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Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

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Published on: June 3, 2017

Epitope Tagging ChIP-Seq of DNA Binding Proteins Using CETCh-Seq.

Sarah K Meadows¹, Laurel A Brandsmeier¹, Kimberly M Newberry¹

¹HudsonAlpha Institute for Biotechnology, Huntsville, AL, USA.

Methods in Molecular Biology (Clifton, N.J.)

|January 22, 2020

Summary

This summary is machine-generated.

Chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-seq) is limited by antibody availability. CRISPR/Cas9-mediated epitope tagging (CETCh-seq) enables ChIP-seq for transcription factors lacking suitable antibodies, expanding genomic analysis capabilities.

Keywords:

CETCh-seq CRISPR ChIP-seq Epitope tagging Gene regulation Genomics Transcription factors

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Area of Science:

Molecular Biology
Genomics
Epigenetics

Background:

Chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-seq) is a key technique for identifying transcription factor (TF) binding sites genome-wide.
The utility of ChIP-seq is often hindered by the lack of high-quality, commercially available antibodies specific for many TFs.
This limitation restricts the comprehensive study of TF functions and their regulatory roles.

Purpose of the Study:

To present a novel method, CRISPR/Cas9-enabled TEnC-ChIP sequencing (CETCh-seq), for performing ChIP-seq when specific antibodies are unavailable.
To provide a generalizable protocol for CETCh-seq applicable to both adherent and nonadherent cell lines.
To overcome the antibody-dependency of traditional ChIP-seq for TF binding analysis.

Main Methods:

Development and application of CRISPR/Cas9 genome editing to introduce epitope tags (e.g., FLAG) into endogenous TF loci.
Performing ChIP using a commercially available antibody against the introduced epitope tag.
Utilizing next-generation DNA sequencing to identify TF binding regions across the genome.

Main Results:

CETCh-seq successfully generated ChIP-seq data for TFs lacking appropriate antibodies.
The protocol is adaptable for various cell types, including both adherent and nonadherent cell lines.
This method expands the scope of TF binding site identification using ChIP-seq.

Conclusions:

CETCh-seq offers a robust alternative to traditional ChIP-seq when suitable antibodies are not available.
This technique significantly broadens the range of TFs that can be studied using genome-wide binding analysis.
CETCh-seq facilitates a more comprehensive understanding of transcriptional regulation.