Project Summary Clustered regularly interspaced short palindromic repeats (CRISPRs) are a recently discovered RNA-based adaptive immune system that protects bacteria and archaea from foreign DNA. CRISPRs and the associated (Cas) proteins have been identified in 90% of archaea and 40% of bacteria, including many human pathogens. These immune systems also play a central role in controlling the horizontal transfer of virulence genes. The central step in CRISPR-Cas adaptive immunity is degradation of foreign DNA by a programmable RNA-guided nuclease. CRISPR-cas nucleases—enzymes that cleave a target DNA or RNA that is complementary to a guide CRISPR-RNA (crRNA)—have also been repurposed for precision gene regulation in many organisms. Despite the intense interest in these enzymes for both research and clinical applications, we still lack a complete understanding of their functions. Our long-term goal is to understand the mechanisms of CRISPR- mediated adaptive immunity. A related goal is to engineer improved CRISPR-associated enzymes for gene regulation and other biotechnological applications. The aims described in this proposal will mechanistically dissect a newly discovered family of RNA-guided nucleases. To achieve these aims, we pioneered high- throughput microscopy techniques that can image multiple enzymes and record their biochemical activities on tens of thousands of distinct DNA substrates. Using an interdisciplinary approach that integrates biophysics, bioinformatics, and micro-/nano-scale engineering, we will investigate how a CRISPR-associated RNA-guided nuclease recognizes and cleaves a target DNA. First, we will determine how the nuclease finds and cleaves a target DNA in the context of chromatin. Second, we will determine the biophysical mechanisms governing DNA binding and cleavage at off-target sites that resemble the on-target sequence. These off-target activities can cause unanticipated mutations that confound research experiments and limit therapeutic applications. Finally, we will engineer and profile novel high fidelity enzymes that reduce off-target activities and expand the genome engineering toolkit. CRISPR-mediated gene silencing and gene editing offers an exciting avenue for genetic manipulation of eukaryotic cells. We anticipate that our results will offer new insights in understanding the mechanisms of CRISPR-associated adaptive immunity and for using these enzymes for precision genome engineering in both scientific and future therapeutic settings.