Fujita Toshitsugu, Yuno Miyuki, Fujii Hodaka
Chromatin Biochemistry Research Group, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Genes Cells. 2016 Apr;21(4):370-7. doi: 10.1111/gtc.12341. Epub 2016 Feb 5.
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.
成簇规律间隔短回文重复序列（CRISPR）系统被广泛应用于包括基因组编辑在内的各种生物学应用中。我们利用CRISPR开发了工程化DNA结合分子介导的染色质免疫沉淀技术（enChIP），以从细胞中分离目标基因组区域用于其生化特性分析。在本研究中，我们利用重组CRISPR核糖核蛋白（RNP）开发了“体外enChIP”技术来分离目标基因组区域。体外enChIP相对于传统enChIP具有很大优势，即不需要在细胞中表达CRISPR复合物。我们首先证明，使用重组CRISPR RNP的体外enChIP可用于从纯化DNA混合物中以序列特异性方式分离目标DNA。此外，我们还表明，该技术可用于高效分离目标基因组区域，同时保留其细胞内分子相互作用，来自无关基因组区域的污染可忽略不计。因此，体外enChIP技术在DNA的序列特异性分离以及结合下游分析鉴定体内与感兴趣基因组区域相互作用的分子方面具有潜在用途。
