Probing the Conformational Dynamics of VeCas9 Using High-Resolution Single-Molecule Junctions

Abstract Gene editing enables precise genetic modifications, revolutionizing disease treatment, crop improvement, and biotechnological innovation. VeCas9, a recently identified CRISPR endonuclease from the Veillonella genus, exhibits a broader protospacer adjacent motif recognition range in comparison with the widely used SpCas9 nuclease. However, its working mechanism and potential applications in gene editing still need to be fully characterized. In this work, by integrating VeCas9 into a graphene-molecule-graphene single-molecule junction platform, we carried out label-free, in situ mechanistic studies of VeCas9 function at the single-molecule level with high temporal resolution. Using this setup, we monitored the dynamic interactions between VeCas9, single-guide RNA, and double-stranded DNA in real time, in both stable and transient binding modes. Through temperature-dependent measurements, we determined the kinetic parameters of key steps in the interaction process. Moreover, a series of double-stranded DNA substrates was employed to examine how various sequence mismatches affect the VeCas9 activity, revealing that its sensitivity depends on the position of the mismatch. More importantly, we directly observed VeCas9-mediated R-loop formation and quantified the time scale of single-base expansion events. These single-molecule observations provide mechanistic insights into the Cas9 function and demonstrate the strong potential of high temporal-resolution single-molecule techniques for studying dynamic biological processes.