Bacteriophages drive the evolution of their bacterial hosts and can impart virulence factors that lead to microbial pathogenesis. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity is a critical line of defense for bacteria and archaea against invasive mobile genetic elements. This system allows the bacterial host to selectively degrade these foreign genetic elements via CRISPR associated (Cas) proteins. Understanding the mechanisms of CRISPR immunity will generate new strategies for targeting pathogenic bacteria, and drive the development of biotechnological tools for genome editing and gene silencing. Despite extensive research in recent years, a thorough understanding of the entire CRISPR-Cas system remains incomplete. Two critical, unresolved questions are how these protein-RNA complexes target foreign nucleic acids for degradation (i.e., DNA interference), and how dynamic assemblies of Cas proteins result in acquisition of foreign DNA sequences for incorporation into the host genome (i.e., primed acquisition). Therefore, the objective of this proposal is to determine how the Type I CRISPR-Cas protein complexes assemble to degrade DNA and acquire foreign DNA sequences. The overarching hypothesis for this proposal is that dynamic and DNA-sequence dependent formation of the Type I CRISPR machinery coordinate both DNA interference and immunity acquisition. Therefore, this proposal aims to determine 1) the mechanism of target recognition by the RNA-guided effector complex called Cascade, 2) how the Cas3 nuclease is recruited to the Cascade complex to initiate DNA interference, and 3) the interactions and functions of Cas1-Cas2 and Cas3 in primed spacer acquisition. This research uses an innovative, high-throughput single-molecule approach called DNA curtains to determine the transient and dynamic interactions of the CRISPR-Cas proteins on lambda phage DNA. Additionally, this proposal characterizes the Thermobifida fusca Type I-E system, a stable thermophilic organism, which contains proteins that are highly amenable to structural and biochemical applications. Completion of these aims will yield a comprehensive understanding of the Type I CRISPR adaptive immune system, including the mechanism of Cas1-Cas2 recruitment during immunity acquisition. Ultimately, this proposal will shed light on how bacteria maintain their genomic integrity by protecting themselves from bacteriophages, and will provide new insights into the CRISPR mechanisms that are rapidly advancing genetic engineering and promising gene therapy techniques.