Identification of Proteins Interacting with Genomic Regions of Interest in vivo Using Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (enChIP)

2016

Gene�Regul Syst Bio

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To isolate specific genomic regions that retain their molecular interactions, allowing direct identification of chromatin-bound molecules, we developed two locus-specific chromatin immunoprecipitation (locus-specific ChIP) technologies, insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP) using the clustered regularly interspaced short palindromic repeats (CRISPR) system or transcription activator-like (TAL) proteins. Essentially, a locus-specific ChIP consists of locus-tagging and affinity purification and can be combined with downstream analyses to identify molecules associated with the target genomic regions. In this review, we discuss the applications of locus-specific ChIP to analyze the genome functions, including transcription and epigenetic regulation.

Section: Discussionmentioning

confidence: 99%

Biochemical Analysis of Genome Functions Using Locus-Specific Chromatin Immunoprecipitation Technologies

2016

Gene�Regul Syst Bio

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“…The engineered DNA-binding molecules that can be used to tag the target locus include transcription activator-like (TAL) proteins [3] and the clustered regularly interspaced short palindromic repeats (CRISPR) system [4,5] consisting of a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9) (see our recent review [6] for the CRISPR-based systems). Locus-tagging can be achieved by expression of engineered DNA-binding molecules (in-cell enChIP) [1,2,[7][8][9][10]. Alternatively, it can be achieved in vitro using recombinant or synthetic engineered DNA-binding molecules (in vitro enChIP) [11,12].…”

Section: Methods Detailsmentioning

confidence: 99%

enChIP systems with MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9

Yuno¹,

2020

Preprint

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Objective: Engineered DNA-binding molecule–mediated chromatin immunoprecipitation (enChIP) is a technology for purifying specific genomic regions to facilitate identification of their associated molecules, including proteins, RNAs, and other genomic regions. In enChIP, the target genomic region is tagged with engineered DNA-binding molecules, e.g., a variant of the clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). In this study, to increase the flexibility of enChIP and expand the range of target cells, we generated murine stem cell virus (MSCV)-based retroviral plasmids for expressing dCas9. Results: We constructed MSCV-based retroviral plasmids expressing Streptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-Sp-dCas9) and various drug resistance genes. We showed that it is feasible to purify target genomic regions with high yields using these plasmids. These systems might give enChIP users greater flexibility in choosing optimal systems for drug selection of transduced cells. In addition, they could be used to analyze different types of target cells.

“…Detailed protocols for enChIP can be obtained at the authors’ homepage ( ), bio-protocol publications [ 27 ], and our published protocol papers [ 28 , 29 ]. Here, we describe general technical considerations in performing enChIP.…”

Section: Technical Considerations In Performing Enchipmentioning

confidence: 99%

Isolation of Specific Genomic Regions and Identification of Their Associated Molecules by Engineered DNA-Binding Molecule-Mediated Chromatin Immunoprecipitation (enChIP) Using the CRISPR System and TAL Proteins

2015

IJMS

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Comprehensive understanding of genome functions requires identification of molecules (proteins, RNAs, genomic regions, etc.) bound to specific genomic regions of interest in vivo. To perform biochemical and molecular biological analysis of specific genomic regions, we developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) to purify genomic regions of interest. In enChIP, specific genomic regions are tagged for biochemical purification using engineered DNA-binding molecules, such as transcription activator-like (TAL) proteins and a catalytically inactive form of the clustered regularly interspaced short palindromic repeats (CRISPR) system. enChIP is a comprehensive approach that emphasizes non-biased search using next-generation sequencing (NGS), microarrays, mass spectrometry (MS), and other methods. Moreover, this approach is not restricted to cultured cell lines and can be easily extended to organisms. In this review, we discuss applications of enChIP to elucidating the molecular mechanisms underlying genome functions.