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

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

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
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 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.

scholarly journals Transgenic mouse lines expressing the 3xFLAG-dCas9 protein for enChIP analysis

2017 ◽  
Author(s):  
Toshitsugu Fujita ◽  
Fusako Kitaura ◽  
Asami Oji ◽  
Naoki Tanigawa ◽  
Miyuki Yuno ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transgenic Mouse ◽  
Genomic Region ◽  
Guide Rna ◽  
Inactive Form ◽  
Primary Mouse ◽  
Mouse Cells ◽  
High Yields ◽  
Genomic Regions ◽  
Mouse Lines

AbstractWe developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology to isolate specific genomic regions while retaining their molecular interactions. In enChIP, the locus of interest is tagged with an engineered DNA-binding molecule, such as a modified form of the clustered regularly interspaced short palindromic repeats (CRISPR) system containing a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). The locus is then affinity-purified to enable identification of associated molecules. In this study, we generated transgenic mice expressing 3xFLAG-tagged Streptococcus pyogenes dCas9 (3xFLAG-dCas9) and retrovirally transduced gRNA into primary CD4+ T cells from these mice for enChIP. Using this approach, we achieved high yields of enChIP at the targeted genomic region. Our novel transgenic mouse lines provide a valuable tool for enChIP analysis in primary mouse cells.

scholarly journals Identification of physical interactions between genomic regions by enChIP-Seq

2016 ◽  
Author(s):  
Toshitsugu Fujita ◽  
Miyuki Yuno ◽  
Yutaka Suzuki ◽  
Sumio Sugano ◽  
Hodaka Fujii
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Erythroid Differentiation ◽  
Genomic Region ◽  
Globin Genes ◽  
Guide Rna ◽  
Inactive Form ◽  
Erythroleukemia Cell ◽  
Physical Interactions ◽  
Genomic Regions

Physical interactions between genomic regions play critical roles in the regulation of genome functions, including gene expression. However, the methods for confidently detecting physical interactions between genomic regions remain limited. Here, we demonstrate 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 CRISPR system consisting of a catalytically inactive form of Cas9 (dCas9) and a single guide RNA (sgRNA). 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 regulates expression of the β-globin genes, 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 directly or indirectly mediated by physical interactions between these loci. Our data suggest that enChIP-Seq is a potentially useful tool for detecting physical interactions between genomic regions in a non-biased manner, which would facilitate elucidation of the molecular mechanisms underlying regulation of genome functions.

scholarly journals Parallel Seed Color Adaptation during Multiple Domestication Attempts of an Ancient New World Grain

2019 ◽  
Vol 37 (5) ◽  
pp. 1407-1419 ◽  
Author(s):  
Markus G Stetter ◽  
Mireia Vidal-Villarejo ◽  
Karl J Schmid
Keyword(s):  
Genomic Region ◽  
Color Variation ◽  
Chromosome 9 ◽  
Seed Color ◽  
Color Adaptation ◽  
Crop Species ◽  
Grain Amaranth ◽  
Amaranth Species ◽  
Genomic Regions ◽  
Selection Of

Abstract Thousands of plants have been selected as crops; yet, only a few are fully domesticated. The lack of adaptation to agroecological environments of many crop plants with few characteristic domestication traits potentially has genetic causes. Here, we investigate the incomplete domestication of an ancient grain from the Americas, amaranth. Although three grain amaranth species have been cultivated as crop for millennia, all three lack key domestication traits. We sequenced 121 crop and wild individuals to investigate the genomic signature of repeated incomplete adaptation. Our analysis shows that grain amaranth has been domesticated three times from a single wild ancestor. One trait that has been selected during domestication in all three grain species is the seed color, which changed from dark seeds to white seeds. We were able to map the genetic control of the seed color adaptation to two genomic regions on chromosomes 3 and 9, employing three independent mapping populations. Within the locus on chromosome 9, we identify an MYB-like transcription factor gene, a known regulator for seed color variation in other plant species. We identify a soft selective sweep in this genomic region in one of the crop species but not in the other two species. The demographic analysis of wild and domesticated amaranths revealed a population bottleneck predating the domestication of grain amaranth. Our results indicate that a reduced level of ancestral genetic variation did not prevent the selection of traits with a simple genetic architecture but may have limited the adaptation of complex domestication traits.

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 RNA-guided Retargeting of Sleeping Beauty Transposition in Human Cells

2019 ◽  
Author(s):  
Adrian Kovač ◽  
Csaba Miskey ◽  
Michael Menzel ◽  
Esther Grueso ◽  
Andreas Gogol-Döring ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Sleeping Beauty ◽  
Specific Gene ◽  
Guide Rna ◽  
Gene Insertion ◽  
Genome Wide ◽  
Asymmetric Pattern ◽  
Integration Sites ◽  
Genomic Regions ◽  
Selection Of

ABSTRACTTwo different approaches of genomic modification are currently used for genome engineering and gene therapy: integrating vectors, which can efficiently integrate large transgenes but are unspecific with respect to their integration sites, and nuclease-based approaches, which are highly specific but not efficient at integrating large genetic cargoes. Here we demonstrate biased genome-wide integration of the Sleeping Beauty (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to influence the target site selection of SB by fusing it to a catalytically inactive Cas9 (dCas9) and by providing a single guide RNA (sgRNA) against the human Alu retrotransposon. Enrichment of transposon integrations was dependent on the sgRNA, occurred in a relatively narrow, ∼200 bp window around the targeted sites and displayed an asymmetric pattern with a bias towards sites that are downstream of the sgRNA targets. Our data indicate that the targeting mechanism specified by CRISPR/Cas9 forces integration into genomic regions that are otherwise poor targets for SB transposition. Future modifications of this technology may allow the development of methods for efficient and specific gene insertion for precision genetic engineering.

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