Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, 565-0871 Osaka, Japan.
Biochem Biophys Res Commun. 2013 Sep 13;439(1):132-6. doi: 10.1016/j.bbrc.2013.08.013. Epub 2013 Aug 11.
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.
为了对分子相互作用保留的特定基因组区域进行生化分析，有必要对其进行分离。在这里，我们建立了一种新的方法，即工程化 DNA 结合分子介导的染色质免疫沉淀（enChIP），用于纯化保留分子相互作用的特定基因组区域。我们表明，使用 CRISPR 系统的 enChIP 可以有效地分离特定的基因组区域。在这种形式的 enChIP 中，特异性基因组区域通过与标签（s）融合的抗体进行免疫沉淀，该标签融合有催化失活形式的 Cas9（dCas9），该 dCas9 与向导 RNA（gRNA）共表达，并识别感兴趣的基因组区域中的内源性 DNA 序列。针对内源性基因座的 enChIP-质谱分析（enChIP-MS）鉴定了相关蛋白。使用 CRISPR 系统的 enChIP 将是一种用于剖析感兴趣的基因组区域染色质结构的便捷而有用的工具。
