Live-cell single-particle tracking photoactivated localization microscopy of Cascade-mediated DNA surveillance.

Abstract

Type I CRISPR-Cas systems utilize small CRISPR RNA (crRNA) molecules to scan DNA strands for target regions. Different crRNAs are bound by several CRISPR-associated (Cas) protein subunits that form the stable ribonucleoprotein complex Cascade. The Cascade-mediated DNA surveillance process requires a sufficient degree of base-complementarity between crRNA and target sequences and relies on the recognition of small DNA motifs, termed protospacer adjacent motifs. Recently, super-resolution microscopy and single-particle tracking methods have been developed to follow individual protein complexes in live cells. Here, we described how this technology can be adapted to visualize the DNA scanning process of Cascade assemblies in Escherichia coli cells. The activity of recombinant Type I-Fv Cascade complexes of Shewanella putrefaciens CN-32 serves as a model system that facilitates comparative studies for many of the diverse CRISPR-Cas systems.