The mechanism of locus-specific ChIP is as follows:

• Target genomic regions are tagged with engineered DNA-binding molecules (enChIP) or exogenous DNA-binding molecules recognizing their binding elements inserted into the target genomic regions (iChIP).
• The resultant cells are stimulated and crosslinked with formaldehyde or other crosslinkers, if necessary.
• The cells are lysed, and DNA is fragmented.
• The complexes including the engineered DB are subjected to affinity purification, such as immunoprecipitation.
• The isolated complexes retain molecules interacting with the genomic region of interest. Reverse crosslinking and subsequent purification of DNA, RNA, proteins, or other molecules allows the identification and characterization of these molecules.

Original Reports

• Fujii H, Fujita T: An enChIP system for the analysis of genome functions in budding yeast. Biol. Methods Protoc. (2022) 7, bpac025.

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• Sarudate A, Fujita T, Nakayama T, Fujii H: enChIP-Seq analyzer: a software program to analyze and interpret enChIP-Seq data for the detection of physical interactions between genomic regions. Genes (2022) 13, 472.

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• Takeda T, Yokoyama Y, Takahashi H, Okuzaki D, Asai K, Itakura H, Miyoshi N, Kobayashi S, Uemura M, Fujita T, Ueno H, Mori M, Doki Y, Fujii H, Eguchi H, Yamamoto H: A stem cell marker KLF5 regulates CCAT1 via three-dimensional genome structure in colorectal cancer cells. Br. J. Cancer (2021) 126, 109-119.

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• Yuno M, Nagata S, Fujita T, Fujii H: MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis. Biol. Methods Protoc. (2021) 6, bpab013.

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• Husain A, Xu J, Fujii H, Nakata M, Kobayashi M, Wang JY, Rehwinkel J, Honjo T, Begum NA: SAMHD1-mediated dNTP degradation is required for efficient DNA repair during antibody class switch recombination. EMBO J. (2020) 39, e102931.

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• Fujita T, Yuno M, Fujii H: An enChIP system for the analysis of bacterial genome functions. BMC Res. Notes (2018) 11, 387.

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• Hamidian A, Vaapil M, von Stedingk K, Fujita T, Persson CU, Eriksson P, Veerla S, De Preter K, Speleman F, Fujii H, Påhlman S, Mohlin S: Promoter-associated proteins of EPAS1 identified by enChIP-MS - A putative role of HDX as a negative regulator. Biochem. Biophys. Res. Commun. (2018) 499, 291-298.

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• Fujita T, Yuno M, Fujii H: enChIP systems using different CRISPR orthologues and epitope tags. BMC Res. Notes (2018) 11, 154.

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• Fujita T, Kitaura F, Oji A, Tanigawa N, Yuno M, Ikawa M, Taniuchi I, Fujii H: Transgenic mouse lines expressing the 3xFLAG-dCas9 protein for enChIP analysis. Genes Cells (2018) 23, 318-325.

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• Fujita T, Kitaura F, Yuno M, Suzuki Y, Sugano S, Fujii H: Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line. DNA Res. (2017) 24, 537–548.

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• Fujita T, Yuno M, Suzuki Y, Sugano S, Fujii H: Identification of physical interactions between genomic regions by enChIP-Seq. Genes Cells (2017) 22, 506-520.

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• Fujita T, Yuno M, Fujii H: Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus. Sci. Rep. (2016) 6, 30485.

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• Fujita T, Yuno M, Fujii H: Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins. Genes Cells (2016) 21, 370-377.

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• Fujita T, Yuno M, Okuzaki D, Ohki R, Fujii H: Identification of non-coding RNAs associated with telomeres using a combination of enChIP and RNA sequencing. PLoS One (2015) 10, e0123387.

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• Fujita T, Kitaura F, and Fujii H: A critical role of the Thy28-MYH9 axis in B cell-specific expression of the Pax5 gene in chicken B cells. PLoS One (2015) 10, e0116579.

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• Fujita T, and Fujii H: Efficient isolation of specific genomic regions retaining molecular interactions by the iChIP system using recombinant exogenous DNA-binding proteins. BMC Mol. Biol. (2014) 15, 26.

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• Fujita T, and Fujii H: Identification of proteins associated with an IFNγ-responsive promoter by a retroviral expression system for enChIP using CRISPR. PLoS One (2014) 9, e103084.

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• Fujita T, Asano Y, Ohtsuka J, Takada Y, Saito K, Ohki R, and Fujii H: Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP). Sci. Rep. (2013) 3, 3171.

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• Fujita T, and Fujii H: Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem. Biophys. Res. Commun. (2013) 439, 132-136.

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• Fujita T, and Fujii H: Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain. Adv. Biosci. Biotechnol. (2012) 3, 626-629.

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• Fujita T, and Fujii H: Direct identification of insulator components by insertional chromatin immunoprecipitation. PLoS One (2011) 6, e26109.

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• Hoshino A, and Fujii H: Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions. J. Biosci. Bioeng. (2009), 108, 446-449.

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Reviews

• Fujita H, Fujita T, and Fujii H: Locus-specific genomic DNA purification using the CRISPR system: methods and applications. CRISPR J. (2021) 4 (2), 290-300.

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• Fujita T, and Fujii H: Purification of specific DNA species using the CRISPR system. Biol. Methods Protoc. (2019) 4 (1), bpz008.

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• Fujita T, and Fujii H: in vitro engineered DNA-binding molecule-mediated chromatin immunoprecipitation (in vitro enChIP) using CRISPR ribonucleoproteins in combination with next-generation sequencing (in vitro enChIP-Seq) for the identification of chromosomal interactions. Bio-protocol (2017) 7 (22), doi: 10.21769/BioProtoc.2612.

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• Fujita T, and Fujii H: Isolation of specific genomic regions and identification of associated molecules by enChIP. JoVE (2016) issue 107, doi: 10.3791/53478.

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• Fujita T, and Fujii H: Biochemical analysis of genome functions using locus-specific chromatin immunoprecipitation technologies. Gene Regul. Syst. Bio. (2016) Suppl. 1, 1-9.

• Fujita T, and Fujii H: Applications of engineered DNA-binding molecules such as TAL proteins and the CRISPR/Cas system in biology research. Int. J. Mol. Sci. (2015) 16, 23143-23164.

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• Fujii H, and Fujita T: 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. Int. J. Mol. Sci. (2015) 16, 21802-21812.

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• Fujita T, and Fujii H: Identification of proteins interacting with genomic regions of interest in vivo using engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP). Bio-protocol (2014) 4 (10), doi: 10.21769/BioProtoc.1124.

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• Fujita T, and Fujii H: Locus-specific biochemical epigenetics/chromatin biochemistry by insertional chromatin immunoprecipitation. ISRN Biochem. (2013) 2013, 913273.

Books

• Fujita T, and Fujii H: New directions for epigenetics: application of engineered DNA-binding molecules to locus-specific epigenetic research. Handbook of Epigenetics, Third Edition, Academic Press (Elsevier), Editor: Trygve O. Tollefsbol, (2023) 843-868.

• Fujita T, and Fujii H: New directions for epigenetics: application of engineered DNA-binding molecules to locus-specific epigenetic research. Handbook of Epigenetics, Second Edition, Academic Press (Elsevier), Editor: Trygve O. Tollefsbol, (2017) 635-652.

• Fujita T, and Fujii H: Isolation of specific genomic regions and identification of associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Methods Mol. Biol. (2015) 1288:43-52. doi: 10.1007/978-1-4939-2474-5_4.

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