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. 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

Daniel Savic  1 , E Christopher Partridge  1 , Kimberly M Newberry  1 , Sophia B Smith  2 , Sarah K Meadows  1 , Brian S Roberts  1 , Mark Mackiewicz  1 , Eric M Mendenhall  3 , Richard M Myers  1
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CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins

Daniel Savic et al. Genome Res. .
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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.

Figures

Figure 1.
Overview of CETCh-seq experimental method. (A) A schematic of the CETCh-seq approach is displayed. Cells are transfected with plasmids containing the Cas9 nuclease, gRNAs, and epitope tag donor constructs, leading to the homologous integration of the Flag tag, P2A linker sequence, and neomycin resistance gene at the 3′ end of the transcription factor in place of the endogenous transcription factor stop codon. Flag-tagged transcription factor and neomycin resistance genes are cotranscribed. Subsequently, a tagged transcription factor and a neomycin resistance protein are generated due to the P2A linker sequence. Cells are selected and colony-forming units are expanded for ChIP-seq experimentation using a Flag antibody. (B) HepG2 DNA-binding protein read enrichment tracks on the UCSC Genome Browser are given. The names of transcription factors are given. WT denotes transcription factor antibody experiments, while Flag Rep 1 and Flag Rep 2 are technical replicates using Flag antibodies for CETCh-seq experiments.
Figure 2.
HepG2 ChIP-seq reproducibility and gene expression analysis. (A) PCR validation of ATF1 homologous recombination. From left to right, the gel image displays ladder, 5′ end, and 3′ end homology PCR products. Ladder band sizes are given at the left. Correct homologous recombination generates 1626-bp (5′ end) and 1433-bp (3′ end) gel bands. (B) IP Western blot validation of epitope-tagged ATF1. An ∼40-kDa protein band (marked by red arrow) corresponding to the predicted size of ATF1 is visible in IP Western blots using a Flag antibody but is absent in a control IgG pulldown. (C) DNA-binding protein read enrichment tracks on the UCSC Genome Browser are shown at distinct genetic loci. Data are given for both CETCh-seq (Tag, lower panels) and standard ChIP-seq using transcription factor antibodies (Ctrl, upper panels). The transcription factor name is displayed at the left of each image. For ATF1, technical CETCh-seq replicates are displayed (Rep1 and Rep2). (D) RAD21 rank correlations of normalized sequence read counts between CRISPR-modified HepG2 cells (Modified cells) and wild-type HepG2 cells (WT), both using a RAD21 antibody (top). (Bottom) RAD21 rank correlations of normalized sequence read counts between CRISPR-modified HepG2 cells using a RAD21 TF antibody (Modified cells) and CETCh-seq results of tagged RAD21 using Flag antibodies (Flag). Average rank correlations for all Flag and RAD21 replicate pairwise comparisons are given in the top left corner of each plot. (E) RNA-seq gene RPKM comparisons between CRISPR-modified HepG2 cells (Modified cells RPKM) and wild-type HepG2 cells (WT RPKM) are plotted for RAD21 (top) and ATF1 (bottom) experiments. Rank correlations and the location of tagged transcription factor RPKM values on each graph are displayed.
Figure 3.
MCF7 ChIP-seq reproducibility and gene expression analysis. (A) PCR validation of RAD21 homologous recombination. From left to right, the gel image displays ladder, 5′ end, and 3′ end homology PCR products. Ladder band sizes are given at the left. Correct homologous recombination generates 1363-bp (5′) and 2278-bp (3′) gel bands. (B) Western blot validation of epitope-tagged RAD21. An ∼130-kDa protein band (marked by red arrow) corresponding to the predicted full-length size of RAD21 is visible in Western blots using a Flag antibody in CRISPR-modified cells but is absent in wild-type HepG2 cells. (C) DNA-binding protein read enrichment tracks on the UCSC Genome Browser are shown at distinct genome distance intervals. Data is given for CETCh-seq RAD21 biological replicates (BR1 and BR2, lower panels) and standard ChIP-seq using a RAD21 antibody (Ctrl, upper panels). (D) MCF7 RAD21 rank correlations of normalized sequence read counts between wild-type MCF7 cells (WT) using a RAD21 antibody and CETCh-seq Flag-tagged RAD21 (Flag) data sets (top). (Bottom) MCF7 RAD21 rank correlations of normalized sequence read counts between CETCh-seq biological replicates (Flag BR1 and Flag BR2). Average rank correlation values for all Flag replicate pairwise comparisons are given in the top left corner of each plot.
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