Locus-Specific Genomic DNA Purification Using the CRISPR System: Methods and Applications

Abstract

A multitude of molecular interactions with chromatin governs various chromosomal functions in cells. Insights into the molecular compositions at specific genomic regions are pivotal to deepen our understanding of regulatory mechanisms and the pathogenesis of disorders caused by the abnormal regulation of genes. The locusspecific purification of genomic DNA using the clustered regularly interspaced short palindromic repeats (CRISPR) system enables the isolation of target genomic regions for identification of bound interacting molecules. This CRISPR-based DNA purification method has many applications. In this study, we present an overview of the CRISPR-based DNA purification methodologies as well as recent applications.

iChIP‐SILAC analysis identifies epigenetic regulators of CpG methylation of the p16INK4A gene

“…To identify molecules that bind to a target genomic region and thus examine the mechanisms of aberrant epigenetic regulation, we developed a locus‐specific chromatin immunoprecipitation (locus‐specific ChIP) method, which can be used to isolate specific genomic regions [5]. Molecules (DNA, RNA, and proteins) bound to the isolated genomic region can be comprehensively identified by downstream analysis using mass spectrometry (MS) and next‐generation sequencing.…”

“…Molecules (DNA, RNA, and proteins) bound to the isolated genomic region can be comprehensively identified by downstream analysis using mass spectrometry (MS) and next‐generation sequencing. Locus‐specific ChIP consists of insertional ChIP (iChIP) [5–7] and engineered DNA‐binding molecule‐mediated ChIP (enChIP) [5,8,9]. The iChIP method consists of three steps: (I) locus targeting by insertion of the binding elements of LexA, a bacterial DNA‐binding protein (LexA BE), in/around a target genomic region in a cell; (II) expression of the 3xFLAG‐tagged DNA‐binding domain of LexA (3xFNLDD) in the same cell; and (III) isolation of the target genomic region by affinity purification of 3xFNLDD.…”

Reverse-ChIP Techniques for Identifying Locus-Specific Proteomes: A Key Tool in Unlocking the Cancer Regulome

“…Beyond the explosion of use for genetic editing and engineering, a great deal of research has focused on the development of catalytically dead Cas9 (dCas9) as a tool for genomics [148]. A variety of strategies have employed the modular RNA-guided binding capabilities of dCas9 as a tool to enable the biochemical purification of specific chromatin regions for use in reverse-ChIP experiments (Figure 5) [149]. These dCas9-based approaches gain the advantages of nucleic acid-based targeting without succumbing to the inefficient purification that plagues PICh and related protocols or the laborious multi-step genetic engineering required for most protein-based methods.…”

An enChIP system for the analysis of genome functions in budding yeast

“…To achieve this goal, we developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology, which enables isolation of a genomic region of interest and identification of its associated molecules [ 1 ]. Engineered DNA-binding molecules that can be used to tag a target locus include transcription activator-like proteins [ 2 ] and the clustered regularly interspaced short palindromic repeats (CRISPR) system [ 3–5 ], which consist of a catalytically inactive form of Cas9 (dCas9) and a guide RNA (gRNA) (see our recent reviews [ 6 , 7 ] for comprehensive lists of publications using CRISPR-based systems). These engineered molecules can be expressed in a cell of interest to tag a locus (in-cell enChIP) [ 1 ] or incubated with fragmented chromatin in vitro ( in vitro enChIP) [ 8 ].…”