An essential primary step of the prokaryotic CRISPR-Cas defense pathwayincludes acquiring foreign spacer DNA into the CRISPR locus. This first step, termed adaptation, provides the necessary immunity against the foreign mobile genetic element from which the DNA was captured. In this dissertation, a number of approaches were used to investigate the adaptation stage of CRISPR-Cas prokaryotic defense systems. The mechanisms of spacer integration into the CRISPR locus was investigated in vitro using two different model organisms, Pyrococcus furiosus and Streptococcus thermophilus. For P. furiosus, it was determined that Cas1 and Cas2 are sufficient to accurately integrate spacers into a minimal CRISPR locus, while Cas1 from S. thermophilus seems to be capable of independent spacer integration. The sequences and their relative spacing in the P. furiosus CRISPR repeat were found to contribute to accurate spacer integration at the sites required for maintaining the proper repeat size as the CRISPR locus grows. The precursor step of spacer generation was also considered here, to investigate how foreign DNA is selected and processed to the appropriate size for the integration of functional spacers. A complex consisting of Cas1, Cas2, Cas4-1, and Cas4-2 from P. furiosus was observed and demonstrated to be active in spacer integration into the CRISPR repeat in vitro. Furthermore, Cas4-2 exhibited in vitro nuclease activity, supporting previous in vivo evidence of Cas4-2 involvement in pre-spacer generation. Together these in vitro studies of Cas protein molecular mechanisms add to the constantly growing model of CRISPR-Cas adaptation.
