Understanding the Molecular Mechanisms of the CRISPR Toolbox Using Single Molecule Approaches

Adaptive immunity against foreign genetic elements conferred by the CRISPR systems in microbial species has been repurposed as a revolutionary technology for wide-ranging biological applications-chiefly genome engineering. Biochemical, structural, genetic, and genomics studies have revealed important insights into their function and mechanisms, but most ensemble studies cannot observe structural changes of these molecules during their function and are often blind to key reaction intermediates. Here, we review the use of single molecule approaches such as fluorescent particle tracking, FRET, magnetic tweezers, and atomic force microscopy imaging in improving our understanding of the CRISPR toolbox.