ENDO-Pore: High-throughput Linked-end Mapping of Single DNA Cleavage Events Using Nanopore Sequencing

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2021 Aug 21

PMID 34417616

Citations 1

Authors

Oscar E Torres Montaguth

Stephen J Cross

Kincaid W A Ingram

Laura Lee

Fiona M Diffin

Mark D Szczelkun

Affiliations

Soon will be listed here.

Abstract

Mapping the precise position of DNA cleavage events plays a key role in determining the mechanism and function of endonucleases. ENDO-Pore is a high-throughput nanopore-based method that allows the time resolved mapping single molecule DNA cleavage events in vitro. Following linearisation of a circular DNA substrate by the endonuclease, a resistance cassette is ligated recording the position of the cleavage event. A library of single cleavage events is constructed and subjected to rolling circle amplification to generate concatemers. These are sequenced and used to produce accurate consensus sequences. To identify the cleavage site(s), we developed CSI (Cleavage Site Investigator). CSI recognizes the ends of the cassette ligated into the cleaved substrate and triangulates the position of the dsDNA break. We firstly benchmarked ENDO-Pore using Type II restriction endonucleases. Secondly, we analysed the effect of crRNA length on the cleavage pattern of CRISPR Cas12a. Finally, we mapped the time-resolved DNA cleavage by the Type ISP restriction endonuclease LlaGI that introduces random double-strand breaks into its DNA substrates.

Citing Articles

Comparison of Nanopore with Illumina Whole Genome Assemblies of the Epstein-Barr Virus in Burkitt Lymphoma.

Kim I, Kim Jr I, Fola A, Puig E, Maina T, Hui S medRxiv. 2025; .

PMID: 40061313 PMC: 11888525. DOI: 10.1101/2025.02.21.25322471.

CRISPR-Cas12a-mediated DNA clamping triggers target-strand cleavage.

Naqvi M, Lee L, Montaguth O, Diffin F, Szczelkun M Nat Chem Biol. 2022; 18(9):1014-1022.

PMID: 35836018 PMC: 9395263. DOI: 10.1038/s41589-022-01082-8.

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