scholarly journals 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 ◽  
Vol 16 (9) ◽  
pp. 21802-21812 ◽  
Author(s):  
Hodaka Fujii ◽  
Toshitsugu Fujita
Keyword(s):  
Dna Binding ◽  
Chromatin Immunoprecipitation ◽  
Crispr System ◽  
Genomic Regions

scholarly journals Purification of specific DNA species using the CRISPR system

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Toshitsugu Fujita ◽  
Hodaka Fujii
Keyword(s):  
Dna Sequences ◽  
Chromatin Immunoprecipitation ◽  
In Vitro Method ◽  
Guide Rna ◽  
Crispr System ◽  
Target Dna ◽  
Genomic Regions ◽  
Generation Sequencing ◽  
Subsequent Identification

AbstractIn 2013, we developed a new method of engineered DNA-binding molecule-mediated chromatin immunoprecipitation that incorporates the clustered regularly interspaced short palindromic repeats (CRISPR) system to purify specific DNA species. This CRISPR-mediated purification can be performed in-cell or in vitro; CRISPR complexes can be expressed to tag target DNA sequences in the cells to be analyzed, or a CRISPR ribonucleoprotein complex consisting of recombinant nuclease-dead Cas9 (dCas9) and synthetic guide RNA can be used to tag target DNA sequences in vitro. Both methods enable purification of specific DNA sequences in chromatin structures for subsequent identification of molecules (proteins, RNAs, and other genomic regions) associated with the target sequences. The in vitro method also enables enrichment of purified DNA sequences from a pool of heterogeneous sequences for next-generation sequencing or other applications. In this review, we outline the principle of CRISPR-mediated purification of specific DNA species and discuss recent advances in the technology.

scholarly journals MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis

2021 ◽  
Author(s):  
Miyuki Yuno ◽  
Toshitsugu Fujita ◽  
Hodaka Fujii
Keyword(s):  
Dna Binding ◽  
Streptococcus Pyogenes ◽  
Chromatin Immunoprecipitation ◽  
Genomic Region ◽  
Target Cells ◽  
Drug Selection ◽  
Guide Rna ◽  
Inactive Form ◽  
Genomic Regions ◽  
Selection Of

Abstract 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. 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 by using these plasmids, it is feasible to purify target genomic regions with yields comparable to those reported using other systems. These systems might give enChIP users greater flexibility in choosing optimal systems for drug selection of transduced cells.

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

2020 ◽  
Author(s):  
Miyuki Yuno ◽  
Toshitsugu Fujita ◽  
Hodaka Fujii
Keyword(s):  
Dna Binding ◽  
Chromatin Immunoprecipitation ◽  
Genomic Region ◽  
Target Cells ◽  
Guide Rna ◽  
Inactive Form ◽  
Different Types ◽  
High Yields ◽  
Genomic Regions ◽  
Selection Of

Abstract 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.

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

2020 ◽  
Author(s):  
Miyuki Yuno ◽  
Toshitsugu Fujita ◽  
Hodaka Fujii
Keyword(s):  
Dna Binding ◽  
Chromatin Immunoprecipitation ◽  
Genomic Region ◽  
Target Cells ◽  
Guide Rna ◽  
Inactive Form ◽  
Different Types ◽  
High Yields ◽  
Genomic Regions ◽  
Selection Of

Abstract 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. 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.

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

2019 ◽  
Author(s):  
Miyuki Yuno ◽  
Toshitsugu Fujita ◽  
Hodaka Fujii
Keyword(s):  
Dna Binding ◽  
Chromatin Immunoprecipitation ◽  
Genomic Region ◽  
Target Cells ◽  
Guide Rna ◽  
Inactive Form ◽  
Different Types ◽  
High Yields ◽  
Genomic Regions ◽  
Selection Of

Abstract 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.

scholarly journals MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis

2021 ◽  
Author(s):  
Miyuki Yuno ◽  
Toshitsugu Fujita ◽  
Hodaka Fujii ◽  
Shoko Nagata
Keyword(s):  
Dna Binding ◽  
Streptococcus Pyogenes ◽  
Chromatin Immunoprecipitation ◽  
Genomic Region ◽  
Target Cells ◽  
Drug Selection ◽  
Guide Rna ◽  
Inactive Form ◽  
Genomic Regions ◽  
Selection Of

Abstract 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. 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 by using these plasmids, it is feasible to purify target genomic regions with yields comparable to those reported using other systems. These systems might give enChIP users greater flexibility in choosing optimal systems for drug selection of transduced cells.

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