Project Summary The proposed research combines biophysics, molecular genetics, and genomics tools to provide high-resolution mechanisms for RNA-dominated biological processes. Our study focuses on two central themes in RNA biology. (1) RNA can serve as an information carrier to guide the action of proteins. Such systems are inherently precise and programmable, therefore carry great potential in biotechnology and medicine, as exemplified by RNAi and CRISPR-Cas. My lab spent more than ten years dissecting the mechanisms of CRISPR-Cas, and more recently has been developing genome editing applications based on our mechanistic insights. (2) RNA can adopt defined tertiary structures to exert important functions. My lab has elucidated the structure, function, and conformation dynamics of many riboswitches. The high-resolution information may enable new antibiotics development. We now aim to combine genetic screen with biochemical purification to systematically identify ligands for orphan riboswitches. Such studies often reveal novel aspects of bacterial physiology, which may even be conserved in eukaryotes. We propose to work on the following four areas of research: 1) Structure, mechanism, and genome editing applications of Type I CRISPR (CRISPR-Cas3); 2) Mechanism of CRISPR immunity establishment by Cas1-Cas2 integrase; 3) Mechanism and genome editing application of CRISPR-Tn7; 4) Dissecting riboswitch- controlled mechanism and bacterial physiology.