CRISPR off-target detection with DISCOVER-seq
- 20 April 2020
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature Protocols
- Vol. 15 (5), 1775-1799
- https://doi.org/10.1038/s41596-020-0309-5
Abstract
DISCOVER-seq (discovery of in situ Cas off-targets and verification by sequencing) is a broadly applicable approach for unbiased CRISPR–Cas off-target identification in cells and tissues. It leverages the recruitment of DNA repair factors to double-strand breaks (DSBs) after genome editing with CRISPR nucleases. Here, we describe a detailed experimental protocol and analysis pipeline with which to perform DISCOVER-seq. The principle of this method is to track the precise recruitment of MRE11 to DSBs by chromatin immunoprecipitation followed by next-generation sequencing. A customized open-source bioinformatics pipeline, BLENDER (blunt end finder), then identifies off-target sequences genome wide. DISCOVER-seq is capable of finding and measuring off-targets in primary cells and in situ. The two main advantages of DISCOVER-seq are (i) low false-positive rates because DNA repair enzyme binding is required for genome edits to occur and (ii) its applicability to a wide variety of systems, including patient-derived cells and animal models. The whole protocol, including the analysis, can be completed within 2 weeks.Funding Information
- NOMIS Stiftung
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (310030_188858)
- Fanconi Anemia Research Fund
- Bill and Melinda Gates Foundation
- Lotte and Adolf Hotz-Sprenger Stiftung
- Department of Health | National Health and Medical Research Council
- Li Ka Shing Foundation
- U.S. Department of Health & Human Services | National Institutes of Health (R01-EY028249, R01-HL130533, R01-HL13535801)
This publication has 29 references indexed in Scilit:
- Collateral damage: benchmarking off-target effects in genome editingGenome Biology, 2019
- Unbiased detection of CRISPR off-targets in vivo using DISCOVER-SeqScience, 2019
- Illuminating the genome-wide activity of genome editors for safe and effective therapeuticsGenome Biology, 2018
- A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cellsNature Medicine, 2018
- Enhanced proofreading governs CRISPR–Cas9 targeting accuracyNature, 2017
- High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effectsNature, 2016
- Rationally engineered Cas9 nucleases with improved specificityScience, 2016
- The MRE11 complex: starting from the endsNature Reviews Molecular Cell Biology, 2011
- Mre11–Rad50–Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin templateThis paper is one of a selection of papers published in this Special Issue, entitled 28th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process.Biochemistry and Cell Biology, 2007
- Molecular Mechanism of the Recruitment of NBS1/hMRE11/hRAD50 Complex to DNA Double-strand Breaks: NBS1 Binds to γ-H2AX through FHA/BRCT DomainJournal of Radiation Research, 2004