The discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems led to creative new applications that are transforming science and medicine. However, the rapid discovery of new CRISPR systems outpaces our understanding of their biological roles in anti-phage defense and their development for novel applications. To acquire immunity to new phages, CRISPR-associated proteins (Cas1 and Cas2) integrate fragments of phage DNA ("spacers") at the "leader-end" of the CRISPR locus, near the transcription start site. But how Cas1-2 recognizes the leader-end of the CRISPR remains poorly understood. Next, the CRISPR locus is transcribed and processed into "guide RNAs" that are loaded into surveillance complexes (i.e., Csm complex). Upon sensing viral RNA, the Csm complex makes cyclic oligonucleotide messengers that regulate CRISPR adaptation and nucleases critical for phage defense. But the biological roles of many of these immune effectors remain understudied. The 1961 discovery of regulatory DNA motifs kindled an interest in the "grammatical rules" of DNA motifs that control the storage and retrieval of genetic information. In Aim 1, I will use a bioinformatic approach to discover DNA motifs in CRISPR leaders with highly conserved sequences and positions, that I hypothesize regulate key steps in CRISPR biology such as integration, transcription, or RNA processing. I will determine the role of novel DNA motifs and host factors in CRISPR integration using in vitro biochemical assays and cryo-EM structural biology. Nucleotide messengers regulate critical cellular processes across the tree of life, including anti-viral immune responses, cell morphology, and motility. To address a growing need for rapid and sensitive diagnostics, I co-invented an innovative RNA-guided Csm system for sensitive and sequence-specific detection of SARS-CoV-2 RNA, that repurposes a nuclease immune effector. However, nucleases represent a fraction of the diversity of enzymatic activities predicted to be activated by nucleotide messengers. In Aim 2, I will determine a biochemical and structural understanding of novel immune proteins that I predict to be activated by CRISPR-generated nucleotide messengers.The long-term objectives of this proposal are to address knowledge gaps in our understanding of how bacterial CRISPR adaptive immune systems function and are regulated, and to repurpose basic mechanistic insights for the development of biotech and medical applications.