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Efficient manipulation of gene dosage in human iPSCs using CRISPR/Cas9 nickases

ABSTRACT: Efficient manipulation of gene dosage in human iPSCs using CRISPR/Cas9 nickases

PROVIDER: PRJNA672661 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12909000_1.fastq.gz	Fastqsanger.gz
	SRR12909000_2.fastq.gz	Fastqsanger.gz
	SRR12908992_1.fastq.gz	Fastqsanger.gz
	SRR12908992_2.fastq.gz	Fastqsanger.gz
	SRR12908993_1.fastq.gz	Fastqsanger.gz

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Efficient manipulation of gene dosage in human iPSCs using CRISPR/Cas9 nickases

Project description:The dysregulation of gene dosage due to duplication or haploinsufficiency is a major cause of autosomal dominant diseases such as Alzheimer’s disease. However, there is no rapid and efficient method for manipulating gene dosage in a human model system such as human induced pluripotent stem cells (iPSCs). Here, we describe a simple and precise method to simultaneously generate iPSC lines with different gene dosages using paired Cas9 nickases. We first generate a Cas9 nickase variant with broader protospacer-adjacent motif specificity to expand the targetability of double-nicking–mediated genome editing. As a proof-of-concept study, we examine the gene dosage effects on Alzheimer’s disease pathogenesis patient-derived iPSC line that carries three copies of APP (amyloid precursor protein). This method enables the rapid and simultaneous generation of iPSC lines with monoallelic, biallelic, or triallelic knockout of APP. The cortical neurons generated from isogenically corrected iPSCs exhibit gene dosage-dependent correction of disease-associated phenotypes of amyloid-beta secretion and Tau hyperphosphorylation. Thus, the rapid generation of iPSCs with different gene dosages using the method described herein can be a useful model system for investigating disease mechanisms and therapeutic development.

2021-02-04 | GSE160224 | GEO

Efficient manipulation of gene dosage in human iPSCs using CRISPR/Cas9 nickases.

Project description:The dysregulation of gene dosage due to duplication or haploinsufficiency is a major cause of autosomal dominant diseases such as Alzheimer's disease. However, there is currently no rapid and efficient method for manipulating gene dosage in a human model system such as human induced pluripotent stem cells (iPSCs). Here, we demonstrate a simple and precise method to simultaneously generate iPSC lines with different gene dosages using paired Cas9 nickases. We first generate a Cas9 nickase variant with broader protospacer-adjacent motif specificity to expand the targetability of double-nicking-mediated genome editing. As a proof-of-concept study, we examine the gene dosage effects on an Alzheimer's disease patient-derived iPSC line that carries three copies of APP (amyloid precursor protein). This method enables the rapid and simultaneous generation of iPSC lines with monoallelic, biallelic, or triallelic knockout of APP. The cortical neurons generated from isogenically corrected iPSCs exhibit gene dosage-dependent correction of disease-associated phenotypes of amyloid-beta secretion and Tau hyperphosphorylation. Thus, the rapid generation of iPSCs with different gene dosages using our method described herein can be a useful model system for investigating disease mechanisms and therapeutic development.

| S-EPMC7881037 | biostudies-literature

Project description:Efficient manipulation of gene dosage in human iPSCs using CRISPR/Cas9 nickases

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Naïve iPSCs Generated from β-thalassemia Fibroblasts Allow Efficient Gene Correction with CRISPR/Cas9

Project description:We used a modified 5i/L/FA system to generate transgene-free naïve iPSCs directly from the fibroblasts of a patient suffering from β-thalassemia and further demonstrated efficient gene correction with a CRISPR/Cas9 system, which provides an improved strategy for personalized treatment of β-thalassemia.

2016-12-01 | GSE69319 | GEO

Site-specific manipulation of Arabidopsis loci using CRISPR-Cas9 SunTag systems

Project description:Understanding genomic functions requires site-specific manipulation of loci via efficient protein effector targeting systems. However, few approaches for targeted manipulation of the epigenome are available in plants. Here, we adapt the dCas9-SunTag system to engineer targeted gene activation and DNA methylation in Arabidopsis. We demonstrate that a dCas9-SunTag system utilizing the transcriptional activator VP64 drives robust and specific activation of several loci, including protein coding genes and transposable elements, in diverse chromatin contexts. In addition, we present a CRISPR-based methylation targeting system for plants, utilizing a SunTag system with the catalytic domain of the Nicotiana tabacum DRM methyltransferase, which efficiently targets DNA methylation to specific loci, including the FWA promoter, triggering a developmental phenotype, and the SUPERMAN promoter. These SunTag systems represent valuable tools for the site-specific manipulation of plant epigenomes.

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X Chromosome Dosage Influences DNA Methylation Dynamics During Reprogramming to Mouse iPSCs

Project description:A dramatic difference in global DNA methylation between male and female cells characterizes mouse embryonic stem cells (ESCs), unlike somatic cells. We analyzed DNA methylation changes during reprogramming of male and female somatic cells and in resulting induced pluripotent stem cells (iPSCs). At an intermediate reprogramming stage, somatic and pluripotency enhancers are targeted for partial methylation and demethylation. Demethylation within pluripotency enhancers often occurs at ESC binding sites of pluripotency transcription factors. Late in reprogramming, global hypomethylation is induced in a female-specific manner. Genome-wide hypomethylation in female cells affects many genomic landmarks, including enhancers and imprint control regions, and accompanies the reactivation of the inactive X-chromosome. The loss of one of the two X-chromosomes in propagating female iPSCs is associated with genome-wide methylation gain. Collectively, our findings highlight the dynamic regulation of DNA methylation at enhancers during reprogramming and reveal that X-chromosome dosage dictates global DNA methylation levels in iPSCs.

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X Chromosome Dosage Influences DNA Methylation Dynamics During Reprogramming to Mouse iPSCs

Project description:X Chromosome Dosage Influences DNA Methylation Dynamics During Reprogramming to Mouse iPSCs

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X Chromosome Dosage Modulates Multiple Molecular and Cellular Properties of Mouse Pluripotent Stem Cells Independently of Global DNA Methylation Levels

Project description:During early mammalian development, the two X-chromosomes in female cells are active. Dosage compensation between XX female and XY male cells is then achieved by X-chromosome inactivation in female cells. Reprogramming female mouse somatic cells into induced pluripotent stem cells (iPSCs) leads to X-chromosome reactivation. The extent to which increased X-chromosome dosage (X-dosage) in female iPSCs leads to differences in the molecular and cellular properties of XX and XY iPSCs is still unclear. We show that chromatin accessibility in mouse iPSCs is modulated by X-dosage. Specific sets of transcriptional regulator motifs are enriched in chromatin with increased accessibility in XX or XY iPSCs. We show that the transcriptome, growth and pluripotency exit are also modulated by X-dosage in iPSCs. To understand the mechanisms by which increased X-dosage modulates the molecular and cellular properties of mouse pluripotent stem cells, we used heterozygous deletions of the X-linked gene Dusp9 in XX embryonic stem cells. We show that X-dosage regulates the transcriptome, open chromatin landscape, growth and pluripotency exit largely independently of global DNA methylation. Our results uncover new insights into X-dosage in pluripotent stem cells, providing principles of how gene dosage modulates the epigenetic and genetic mechanisms regulating cell identity.

2018-03-31 | GSE110215 | GEO

Next-generation sequencing facilitates quantitative analysis of the transcriptomes of FOXG1 100% dosage, FOXG1 60% dosage, FOXG1 30% dosage, and FOXG1 0% dosage GABA interneurons derived from human embryonic stem cells

Project description:Next-generation sequencing facilitates quantitative analysis of the transcriptomes of FOXG1 100% dosage GABA interneurons, FOXG1 60% dosage GABA interneurons, FOXG1 30% dosage GABA interneurons, and FOXG1 0% dosage GABA interneurons derived from human embryonic stem cells. We report a genetic manipulation system that enable precise dosage control of FOXG1 protein in human pluripotent stem cells (hPSCs). Using this system, we explored how the various reduced dosage affect human ventrol GABA interneuron development. We employed RNA seq on hPSC-derived GABA interneurons (day 60) to invest the expression pattern under different FOXG1 dosage conditions. RNA-Seq on GABA interneurons (Day 60) indicates that compared to the FOXG1 100% group, variable insufficiency of FOXG1 produces more than 1000 differently expressed genes (DEGs), and more DEGs in the group with less FOXG1 dosage. Heat map on Pearson Correlation indicates that groups with more discriminated FOXG1 exhibit much weaker correlation. Venn diagram reveals that each group has a set of distinct DEGs, suggesting that each FOXG1 protein dosage could results in different expression pattern during differentiation. The DEGs can be divided into two clusters, with one showing dosage-dependent regulation by FOXG1 and the other one typical binary. Key regulatory genes for GABA interneuron induction (NKX2-1, NKX6-2, GAD1, etc.) and for functional GABAergic-speciﬁc synapse formation (GABBR1, GABRA1, GABRB1, GABRG1, GABRQ, SHANK1, etc.) are down regulated along with reduction of FOXG1 protein.

2018-09-18 | GSE120079 | GEO

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