Since its discovery, the CRISPR–Cas9 system has been in the center of attention for its promising applications in genome editing. However, in order to apply this system successfully in genetic engineering, all aspects of its molecular mechanism have to be well understood. One of the best ways to investigate the intricacies of the molecular mechanism is single-molecule studies as they allow real-time observation of the kinetic processes and provide high spatiotemporal resolution. This chapter describes single-molecule fluorescence resonance energy transfer experiments carried out to investigate the Cas9 protein from Streptococcus pyogenes, providing information on the effects of target truncation, Cas9-induced double-stranded DNA dynamics, and target search.
- Target search
- TIRF microscopy