Identification of physical interactions between genomic regions by enCh<scp>IP</scp>‐Seq

Fujita, Toshitsugu; Yuno, Miyuki; Suzuki, Yutaka; Sugano, Sumio; Fujii, Hodaka

doi:10.1111/gtc.12492

Cited by 28 publications

(24 citation statements)

References 49 publications

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“…dCas9 and guide RNA here were used to locate an interested genomic region. Next, cell lysis was crosslinked and sonicated to yield chromatin fragments, the dCas9-gRNA tagged of which was pulled down for further RNA-seq or mass spectrometry analysis [ 69 , 70 ].…”

Section: Introductionmentioning

confidence: 99%

Novel Epigenetic Techniques Provided by the CRISPR/Cas9 System

Xie

Zhou

Sun

et al. 2018

Stem Cells International

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Epigenetics classically refers to the inheritable changes of hereditary information without perturbing DNA sequences. Understanding mechanisms of how epigenetic factors contribute to inheritable phenotype changes and cell identity will pave the way for us to understand diverse biological processes. In recent years, the emergence of CRISPR/Cas9 technology has provided us with new routes to the epigenetic field. In this review, novel epigenetic techniques utilizing the CRISPR/Cas9 system are the main contents to be discussed, including epigenome editing, temporal and spatial control of epigenetic effectors, noncoding RNA manipulation, chromatin in vivo imaging, and epigenetic element screening.

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Section: Introductionmentioning

confidence: 99%

Novel Epigenetic Techniques Provided by the CRISPR/Cas9 System

Xie

Zhou

Sun

et al. 2018

Stem Cells International

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“…A method called engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) was developed for site-specific analysis of protein or DNA. In that method, tag-fused dCas9 and bound target DNA are formaldehyde fixed and isolated for either mass spectrometry to detect proteins interacting with the target DNA (enChIP-MS) (39) or NGS to detect physical interactions between the target DNA and genomic regions (enChIP-Seq) (40). Other applications used the CRISPR system to remove non-target DNAs from DNA libraries.…”

Section: Introductionmentioning

confidence: 99%

CRISPR-Cap: multiplexed double-stranded DNA enrichment based on the CRISPR system

Lee

Lim

Jang

et al. 2018

Nucleic Acids Research

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Existing methods to enrich target regions of genomic DNA based on PCR, hybridization capture, or molecular inversion probes have various drawbacks, including long experiment times and low throughput and/or enrichment quality. We developed CRISPR-Cap, a simple and scalable CRISPR-based method to enrich target regions of dsDNA, requiring only two short experimental procedures that can be completed within two hours. We used CRISPR-Cap to enrich 10 target genes 355.7-fold on average from Escherichia coli genomic DNA with a maximum on-target ratio of 81% and high enrichment uniformity. We also used CRISPR-Cap to measure gene copy numbers and detect rare alleles with frequencies as low as 1%. Finally, we enriched coding sequence regions of 20 genes from the human genome. We envision that CRISPR-Cap can be used as an alternative to other widely used target-enrichment methods, which will broaden the scope of CRISPR applications to the field of target enrichment field.

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“…For specific and efficient affinity purification, we usually use 3xFLAG-tag in conjunction with antibody (Ab) against the epitope tag. Locus-tagging can be done in the cell by expressing engineered DNA-binding molecules [ 1 , 2 , 6 – 9 ] or in vitro using recombinant or synthetic engineered DNA-binding molecules [ 10 , 11 ]. Combination of enChIP with mass spectrometry (MS), RNA sequencing, and next-generation sequencing (NGS) has enabled us to identify proteins [ 1 , 2 , 6 ], RNAs [ 7 ], and genomic regions [ 9 , 11 ] interacting with specific genomic regions of interest in a non-biased manner.…”

Section: Introductionmentioning

confidence: 99%

enChIP systems using different CRISPR orthologues and epitope tags

2018

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ObjectivePreviously, we developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology, which isolates specific genomic regions while preserving their molecular interactions. In enChIP, the locus of interest is tagged with engineered DNA-binding molecules such as the clustered regularly interspaced short palindromic repeats (CRISPR) system, consisting of a catalytically inactive form of Cas9 (dCas9) and guide RNA, followed by affinity purification of the tagged locus to allow identification of associated molecules. In our previous studies, we used a 3xFLAG-tagged CRISPR system from Streptococcus pyogenes (S. pyogenes). In this study, to increase the flexibility of enChIP, we used the CRISPR system from Staphylococcus aureus (S. aureus) along with different epitope tags.ResultsWe generated a plasmid expressing S. aureus dCas9 (Sa-dCas9) fused to a nuclear localization signal (NLS) and a 3xFLAG-tag (Sa-dCas9-3xFLAG). The yields of enChIP using Sa-dCas9-3xFLAG were comparable to those using S. pyogenes dCas9 fused with an NLS and a 3xFLAG-tag (3xFLAG-Sp-dCas9). We also generated another enChIP system using Sp-dCas9 fused with an NLS and a 2xAM-tag (Sp-dCas9-2xAM). We obtained high enChIP yields using this system as well. Our findings indicate that these tools will increase the flexibility of enChIP analysis.

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Identification of physical interactions between genomic regions by enChIP‐Seq

Cited by 28 publications

References 49 publications

Novel Epigenetic Techniques Provided by the CRISPR/Cas9 System

Novel Epigenetic Techniques Provided by the CRISPR/Cas9 System

CRISPR-Cap: multiplexed double-stranded DNA enrichment based on the CRISPR system

enChIP systems using different CRISPR orthologues and epitope tags

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