Chau, C.C.C. orcid.org/0000-0002-3134-6798, Weckman, N.E. orcid.org/0000-0001-5894-4926, Thomson, E.E. orcid.org/0000-0002-0266-7090 et al. (1 more author) (2025) Solid-state nanopore real-time assay for monitoring Cas9 endonuclease reactivity. ACS Nano, 19 (3). pp. 3839-3851. ISSN 1936-0851
Abstract
The field of nanopore sensing is now moving beyond nucleic acid sequencing. An exciting avenue is the use of nanopore platforms for the monitoring of biochemical reactions. Biological nanopores have been used for this application, but solid-state nanopore approaches have lagged. This is due to the necessity of using higher salt conditions (e.g., 4 M LiCl) to improve the signal-to-noise ratio which completely abolish the activities of many biochemical reactions. We pioneered a polymer electrolyte solid-state nanopore approach that maintains a high signal-to-noise ratio even at a physiologically relevant salt concentration. Here, we report the monitoring of the restriction enzyme SwaI and CRISPR-Cas9 endonuclease activities under physiological salt conditions and in real time. We investigated the dsDNA cleavage activity of these enzymes in a range of digestion buffers and elucidated the off-target activity of CRISPR-Cas9 ribonucleoprotein endonuclease in the presence of single base pair mismatches. This approach enables the application of solid-state nanopores for the dynamic monitoring of biochemical reactions under physiological salt conditions.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2025 The Authors. Published by American Chemical Society. This publication is licensed under CC-BY 4.0. (https://creativecommons.org/licenses/by/4.0/) |
Keywords: | CRISPR; endonuclease; nanopore; polymer electrolyte; single molecule |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) |
Funding Information: | Funder Grant number BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL BB/X003086/1 |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 30 Jan 2025 17:25 |
Last Modified: | 30 Jan 2025 17:25 |
Status: | Published |
Publisher: | American Chemical Society (ACS) |
Refereed: | Yes |
Identification Number: | 10.1021/acsnano.4c15173 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:222545 |