Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus

Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

doi:10.1038/srep30485

Cited by 35 publications

(29 citation statements)

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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%

“…An alternative approach to detecting physical interactions between genomic regions is to purify specific genomic regions engaged in molecular interactions and then analyze the genomic DNA in the purified complexes. To purify specific genomic regions, we recently developed two locus-specific chromatin immunoprecipitation (locus-specific ChIP) technologies, insertional ChIP (iChIP) (Hoshino & Fujii 2009;Fujita & Fujii 2011, 2012a, 2014aFujita et al 2015a) (see review (Fujita & Fujii 2016)) and engineered DNA-binding molecule-mediated ChIP (enChIP) , 2014bFujita et al , 2015bFujita et al , 2016a) (see reviews ). enChIP consists of the following steps ( Fig.…”

Section: Introductionmentioning

confidence: 99%

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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%

Section: Introductionmentioning

confidence: 99%

Identification of physical interactions between genomic regions by enChIP‐Seq

et al. 2017

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“…9A). 17 , 23 We designed an ORN, ORN_Gx5, complementary to the sequence of the G-insertion allele (Gx5) but with a single-nucleotide mismatch at the 3′ position with the corresponding DNA sequence in the other allele (Gx4) (Fig. 9A).…”

Section: Resultsmentioning

confidence: 99%

“…To construct a Cas9 plus sgRNA expression plasmid targeting the human CDKN2A(p16) locus, the sgRNA expression cassette for CDKN2A(p16) (Gx4 no. 2) 17 was cloned upstream of the Cas9 expression cassette in the Cas9 expression plasmid.…”

Section: Methodsmentioning

confidence: 99%

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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“…However, Cas9, being prokaryotic in origin, did not evolve to cope with the complex chromatinised environment of the eukaryotic genome. Despite prior studies in this area [4,7–14], the extent to which epigenetic properties of the target site, including DNA and histone modifications, influence mutation frequency and DNA repair outcome remains incompletely understood. Stably positioned nucleosomes act as a barrier to Cas9 binding and function on synthetic chromatin fibres [7,8,11], and in vivo [7], yet catalytically dead (d)Cas9 can open previously inaccessible regions of chromatin [15,16].…”

Section: Introductionmentioning

confidence: 99%

Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence repair outcome

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Taylor

Meynert

et al. 2018

Preprint

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25CRISPR-Cas9 genome editing occurs in the context of chromatin, which is heterogeneous in 26 structure and function across the genome. Chromatin heterogeneity is thought to affect 27 genome editing efficiency, but this has been challenging to quantify due to the presence of 28 confounding variables. Here, we develop a method that exploits the allele-specific chromatin 29 status of imprinted genes in order to address this problem. Because maternal and paternal 30 alleles of imprinted genes have identical DNA sequence and are situated in the same 31 nucleus, allele-specific differences in the frequency and spectrum of Cas9-induced mutations 32 can be attributed unequivocally to epigenetic mechanisms. We found that heterochromatin 33 can impede mutagenesis, but to a degree that depends on other key experimental 34 parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and 35 when intracellular Cas9 expression was low. Surprisingly, the outcome of mutagenic DNA 36 repair was independent of chromatin state, with similar efficiencies of homology directed 37 repair and deletion spectra on maternal and paternal chromosomes. Combined, our data 38show that heterochromatin imposes a permeable barrier that influences the kinetics, but not 39 the endpoint of CRISPR-Cas9 genome editing, and suggest that therapeutic applications 40 involving low-level Cas9 exposure will be particularly affected by chromatin status. 41 42 43 44 45 46 47 48

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Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus

Cited by 35 publications

References 45 publications

Identification of physical interactions between genomic regions by enChIP‐Seq

Identification of physical interactions between genomic regions by enChIP‐Seq

Detection of genome-edited cells by oligoribonucleotide interference-PCR

Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence repair outcome

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