期刊
NATURE PROTOCOLS
卷 15, 期 5, 页码 1775-1799出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/s41596-020-0309-5
关键词
-
资金
- NOMIS Foundation
- Lotte and Adolf Hotz-Sprenger Stiftung
- Swiss National Science Foundation [310030_188858]
- Fanconi Anemia Research Foundation
- Bill and Melinda Gates Foundation
- NHMRC Early Career Fellowship
- Gladstone Institutes
- NIH [R01EY028249, R01-HL130533, R01-HL13535801, S10 OD018174]
- Li Ka Shing Foundation
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. 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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据