Nanopore assay for fingerprinting DNA binding and quantifying real-time cleavage by catalytically active Cas9 enzyme

Abstract

Nanopore sensing, a high-resolution DNA sequencing technology, is rapidly expanding into novel and exciting directions of probing specific DNA-enzyme interactions. Although proven excellent for the detecting structural features of bare DNA, quantitative measurements on enzyme-DNA complexes and their real-time activity are lagging and only starting to emerge for long DNA templates. Signal-to-noise requirement and high translocation speeds make it difficult to detect protein bound on biologically relevant plasmid-length DNA. To this end we report accurate position detection of a catalytically active Cas9 bound to its single or multiple target sites on the DNA. Protein position is fingerprinted using event charge deficit (ECD) based analysis of the high signal-to-noise electrical signals as the complex translocates through a glass nanopore. Using a time-dependent assay, we quantify the kinetics of the released products upon enzymatic cleavage of the target DNA by the wild-type Cas9 nuclease. Our approach enables the nanopore-based single-molecule sensing of DNA-protein complexes, for real-time monitoring of biochemical reactions. This may help understand protein binding & localization as well as improve Cas9-based targeting in genome engineering applications.