Single-Molecule Characterization of a Nanopore-Coupled Cas9 Protein on an Electrode Array

Nanopore sequencing technology is an emerging method for achieving long sequence reads on single DNA molecules without prior sample amplification. Detection involves changes in current across a membrane through a self-assembling protein nanopore complex. Each pore is associated with a single electrode within a complementary metal-oxide semiconductor (CMOS) array, enabling detection of single-molecule events. Extending these capabilities, we describe here a nanopore-based method to detect specific DNA molecules through binding to a Cas9:gRNA complex. Specifically, we generated a recombinant protein tool for the assembly of a functional Cas9 or dCas9 molecules on a nanopore array. To date, we have demonstrated that the construct is functional, recruits appropriately designed gRNA molecules, and binds to target DNA molecules while failing to bind non-target DNA. We believe that our Cas9-functionalized nanopore method may have utility in both basic research and clinical diagnostic applications by enabling single-molecule kinetic characterization of the enzyme, potentially offering novel insights into the mechanism of Cas9 catalytic cycle.