Solid-State Nanopore Real Time Assay for Monitoring Cas9 Endonuclease Reactivity

Abstract

The field of nanopore sensing is now moving beyond nucleic acid sequencing. An exciting new avenue is the use of nanopore platforms for the monitoring of biochemical reactions. Biological nanopores have been used to investigate the dynamics of enzymatic reactions but solid-state nanopore approaches have lagged. This is partially due to the necessity of using higher salt conditions (e.g. 4M LiCl) to improve the signal-to-noise ratio. These conditions are often incompatible with monitoring biological activities such as restriction enzyme and CRISPR-Cas9 endonuclease activities, as their abilities to digest DNA are largely abolished under high salt conditions. We pioneered a polymer electrolyte based solid-state nanopore approach that can maintain a high signal-to-noise ratio even at a physiologically relevant salt concentration. Based on this, we report monitoring the endonuclease activities at physiological salt conditions in real time with a polymer electrolyte nanopore. We successfully monitor the dsDNA cleavage activity of the restriction enzyme SwaI in in a range of digestion buffers and the off-target activity of CRISPR-Cas9 RNP endonuclease in the presence of single base pair mismatches. This approach enables solid-state nanopore technology for the dynamic monitoring of biochemical reactions at single molecule resolution.