Dissecting Locus-Specific Chromatin Interactions by CRISPR CAPTURE.
Dissecting Locus-Specific Chromatin Interactions by CRISPR CAPTURE.
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通过 CRISPR CAPTURE 剖析位点特异性染色质相互作用。
DOI：
10.1007/978-1-0716-2847-8_7
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Xu,Jian
中科院分区：
文献类型：
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作者：
Botten,GiovanniA;LeeJr,Michael;Xu,Jian
组织特异性基因表达的时空调控是由顺式调控元件（cis-regulatory elements，克雷斯）和相关的反式作用因子协调的。尽管在候选克雷斯的全基因组注释方面取得了重大进展，但绝大多数克雷斯的原位调控组成仍然未知。为了应对这一挑战，我们开发了CRISPR亲和纯化原位调节元件（CAPTURE）工具箱，该工具箱采用体内生物素化核酸酶缺陷型Cas9（dCas 9）蛋白和可编程单向导RNA（sgRNA）来识别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协议，用于实施基于CRISPR的CAPTURE方法的最新迭代，以询问哺乳动物基因组中基因座特异性染色质复合物和构型的分子组成。
The spatiotemporal control of tissue-specific gene expression is coordinated bycis-regulatory elements (CREs) and associatedtrans-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 theCRISPRaffinitypurification in situofregulatoryelements (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.