Every transcription factor deserves its map: Scaling up epitope tagging of proteins to bypass antibody problems.
Genome-wide identification of transcription factor binding sites with the ChIP-seq method is an extremely important scientific endeavor - one that should ideally be performed for every transcription factor in as many cell types as possible. A major hurdle on the way to this goal is the necessity for a specific, ChIP-grade antibody for each transcription factor of interest, which is often not available. Here, we describe CETCh-seq, a recently published method utilizing genome engineering with the CRISPR/Cas9 system to circumvent the need for a specific antibody. Using the CETCh-seq method, targeted genomic editing results in an epitope-tagged transcription factor, which is recognized by a well-characterized, standard antibody, efficacious for ChIP-seq. We have used CETCh-seq in human cancer cell lines as well as mouse embryonic stem cells. We find that roughly 60% of transcription factors tagged using CETCh-seq produce a high quality ChIP-seq map, a significant improvement over traditional antibody-based methods.
HudsonAlpha Institute for Biotechnology, Huntsville, AL, USA.
E Christopher
EC
Partridge
University of Alabama in Huntsville, AL, USA.
Timley A
TA
Watkins
HudsonAlpha Institute for Biotechnology, Huntsville, AL, USA.|University of Alabama in Huntsville, AL, USA.
Eric M
EM
Mendenhall
D000818:Animals; D064113:CRISPR-Cas Systems; D047369:Chromatin Immunoprecipitation / Q000379:methods; D004247:DNA / Q000378:metabolism; D000939:Epitopes; D015894:Genome, Human; D023281:Genomics / Q000379:methods*; D006801:Humans; D051379:Mice; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA / Q000379:methods; D014157:Transcription Factors / Q000276:immunology / Q000378:metabolism*
D000939:Epitopes; D014157:Transcription Factors; D004247:DNA
CRISPR; gene regulation; genomics; transcription factors