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

Area of Science:

• Molecular Biology
• Genomics
• Epigenetics

Background:

• Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is vital for mapping transcription factor (TF) binding sites genome-wide.
• A major limitation of ChIP-seq is the scarcity of high-quality antibodies for many TFs, hindering comprehensive analysis.
• Existing ChIP-seq protocols often yield unreliable data due to antibody quality issues.

Purpose of the Study:

• To develop a robust ChIP-seq methodology that circumvents the need for specific TF antibodies.
• To introduce CRISPR epitope tagging ChIP-seq (CETCh-seq) as a reliable alternative for mapping DNA-binding protein interactions.
• To validate CETCh-seq across different cell types and expression levels.

Main Methods:

• Utilized CRISPR/Cas9 genome editing to create epitope-tagged endogenous DNA-binding proteins.
• Performed CETCh-seq on HepG2 (liver cancer) cells, tagging multiple TFs with varying expression levels.
• Validated CETCh-seq results by comparing replicate data and standard ChIP-seq using TF antibodies.

Main Results:

• CETCh-seq demonstrated strong correlations between biological replicates and with conventional ChIP-seq.
• The technique showed minimal impact on cellular transcriptome and tagged TF expression.
• Successful application of CETCh-seq in MCF7 (breast cancer) cells and mouse embryonic stem cells confirmed its robustness.

Conclusions:

• CETCh-seq provides a reliable and straightforward method for accurately defining genome-wide binding profiles of DNA-binding proteins.
• This approach significantly expands the scope of ChIP-seq analysis, particularly for TFs lacking suitable antibodies.
• CETCh-seq facilitates the study of a broader range of TFs, potentially covering the entire TF repertoire.