RRC ID 66040
著者 Fujita T, Yuno M, Fujii H.
タイトル Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins.
ジャーナル Genes Cells
Abstract The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for various biological applications, including genome editing. We developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR to isolate target genomic regions from cells for their biochemical characterization. In this study, we developed 'in vitro enChIP' using recombinant CRISPR ribonucleoproteins (RNPs) to isolate target genomic regions. in vitro enChIP has the great advantage over conventional enChIP of not requiring expression of CRISPR complexes in cells. We first showed that in vitro enChIP using recombinant CRISPR RNPs can be used to isolate target DNA from mixtures of purified DNA in a sequence-specific manner. In addition, we showed that this technology can be used to efficiently isolate target genomic regions, while retaining their intracellular molecular interactions, with negligible contamination from irrelevant genomic regions. Thus, in vitro enChIP technology is of potential use for sequence-specific isolation of DNA, as well as for identification of molecules interacting with genomic regions of interest in vivo in combination with downstream analysis.
巻・号 21(4)
ページ 370-7
公開日 2016-4-1
DOI 10.1111/gtc.12341
PMID 26848818
MeSH Chromatin Immunoprecipitation / methods* Clustered Regularly Interspaced Short Palindromic Repeats* HEK293 Cells Humans Recombinant Proteins / metabolism Ribonucleoproteins / metabolism
IF 1.655
リソース情報
ヒト・動物細胞 DT40(RCB1464)