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

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 ChIPseq 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.

Decoding transcriptional enhancers: Evolving from annotation to functional interpretation

“…For instance, ChIP-seq relies on the availability of suitable ChIP-seq grade antibodies [152]; current estimates from the ENCODE Project indicate that less than 10% of commercial antibodies are sufficiently specific or efficacious enough to be useful in ChIP-seq assays (our unpublished observations). To circumvent these problems, epitope tagging techniques have been applied where diverse DNA-binding proteins can be annotated using a single, high-quality antibody [153]. To further combat the difficulties of working with primary tissues, alternative preparation strategies have been used for downstream ChIP-seq characterizations [154].…”

CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins

“…We further note the presence of tag-specific background signal when anti-tag peptides are used in wild-type cells, indicating that paired control experiments in which anti-tag antibody is used in wild-type cells is an essential control to such TAG-eCLIP experiments. These results should aid in the design and implementation of eCLIP experiments, particularly for poorly characterized RBPs, in the same way that validation of peptide tag usage for DNA binding proteins provided a boost to the study of transcription factors (19). …”

“…A recent method to perform endogenous tagging followed by ChIP-seq (CETCh-seq) demonstrated successful use of the CRISPR-Cas9 system to introduce a 3xFLAG tag at the 3′ end of transcription factors (19). Specifically, ChIP-seq using the FLAG tag yielded substantially similar binding site identification to parallel experiments performed with antibodies targeting native proteins, confirming this approach as a general scheme for profiling DNA binding proteins lacking antibodies.…”

Gene editing (CRISPR-Cas) technology and fisheries sector

“…Here, the donor vector stimulates DNA repair through homologous recombination pathway [31]. This method has been used successfully for multiple applications [32] including gene knock out, delivery of therapeutic genes [15,33] or for tagging endogenous proteins [34][35][36].…”