PT  - JOURNAL ARTICLE
AU  - Toshitsugu Fujita
AU  - Miyuki Yuno
AU  - Hodaka Fujii
TI  - Efficient sequence-specific isolation of DNA fragments and chromatin by <em>in vitro</em> enChIP technology using recombinant CRISPR ribonucleoproteins
AID  - 10.1101/033241
DP  - 2015 Jan 01
TA  - bioRxiv
PG  - 033241
4099  - http://biorxiv.org/content/early/2015/11/30/033241.short
4100  - http://biorxiv.org/content/early/2015/11/30/033241.full
AB  - The clustered regularly interspaced short palindromic repeat (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 demonstrate 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 show that this technology can be employed 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.