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

## TL;DR

This paper introduces a nanopore-based method to detect and quantify the activity of Cas9 enzymes on DNA in real time.

## Contribution

The study presents a novel nanopore assay for fingerprinting DNA binding and measuring real-time cleavage by active Cas9.

## Key findings

- The method accurately detects the position of Cas9 on DNA using event charge deficit analysis.
- It quantifies the kinetics of DNA cleavage by wild-type Cas9 in real time.
- The approach enables single-molecule sensing of DNA-protein interactions for biochemical monitoring.

## 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.

The online version contains supplementary material available at 10.1186/s12951-025-03837-6.