Csm6 was found to be dependent upon the C-terminal Higher Eukaryotes and Prokaryotes Nucleotide Binding (HEPN) motif that is common to a large family of ribonucleases. Functioning of the defense response for all three Csm systems, in vivo, was found to be dependent upon the ribonuclease activity of Csm6. Characterization of the Type III-B system was conducted using the Cmr system of Pyrococcus furiosus (Pfu). In Pfu, the role of Csx1 in defense was explored by reconstituting the recently discovered cyclic oligoadenylate (cOA) production activity of Type III systems. Csx1 was found to be an important component of the Type III-B response of Pfu. Production of cOA molecules by the Cmr effector complex activates Pfu Csx1 endoribonuclease activity through first binding to the Cas Rossman Fold (CARF) domain. A component of the target RNA, the protospacer flanking sequence (PFS), was found to be essential for triggering both the DNA and RNA nuclease activities of the Type III-B system. The results presented in this dissertation provide important insight into the mechanism of action and regulation of Type III CRISPR-Cas systems and solidifies the role of highly related Csm6 and Csx1 ribonucleases in CRISPR-Cas immunity.
