Project Summary Viral resistance is essential in all kingdoms of life, although diverse organisms have evolved equally diverse mechanisms for combatting infection. In bacteria and archaea, the CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system clears invading DNA during infection through a small-RNA guided interference mechanism. CRISPR immunity proceeds through two stages: adaptation, in which fragments of invasive DNA from bacteriophages or plasmids are inserted as spacers within the CRISPR locus of the host genome and subsequently serve as templates for the production of small guide CRISPR (cr)RNAs;; and interference, during which the crRNA and its effector CRISPR associated (Cas) proteins bind complementary target regions of the invading DNA, leading to its destruction by a Cas endonuclease. Our goal is to define how bacteria maximize their immune capacity to gain an advantage in the molecular arms race against their invaders. Our first goal is to understand the sequence-dependence of immune system evasion through the development of point mutations within the invading DNA. Our previous studies have revealed that spacer sequence greatly influences the effectiveness of these “escape” mutations, suggesting for the first time that some spacer sequences provide stronger immunity than others. In addition, we have discovered that during initial infection, bacteria use a two-tiered defensive system to broaden their adaptation capacity. We will evaluate the impact of this tactic on host immunity and elucidate the molecular mechanisms underlying this defense strategy. Finally, we will determine the structural basis for rapid adaptation triggered when the CRISPR machinery senses non- canonical target sequences. Our studies will have major implications on the understanding of host-virus interactions and co-evolution, an important determinant of the compositional dynamics within complex ecological systems including the human microbiome.