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Genome Res. 2015 Oct;25(10):1581-9. doi: 10.1101/gr.193540.115. Epub 2015 Sep 9.

CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins.

Author information

1
HudsonAlpha Institute for Biotechnology, Huntsville, Alabama 35806, USA;
2
University of Alabama in Huntsville, Huntsville, Alabama 35899, USA.
3
HudsonAlpha Institute for Biotechnology, Huntsville, Alabama 35806, USA; University of Alabama in Huntsville, Huntsville, Alabama 35899, USA.

Abstract

Chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-seq) is a widely used technique for identifying transcription factor (TF) binding events throughout an entire genome. However, ChIP-seq is limited by the availability of suitable ChIP-seq grade antibodies, and the vast majority of commercially available antibodies fail to generate usable data sets. To ameliorate these technical obstacles, we present a robust methodological approach for performing ChIP-seq through epitope tagging of endogenous TFs. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing technology to develop CRISPR epitope tagging ChIP-seq (CETCh-seq) of DNA-binding proteins. We assessed the feasibility of CETCh-seq by tagging several DNA-binding proteins spanning a wide range of endogenous expression levels in the hepatocellular carcinoma cell line HepG2. Our data exhibit strong correlations between both replicate types as well as with standard ChIP-seq approaches that use TF antibodies. Notably, we also observed minimal changes to the cellular transcriptome and to the expression of the tagged TF. To examine the robustness of our technique, we further performed CETCh-seq in the breast adenocarcinoma cell line MCF7 as well as mouse embryonic stem cells and observed similarly high correlations. Collectively, these data highlight the applicability of CETCh-seq to accurately define the genome-wide binding profiles of DNA-binding proteins, allowing for a straightforward methodology to potentially assay the complete repertoire of TFs, including the large fraction for which ChIP-quality antibodies are not available.

PMID:
26355004
PMCID:
PMC4579343
DOI:
10.1101/gr.193540.115
[Indexed for MEDLINE]
Free PMC Article

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