Implementation of single molecule FRET for visualizing intramolecular movement in CRISPR-Cas9

Abstract

SHORT ABSTRACTThis paper summarizes how to visualize the flexible inter-domain movements of CRISPR-associated protein Cas9 using single molecule FRET LONG ABSTRACTThe CRISPR-associated protein Cas9 is widely used as a genome editing tool because of its ability to be programmed to cleave any DNA sequence that is followed by a protospacer adjacent motif. The continuing expansion of Cas9 technologies has stimulated studies regarding the molecular basis of the Cas9 catalytic process. Here we summarize methods for single molecule FRET (smFRET) to visualize the inter-domain movements of Cas9 protein. Our measurements and analysis demonstrate flexible and reversible movements of the Cas9 domains. Such flexible movements allow Cas9 to adopt transient conformations beyond those solved by crystal structures and play important roles in the Cas9 catalytic process. In addition to the smFRET measurement itself, to obtain precise results, it is necessary to validate Cas9 catalytic activity. Also, fluorescence anisotropy data are required to interpret smFRET data properly. Thus, in this paper, we describe the details of these important additional experiments for smFRET measurements.