Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line

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

doi:10.1093/dnares/dsx023

Cited by 17 publications

(22 citation statements)

References 62 publications

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“…S7B and C and Figs 4 and 5). crRNA_NC, a crRNA targeting an irrelevant locus (chicken Pax5 locus), 22 did not affect amplification (Supplementary Fig. S7B and C).…”

Section: Resultsmentioning

confidence: 95%

Detection of genome-edited cells by oligoribonucleotide interference-PCR

et al. 2018

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Genome editing by engineered sequence-specific nucleases, such as the clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for analysis of gene functions. Several techniques have been developed for detection of genome-edited cells, but simple, cost-effective, and positive detection methods remain limited. Recently, we developed oligoribonucleotide (ORN) interference-PCR (ORNi-PCR), in which hybridization of an ORN with a complementary DNA sequence inhibits amplification across the sequence. Here, we investigated whether ORNi-PCR can be used to detect genome-edited cells. First, we showed that ORNs that hybridize to a CRISPR target site in the THYN1 locus inhibited amplification across the target site, but no longer inhibited amplification after the target site was edited, resulting in mismatches. Importantly, ORNi-PCR could distinguish even single-nucleotide differences. These features of ORNi-PCR enabled detection of genome-edited cells by positive PCR amplification. In addition, ORNi-PCR was successful in discriminating genome-edited cells from wild-type cells, and multiplex ORNi-PCR simultaneously detected indel mutations at multiple loci. However, endpoint ORNi-PCR may not be able to distinguish between mono- and bi-allelic mutations, which may limit its utility. Taken together, these results demonstrate the potential utility of ORNi-PCR for the screening of genome-edited cells.

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“…S7B and C and Figs 4 and 5). crRNA_NC, a crRNA targeting an irrelevant locus (chicken Pax5 locus), 22 did not affect amplification (Supplementary Fig. S7B and C).…”

Section: Resultsmentioning

confidence: 95%

Detection of genome-edited cells by oligoribonucleotide interference-PCR

et al. 2018

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View full text Add to dashboard Cite

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“…Locus-tagging can be achieved by expression of engineered DNA-binding molecules (in-cell enChIP) [1,2,[7][8][9][10]. Alternatively, it can be achieved in vitro using recombinant or synthetic engineered DNA-binding molecules (in vitro enChIP) [11,12]. After a target locus is tagged using an engineered DNAbinding molecule, it is isolated by a nity puri cation.…”

Section: Methods Detailsmentioning

confidence: 99%

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

Yuno¹,

Fujita

Fujii

2020

Preprint

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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.

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“…Locus-tagging can be achieved by expression of engineered DNA-binding molecules (in-cell enChIP) (1,2,7-10). Alternatively, it can be achieved in vitro using recombinant or synthetic engineered DNA-binding molecules (in vitro enChIP) (11,12). After a target locus is tagged using an engineered DNAbinding molecule, it is isolated by a nity puri cation.…”

Section: Introductionmentioning

confidence: 99%

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

Yuno¹,

Fujita

Fujii

2020

Preprint

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line

Cited by 17 publications

References 62 publications

Detection of genome-edited cells by oligoribonucleotide interference-PCR

Detection of genome-edited cells by oligoribonucleotide interference-PCR

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

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

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