CRISPR/Cas9-mediated Chromatin Immunoprecipitation
Purification of specific genomic loci is vital for the characterization of chromatin-associated proteins and RNAs. Modifications to the CRISPR/Cas9 system allow for flexible targeting and isolation of these genomic regions. A nuclear localization signal and epitope tag can be introduced into catalytically inactive Cas9 (dCas9) to create a DNA-binding protein that can be targeted by CRISPR guide RNAs.
Existing CRISPR gRNA databases and design tools allow for targeting to any gene of interest. The CRISPR/Cas9-chromatin complex can then be purified with traditional chromatin immunoprecipitation (CHiP) techniques and exposed to mass spectrometry for further characterization.
CRISPR-mediated CHiP techniques have been utilized to identify proteins associated with the interferon regulatory factor-1 (IRF-1) promoter region in response to interferon γ stimulation. In this study, researchers purified 15 associated proteins including histone deacetylase complex proteins, which have previously been implicated in interferon γ-mediated gene expression, as well as transcription factors, histones and other DNA-associated proteins.
CRISPR-mediated CHiP holds a number of advantages over traditional CHiP methods. While large scale assays require the use of multiple antibodies against each DNA-binding protein or the creation and expression of epitope-tagged proteins, the modular nature of the CRISPR/Cas9 system requires only a single antibody against the tagged-Cas9 protein for purification. In addition, the CRISPR/Cas9 system is unaffected by issues stemming from low, differential or toxic gene expression.
