PROJECT SUMMARY/ABSTRACT 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. My preliminary bioinformatics analysis of CRISPR leaders (upstream DNA) has revealed subtype- and microbe-specific motifs. These conserved motifs are found in a tight distribution of positions relative to the leader-CRISPR junction, and positional variants are shifted by a helical turn of DNA that would preserve the presentation of these motifs on the same "face" of DNA. In Aim 1, I will determine the structures of biochemically trapped CRISPR integration complexes to determine the roles of leader motifs and host proteins in regulating integration. In Aim 2, I will determine a biochemical and structural understanding of immune proteins that I predict to be activated by CRISPR-generated nucleotide messengers. This tailored application will support my transition to an independent academic position through: i) Structural biology training, ii) Mentoring, communication, and grant writing training, iii) Identification of novel CRISPR adaptation regulatory motifs and, iv) Identification of novel immune effectors. I will take formal cryo-EM coursework provided at Montana State 'University's (MSUs) cryo-EM facility by co-advisor Dr. Lawrence and co-mentor Dr. Lander, supplemented by coursework at NIH's National Center for Cryo-EM Access and Training. I will further develop my mentoring, communication, and grant writing skills through coursework provided by 'MSU's Center for Faculty excellence and by ASBMB's UE5 component. I have recruited a mentoring and advisory committee of six scientists with complementary expertise in CRISPR biology, the development of CRISPR-based applications, cryo-EM methodology, virology, and infectious diseases - relevant to guiding me as I pursue my research and career goals. The proposed project provides me with training in cryo-EM structural biology. It provides me with foundational bioinformatic and biochemical data that will serve as a springboard for my independent lab's interdisciplinary research on CRISPR biology and its appl...