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CRISPR off-target detection with DISCOVER-seq.
Journal article

CRISPR off-target detection with DISCOVER-seq.

  • Wienert B Innovative Genomics Institute, University of California, Berkeley, Berkeley, California, USA. beeke.wienert@gladstone.ucsf.edu.
  • Wyman SK Innovative Genomics Institute, University of California, Berkeley, Berkeley, California, USA.
  • Yeh CD Department of Biology, ETH Zurich, Zurich, Switzerland.
  • Conklin BR Innovative Genomics Institute, University of California, Berkeley, Berkeley, California, USA.
  • Corn JE Department of Biology, ETH Zurich, Zurich, Switzerland. jacob.corn@biol.ethz.ch.
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  • 2020-04-22
Published in:
  • Nature protocols. - 2020
Animals
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
DNA Breaks, Double-Stranded
DNA Repair
Gene Editing
Humans
K562 Cells
Mice
Sequence Analysis, DNA
English 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.
Language
  • English
Open access status
green
Identifiers
Persistent URL
https://sonar.ch/global/documents/3077
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