CRISPR off-target detection with DISCOVER-seq
| Autor: | Beeke Wienert, Stacia K. Wyman, Charles D. Yeh, Bruce R. Conklin, Jacob E. Corn |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
DNA Repair
Computer science Sequence analysis DNA repair Bioinformatics genetic processes Computational biology Genome Medical and Health Sciences Article General Biochemistry Genetics and Molecular Biology 03 medical and health sciences chemistry.chemical_compound Mice Double-Stranded 0302 clinical medicine Genome editing Genetics CRISPR Animals Humans DNA Breaks Double-Stranded Clustered Regularly Interspaced Short Palindromic Repeats 030304 developmental biology Gene Editing 0303 health sciences Targeted Gene Repair DNA Breaks Human Genome Sequence Analysis DNA DNA Biological Sciences Stem Cell Research Emerging Infectious Diseases chemistry Chemical Sciences CRISPR-Cas Systems K562 Cells Chromatin immunoprecipitation Sequence Analysis 030217 neurology & neurosurgery |
| Zdroj: | Nature Protocols, 15 (5) Nature protocols, vol 15, iss 5 Nat Protoc |
| Popis: | 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. The authors describe DISCOVER-seq, a method to detect off-targets of CRISPR–Cas genome editing based on ChIP-seq analysis of MRE11 recruitment to DSBs, and subsequent bioinformatics analysis of sequencing data using the BLENDER pipeline. |
| Databáze: | OpenAIRE |
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