Dissecting Locus-Specific Chromatin Interactions by CRISPR CAPTURE

Abstract

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.

Keywords: Biotinylated dCas9; CAPTURE; CAPTURE-3C-seq; CAPTURE-Proteomics; CRISPR; Chromatin looping; Enhancer; Gene regulation; Noncoding genome; cis-regulatory elements.