Mechanisms of bacterial CRISPR immunity and a phage counter-defense

Bacteriophages are constantly evolving to overcome their bacterial hosts. To counteract these attacks, bacteria developed multiple defense systems. One critical line of defense is the adaptive immune system CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). Upon infection, the bacterial host uptakes a genetic memory from the bacteriophage to incorporate into its genome. This molecular memory is used to target CRISPR-associated (Cas) proteins to destroy the phage. In response, phages evolved anti-CRISPR proteins (Acrs) to inactivate this bacterial immune system. Since Cas nucleases can be programmed to target and cut specific genetic material, these molecular scissors have become a valuable biotechnological and genome editing tool. Acrs have the potential to be used to regulate CRISPR-Cas activity in both bacteria and human cells. Thus, investigating the mechanisms of CRISPR and anti-CRISPR proteins gives insight into bacteria-phage dynamics and drives the development of new biotechnological tools for applications such as genome editing and gene regulation. Here, we investigate two key steps of CRISPR-Cas adaptive immunity, interference and primed acquisition, using a high-throughput single-molecule assay (Chapter 2). We also detail a protocol for fluorescently tagging CRISPR-Cas protein subunits within a complex using a sortase-mediated labeling strategy (Chapter 3). Finally, we determine how the anti-CRISPR protein AcrIIA11 inhibits diverse Cas9 orthologs (Chapter 4).