Project A - Structural Studies of Type III CRISPR-Cas Surveillance Complexes Jennifer Doudna PROJECT SUMMARY/ABSTRACT CRISPR-Cas systems provide bacteria and archaea with adaptive immunity to viruses and plasmids using RNA-guided degradation of target sequences. Among the three CRISPR-Cas systems that have been identified, the Type III systems may be uniquely capable of both RNA and DNA target binding and cleavage. We aim to determine the molecular mechanisms by which Type III CRISPR-Cas systems recognize nucleic acids and trigger site-specific cleavage. The results of this project will both provide fundamental insights into CRISPR-Cas biology and enable application of these systems for programmable target modification or elimination. The Type III CRISPR-Cas surveillance complexes are stable and structurally homogeneous, making them particularly well-suited to study by electron microscopy. At the same time, these complexes are relatively small (~350?400 kDa) and can bind to different kinds of substrate sequences, necessitating use of state-of-the-art EM methods and development of new strategies for imaging and analyzing single particles. Thus, this project will benefit enormously from the overall Program and the insights to come from the Core and associated Projects. The project will focus on two types of RNA-protein complexes that constitute the nucleic acid targeting machinery of the Type III CRISPR systems. In Type III-A CRISPR-Cas systems, the Csm complex comprising five protein subunits and a CRISPR RNA (crRNA) finds and degrades target nucleic acids. To determine how RNA versus DNA substrates are identified and degraded, we will extend preliminary studies using electron microscopy to solve Csm complex structures and to define how target sequences are cleaved in a site-specific manner. In Type III-B CRISPR-Cas systems, the Cmr complex containing six distinct subunit proteins and a crRNA locates and cleaves target sequences that can base pair with the crRNA. Although functionally analogous to RNA-induced silencing complexes (RISCs) that operate during RNA interference in eukaryotic cells, the Cmr complex comprises entirely distinct components and operates by an unknown mechanism. We will continue ongoing studies to determine the molecular structure of the Cmr complex and to understand how it cleaves target sequences in a site-specific manner.