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

Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is a high throughput sequencing technique to detect the protein-DNA binding events and chemical modifications of histone proteins. Followed by recognition and direct sequencing, more comprehensive sequence information can be obtained in a high throughput fashion. The quality of any ChIP data is highly dependent on antibodies. Specific antibodies are required for enrichment and the increase of signal contrast over background. However, commercial antibodies for TFs in human genome are very limited. To overcome this major limitation, researchers have utilized an epitope tag to create a fusion protein through plasmid overexpression or bacterial artificial chromosome integration for tagged gene expression and allow the tracking of a TF with an antibody against the tag sequence. We have developed a new method to directly modify endogenous genomic sequence and enable fusion protein expression in the native context for ChIP-seq analysis. Specifically, we combine the clustered regularly interspaced short palindromic repeats (CRISPRs) technology with microhomology mediated end joining (MMEJ) to insert an 3×FLAG-tag with screening markers to the ends of TFs. Current strategies for chromosomal integration typically rely on CRISPR mediated integration vis homologous recombination (HR) or non-homologous end joining (NHEJ). Scaling HR mediated integration on human genome is less feasible; and the reported accurate NHEJ integration efficiency is low. MMEJ provides novel cellular repair mechanism with more precise integration over NHEJ. Using the CRISPR-MMEJ mediated tagging approach, we have enabled the ChIP-seq analysis towards both tagged monoclones and polyclones for three TFs including SP1, MYC and TCF7L2. Our method leverages the scalability of the knock-in process with easy donor construction in contrast to previously reported homologous recombination mediated strategy. We envision the power of our strategy to improve and broaden ChIP-seq technology on mapping the genomes of human beings and any other organisms on a genome-scale.