Liu Xin, Zhang Yuannyu, Chen Yong, Li Mushan, Shao Zhen, Zhang Michael Q, Xu Jian
Children's Medical Center Research Institute, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas.
Department of Biological Sciences, Center for Systems Biology, University of Texas at Dallas, Richardson, Texas.
Curr Protoc Mol Biol. 2018 Jul;123(1):e64. doi: 10.1002/cpmb.64. Epub 2018 Jun 19.
Cis-regulatory elements (CREs) play a pivotal role in spatiotemporal control of tissue-specific gene expression, yet the molecular composition of the vast majority of CREs in native chromatin remains unknown. In this article, we describe the clustered regularly interspaced short palindromic repeats (CRISPR) affinity purification in situ of regulatory elements (CAPTURE) approach to simultaneously identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated nuclease-deficient Cas9 (dCas9) protein and programmable single guide RNAs (sgRNAs), this approach allows for high-resolution and locus-specific isolation of protein complexes and long-range chromatin looping associated with single copy CREs in mammalian cells. Unbiased analysis of the compositional structure of developmentally regulated or disease-associated CREs identifies new features of transcriptional regulation. Hence, CAPTURE provides a versatile platform to study genomic locus-regulating chromatin composition in a mammalian genome. © 2018 by John Wiley & Sons, Inc.
顺式作用调控元件（CREs）在组织特异性基因表达的时空控制中起着关键作用，但天然染色质中绝大多数CREs的分子组成仍不清楚。在本文中，我们描述了一种用于原位亲和纯化调控元件的成簇规律间隔短回文重复序列（CRISPR）方法（CAPTURE），以同时鉴定位点特异性染色质调控蛋白复合物和长程DNA相互作用。利用体内生物素化的核酸酶缺陷型Cas9（dCas9）蛋白和可编程的单向导RNA（sgRNAs），该方法能够在哺乳动物细胞中对与单拷贝CREs相关的蛋白复合物和长程染色质环进行高分辨率和位点特异性分离。对发育调控或疾病相关CREs的组成结构进行无偏分析，可确定转录调控的新特征。因此，CAPTURE为研究哺乳动物基因组中调控染色质组成的基因组位点提供了一个通用平台。© 约翰威立国际出版公司2018年版权所有
