CRISPR−Cas9 Mediated DNA Unwinding Detected Using Site-Directed Spin Labeling and Molecular Dynamics*

The RNA-guided CRISPR−associated Cas9 has revolutionized genome engineering, yet its mechanism for DNA target selection is not fully understood. A crucial step in Cas9 target recognition involves unwinding of the DNA duplex. Our work demonstrates direct detection of Cas9-mediated DNA unwinding by a combination of site-directed spin labeling experiments and molecular dynamics simulations. The results support a model in which the unwound nontarget strand is stabilized by a positively charged patch between the two nuclease domains of Cas9 and reveal uneven increase in flexibility along the unwound nontarget strand upon scissions of the DNA backbone. Analysis of Cas9 with mutations along the positive patch reveal the role of this protein domain in specificity. This work establishes the synergistic combination of spin-labeling and molecular dynamics to directly monitor Cas9-mediated DNA conformational changes and yields information on the target DNA in different stages of Cas9 function, thus advancing mechanistic understanding of CRISPR−Cas9 and aiding future technological development.