通过 CRISPR 捕获技术解析基因座特异性染色质相互作用
Dissecting Locus-Specific Chromatin Interactions by CRISPR CAPTURE.
机构信息
Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Department of Pediatrics, Harold C. Simmons Comprehensive Cancer Center, and Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA.
出版信息
Methods Mol Biol. 2023;2599:69-97. doi: 10.1007/978-1-0716-2847-8_7.
The spatiotemporal control of tissue-specific gene expression is coordinated by cis-regulatory elements (CREs) and associated trans-acting factors. Despite major advances in genome-wide annotation of candidate CREs, the in situ regulatory composition of the vast majority of CREs remain unknown. To address this challenge, we developed the CRISPR affinity purification in situ of regulatory elements (CAPTURE) toolbox that employs an in vivo biotinylated nuclease-deficient Cas9 (dCas9) protein and programmable single-guide RNAs (sgRNAs) to identify CRE-associated macromolecular complexes and chromatin looping. In this chapter, we provide a detailed protocol for implementing the latest iteration of the CRISPR-based CAPTURE methods to interrogate the molecular composition of locus-specific chromatin complexes and configuration in a mammalian genome.
组织特异性基因表达的时空调控是由顺式调控元件（CREs）和相关的反式作用因子协调的。尽管在候选 CREs 的全基因组注释方面取得了重大进展，但绝大多数 CREs 的原位调控组成仍然未知。为了解决这一挑战，我们开发了 CRISPR 亲和纯化原位调控元件（CAPTURE）工具包，该工具包使用体内生物素化的核酸酶缺陷 Cas9（dCas9）蛋白和可编程的单指导 RNA（sgRNA）来鉴定与 CRE 相关的大分子复合物和染色质环。在本章中，我们提供了一个详细的实施基于 CRISPR 的 CAPTURE 方法的最新迭代的方案，以研究哺乳动物基因组中特定基因座染色质复合物的分子组成和结构。
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