Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR.
Isolation of specific genomic regions retaining molecular interactions is necessary for their biochemical analysis. Here, we established a novel method, engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP), for purification of specific genomic regions retaining molecular interactions. We showed that enChIP using the CRISPR system efficiently isolates specific genomic regions. In this form of enChIP, specific genomic regions are immunoprecipitated with antibody against a tag(s), which is fused to a catalytically inactive form of Cas9 (dCas9), which is co-expressed with a guide RNA (gRNA) and recognizes endogenous DNA sequence in the genomic regions of interest. enChIP-mass spectrometry (enChIP-MS) targeting endogenous loci identified associated proteins. enChIP using the CRISPR system would be a convenient and useful tool for dissecting chromatin structure of genomic regions of interest.
Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, 565-0871 Osaka, Japan.
Toshitsugu
T
Fujita
Hodaka
H
Fujii
D002843:Chromatin / Q000737:chemistry*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004247:DNA / Q000032:analysis*; D023281:Genomics; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010957:Plasmids / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D058727:RNA, Small Untranslated
D002843:Chromatin; D004247:DNA; D058727:RNA, Small Untranslated
CRISPR; Locus-specific biochemical epigenetics/chromatin biochemistry; Mass spectrometry; dCas9; enChIP; gRNA