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Chromatin immunoprecipitation (ChIP) assay is a powerful and versatile immunoprecipitation assay for detecting protein-DNA interactions in the natural chromatin environment of cells. The assay can be used to identify a variety of proteins associated with a particular region of the genome, such as a transcription factor at a promoter or other DNA binding site, and possibly a cis protein, or conversely, to identify a genome associated with a particular protein. In addition, ChIP assays can be used to define spatial and temporal relationships for specific protein-DNA interactions.
Using CRISPR, the researchers expanded chromatin immunoprecipitation (ChIP) to purify any genomic sequence specified by a particular gRNA. In the enChIP (engineered DNA binding molecule mediated ChIP) system, catalytically inactive dCas9 is used to purify genomic DNA bound by gRNA. Epitope tags can be fused to dCas9 or gRNA for efficient purification, including 3xFLAG-tags, various epitope tags for PA and biotin tags can be used for enChIP, as well as anti-Cas9 antibodies. Biotin labeling of dCas9 can be achieved by fusing a biotin receptor site to dCas9 and co-expressing BirA biotin ligase. The locus is then isolated by affinity purification against the epitope tag. After purification of the locus, the locus-associated molecules can be identified by mass spectrometry (protein), RNA sequencing (RNA) and NGS (other genomic regions) (Figure 1).
Figure 1: Methods Overview
CRISPR-mediated ChIP technology has been used to identify proteins associated with interferon gamma-stimulated promoters associated with the interferon regulatory factor-1 (IRF-1) promoter region and to purify 15 related proteins, including histone deacetylases complex proteins, these proteins have previously been implicated in interferon gamma-mediated gene expression, as well as transcription factors, histones, and other DNA-related proteins. The CRISPR-mediated ChIP has many advantages over the traditional ChIP method. Large-scale assays using classical ChIP require the production and expression of multiple antibodies or epitope-tagged proteins for each DNA-binding protein, but the modular nature of the CRISPR/Cas9 system requires the only purification of a single antibody against the labeled Cas9 protein, it also directly recognizes molecules associated with genomic regions of interest in the body. Furthermore, the CRISPR/Cas9 system is not affected by low, differential or toxic gene expression. In summary, the CRISPR/Cas system represents a revolution that goes far beyond gene editing/genomic engineering methods. By inactivating the catalytic activity of Cas9, the enzyme has become a universal and site-specific recruitment platform, opening up a number of new applications for basic and medical research.
CRISPR/Cas9 platformCB, a global leading gene editing company, is dedicated to offering customers comprehensive genetic editing services and products and helping to solve the challenges of CRISPR technology application. We offer custom CRISPR-mediated ChIP solutions, including dCas9 fusion strategy design, and synthesis of gRNAs designed for specific loci, as well as proteomic analysis, DNA/RNA sequencing suitable for any human, mouse or rat gene. We can also provide CRISPR-mediated ChIP construction for other mammalian species based on your needs. If you have any projects that require CRISPR/Cas9 technology, don’t hesitate to contact us.