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

Fujii, Hodaka; Fujita, Toshitsugu

doi:10.3390/ijms160921802

Cited by 14 publications

(10 citation statements)

References 38 publications

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“…) (see review (Fujita & Fujii )) and engineered DNA‐binding molecule‐mediated ChIP (enChIP) (Fujita & Fujii , ; Fujita et al . , , ,b) (see reviews (Fujii & Fujita ; Fujita & Fujii )). enChIP consists of the following steps (Fig.…”

Section: Introductionmentioning

confidence: 99%

Identification of physical interactions between genomic regions by enChIP‐Seq

et al. 2017

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Physical interactions between genomic regions play critical roles in the regulation of genome functions, including gene expression. Here, we show the feasibility of using engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) in combination with next-generation sequencing (NGS) (enChIP-Seq) to detect such interactions. In enChIP-Seq, the target genomic region is captured by an engineered DNA-binding complex, such as a clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a catalytically inactive form of Cas9 and a single guide RNA. Subsequently, the genomic regions that physically interact with the target genomic region in the captured complex are sequenced by NGS. Using enChIP-Seq, we found that the 5'HS5 locus, which is involved in the regulation of globin genes expression at the β-globin locus, interacts with multiple genomic regions upon erythroid differentiation in the human erythroleukemia cell line K562. Genes near the genomic regions inducibly associated with the 5'HS5 locus were transcriptionally up-regulated in the differentiated state, suggesting the existence of a coordinated transcription mechanism mediated by physical interactions between these loci. Thus, enChIP-Seq might be a potentially useful tool for detecting physical interactions between genomic regions in a nonbiased manner, which would facilitate elucidation of the molecular mechanisms underlying regulation of genome functions.

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Section: Introductionmentioning

confidence: 99%

Identification of physical interactions between genomic regions by enChIP‐Seq

et al. 2017

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“…By integrating epitope tags in different exons of alternatively spliced genes of interest, this method can also be employed to study DNA-binding patterns of specific splice isoforms of a transcription factor (Li et al, 2015). Even more scalable, as it does not require genetic editing, is the use of sgRNAs to target dCas9 to a locus of interest, and then performing ChIP experiments to dCas9 itself or a protein-tagged dCas9 (Fujii and Fujita, 2015). This approach enables profiling of all proteins interacting at single genomic locus, and has already been used to characterize site-specific proteomic binding patterns by combining dCas9-ChIP with by mass spectrometry (Zhang et al, 2016).…”

Section: Future Directionsmentioning

confidence: 99%

Application of CRISPR/Cas9 to the study of brain development and neuropsychiatric disease

Powell

Gregory

Akbarian

et al. 2017

Molecular and Cellular Neuroscience

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CRISPR/Cas9 technology has transformed our abilities to manipulate the genome and epigenome, as applications have expanded from efficient genomic editing to include the targeted localization of effectors to specific genomic loci. By facilitating the manipulation of DNA- and histone-modifying enzyme activities, activation or repression of gene expression, and targeting of transcriptional regulators to defined loci, it is now possible to directly probe the role of gene-regulatory and epigenetic pathways in order to examine their roles in basic biology and disease processes. Here we discuss these emerging CRISPR-based methodologies, with specific consideration of neurobiological applications using human induced pluripotent stem cell (hiPSC)-based models.

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“…; Hsu et al . ; Fujii & Fujita ; Fujita & Fujii ). Among these engineered DNA‐binding molecules, the CRISPR system is the most convenient, economical, and time‐efficient and has rapidly risen to prevail over technologies with similar functions since its introduction for use in genome editing.…”

Section: Introductionmentioning

confidence: 99%

Efficient sequence‐specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins

2016

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The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for various biological applications, including genome editing. We developed engineered DNAbinding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR to isolate target genomic regions from cells for their biochemical characterization. In this study, we developed 'in vitro enChIP' using recombinant CRISPR ribonucleoproteins (RNPs) to isolate target genomic regions. in vitro enChIP has the great advantage over conventional enChIP of not requiring expression of CRISPR complexes in cells. We first showed that in vitro enChIP using recombinant CRISPR RNPs can be used to isolate target DNA from mixtures of purified DNA in a sequence-specific manner. In addition, we showed that this technology can be used to efficiently isolate target genomic regions, while retaining their intracellular molecular interactions, with negligible contamination from irrelevant genomic regions. Thus, in vitro enChIP technology is of potential use for sequence-specific isolation of DNA, as well as for identification of molecules interacting with genomic regions of interest in vivo in combination with downstream analysis.

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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

Cited by 14 publications

References 38 publications

Identification of physical interactions between genomic regions by enChIP‐Seq

Identification of physical interactions between genomic regions by enChIP‐Seq

Application of CRISPR/Cas9 to the study of brain development and neuropsychiatric disease

Efficient sequence‐specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins

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