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Abstract
CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are adaptive immune systems found in prokaryotes that defend against viruses and other foreign genetic elements. The first step of CRISPR-Cas defense, termed adaptation, involves two phases: the acquisition of foreign DNA as protospacers, and the incorporation of the DNA as spacers into the CRISPR array. In this dissertation, in vitro and in vivo approaches were utilized to investigate both phases of adaptation in the Type II-A CRISPR-Cas system of Streptococcus thermophilus. First, the mechanism of spacer incorporation into the CRISPR array was investigated by reconstituting the integration reaction in vitro. It was determined that Cas1 and Cas2 proteins accurately integrate spacer DNA into a CRISPR locus. Sequences in the CRISPR leader and repeat were identified as important DNA elements that dictate the first site of integration at the leader-repeat junction. Additionally, second-site integration at the repeat-spacer junction was found to be dependent on multiple determinants including a length-defining mechanism that relies on a repeat element proximal to the second site of integration. The protospacer selection phase of adaptation was also addressed to investigate how foreign DNA is acquired and discriminated towards PAM (protospacer adjacent motif)-adjacent sequences to generate functional spacers. Here, we demonstrate that Csn2 influences the selection of PAM-adjacent sequences for integration by Cas1-Cas2. Additional genetic analyses revealed that loss of a component of the Cas9 ribonucleoprotein, tracrRNA (the trans-activating CRISPR RNA), reduced spacer duplication events observed within the CRISPR array. Furthermore, loss of nuclease activity of DNA repair proteins RexAB was found to negatively impact adaptation frequency, presumably through the reduction in protospacer generation. Lastly, spontaneous mutations in the S. thermophilus FtsH protein leads to phage resistance in the absence of a functional CRISPR-Cas system and this effect was bypassed by mutations in a phage tail chaperonin protein. These results provide valuable insight into the mechanism and regulation of CRISPR adaption in the Type II-A CRISPR-Cas system of S. thermophilus and contributes to the overall understanding of adaptive immunity against foreign elements and the host-phage evolutionary arms race.