A Scalable Epitope Tagging Approach for High Throughput ChIP-Seq Analysis

Abstract

Eukaryotic transcriptional factors (TFs) typically recognize short genomic sequences alone or together with other proteins to modulate gene expression. Mapping of TF-DNA interactions in the genome is crucial for understanding the gene regulatory programs in cells. While chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is commonly used for this purpose, its application is severely limited by the availability of suitable antibodies for TFs. To overcome this limitation, we developed an efficient and scalable strategy named cmChIP-Seq that combines the clustered regularly interspaced short palindromic repeats (CRISPR) technology with microhomology mediated end joining (MMEJ) to genetically engineer a TF with an epitope tag. We demonstrated the utility of this tool by applying it to four TFs in a human colorectal cancer cell line. The highly scalable procedure makes this strategy ideal for ChIP-Seq analysis of TFs in diverse species and cell types.

Keywords: CRISPR/Cas9; ChIP-Seq; FLAG tagging; genome engineering; microhomology mediated end joining.

Figure 1

Schematic depiction of the MMEJ-mediated TF tagging strategy named cmChIP-seq for high throughput ChIP-Seq analysis. Cells are transfected with plasmids containing the Cas9 nuclease, gRNAs, and epitope tag donor constructs, leading to the integration of the FLAG tag, 2A linker sequences, mNeonGreen and puromycin resistance gene at the 3′ end of the target transcription factor.

Figure 2

Genotyping analyses for SP1 monoclonal samples. (A) Junction sequencing results. The intended knocked-in sequence is shown at the top. Blue: microhomologous arm; Yellow: initial sequence of 3 × Flag; Green: inserted nucleotides. (B) PCR check for selected samples no. 1, 2, 3, 8. 3′ junction check used forward primer targeting puromycin and reverse primer targeting genomic region after the double-strand break. Genome check applied both primers targeting the genome. Triangular points refer to expected sized amplicons and the asterisk refers to wild type product.

Figure 3

(A) Representative DNA-binding protein read enrichment tracks on the Integrative Genomics Viewer (IGV) for SP1 monoclonal samples. (B) Motif analyses on the top 500 peaks of each SP1 monoclonal sample identified SP1 binding motif. MEME de novo software analyzed 500 top peaks with 100 bp surrounding the peak summit and gave all SP1 motif enrichment validation.

Figure 4

(A) Representative DNA-binding protein read enrichment tracks on the IGV for MYC and TCF7L2 monoclonal samples. (B) Top motif identified for TCF7L2 monoclonal samples matched known TCF7L2 motif. (C) Motif discovery for MYC monoclonal samples identified MYC motif as the top hit.

Figure 5

(A) Genotyping analyses for SP1 pooled cell samples. Whole cassette analysis used both primers annealing to genomic DNA and junction check involved one primer targeting genome and the other recognizing the insert. Triangular points refer to expected amplicon sizes with insertions and the asterisk refers to wild type product. (B) Representative DNA-binding protein read enrichment tracks on the IGV for SP1 pooled cell samples. (C) Motif discovery for SP1 pooled cell samples before fluorescence-activated cell sorting (FACS). (D) Motif discovery for SP1 pooled cell samples after FACS. (E) Read enrichment heatmap of the 3000 bp regions centered on the SP1 ChIP-Seq peaks generated by ENCODE. Each row is a peak and the x-axis denotes the whole 3000 bp genomic regions. Each column is a TF-tagged SP1 sample.

Figure 6

(A) Representative DNA-binding protein read enrichment tracks on the IGV for MYC and CTCF pooled cell samples. (B) Motif discovery for MYC pooled cell samples before FACS. (C) Read enrichment heatmap of the 3000 bp regions centered on the MYC peaks generated by Seitz and co-workers. Each row is a peak and the x-axis denotes the whole 3000 bp genomic regions. Each column is a TF-tagged MYC sample. (D) Motif discovery for CTCF pooled cell samples before FACS. (E) Read enrichment heatmap of the 3000 bp regions centered on the CTCF ChIP-Seq peaks generated by ENCODE. Each row is a peak and the x-axis denotes the whole 3000 bp genomic regions. Each column is a TF-tagged CTCF sample.