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Multiplex CRISPR/Cas9-Based Genome Editing of Mouse Hematopoietic Stem Cells Recapitulates Acute Erythroid Leukemia and Identifies Therapeutic Targets [Bisulfite-Seq]

ABSTRACT: Multiplex CRISPR/Cas9-Based Genome Editing of Mouse Hematopoietic Stem Cells Recapitulates Acute Erythroid Leukemia and Identifies Therapeutic Targets [Bisulfite-Seq]

PROVIDER: PRJNA613910 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11364800_1.fastq.gz	Fastqsanger.gz
	SRR11364800_2.fastq.gz	Fastqsanger.gz
	SRR11364801_1.fastq.gz	Fastqsanger.gz
	SRR11364801_2.fastq.gz	Fastqsanger.gz
	SRR11364802_1.fastq.gz	Fastqsanger.gz

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Multiplex CRISPR/Cas9-Based Genome Editing of Mouse Hematopoietic Stem Cells Recapitulates Acute Erythroid Leukemia and Identifies Therapeutic Targets [Bisulfite-Seq]

Project description:Acute erythroid leukemia (AEL) is a high-risk leukemia with a distinct mutational spectrum. Here, we examined the cooperativity of loss of function alterations in leukemogenesis and tested the therapeutic tractability of each model of AEL. Using CRISPR/Cas9 genome editing, we generated six pools of lentiviral vectors with different combinations of guide RNAs and transduced Cas9-eGFP-mouse mouse hematopoietic stem and progenitor cells (HSPCs) that were used in transplant assays. Induced tumors were characterized by phenotype, genomic/epigenomic analysis and tested for sensitivity to 222 therapeutic agents. Tumor genotype determined tumor phenotype with Bcor and Tp53 driving erythroid leukemogenesis. Tumors showed acquisition of additional somatic mutations during serial passaging and by transcriptomic analysis AEL models had overexpression of transcription factors required for differentiation of megakaryocyte-erythroid progenitors and recapitulated human AEL. Dnmt3a-mutated models exhibited global hypomethylation, but hypermethylation when co-occurred with Tet2 mutations. Drug response was associated with genotype. Tp53 wild-type but Dnmt3a/Tet2 double mutant cells were the most chemo-sensitive models. ViceversaTp53-mutated AEL subtypes were chemo-resistant to several conventional chemotherapeutics but highly sensitivity to PARP inhibitors, including talazoparib. Sensitivity to talazoparib was confirmed in vivo when combined with decitabine. In conclusion, combinations of recurrently mutated genes are critical determinants of leukemia phenotype, with central roles of Tp53 and Bcor mutations in driving the erythroid leukemogenesis. Using these models as preclinical tools, we have shown the potential for bromodomain and PARP inhibition as new therapeutic strategies in AEL.

2021-02-10 | GSE147343 | GEO

Multiplex CRISPR/Cas9-Based Genome Editing of Mouse Hematopoietic Stem Cells Recapitulates Acute Erythroid Leukemia and Identifies Therapeutic Targets

Project description:Multiplex CRISPR/Cas9-Based Genome Editing of Mouse Hematopoietic Stem Cells Recapitulates Acute Erythroid Leukemia and Identifies Therapeutic Targets

| PRJNA613914 | ENA

Multiplex CRISPR/Cas9-Based Genome Editing of Mouse Hematopoietic Stem Cells Recapitulates Acute Erythroid Leukemia and Identifies Therapeutic Targets [RNA-Seq]

Project description:Multiplex CRISPR/Cas9-Based Genome Editing of Mouse Hematopoietic Stem Cells Recapitulates Acute Erythroid Leukemia and Identifies Therapeutic Targets [RNA-Seq]

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Multiplex CRISPR/Cas9-Based Genome Editing of Mouse Hematopoietic Stem Cells Recapitulates Acute Erythroid Leukemia and Identifies Therapeutic Targets [RNA-Seq]

2021-02-10 | GSE147265 | GEO

Exemplary multiplex bisulfite amplicon data used to demonstrate the utility of Methpat.

Project description:DNA methylation is a complex epigenetic marker that can be analysed using a wide variety of methods. Interpretation and visualisation of DNA methylation data can mask complexity in terms of methylation status at each CpG site, cellular heterogeneity of samples and allelic DNA methylation patterns within a given DNA strand. Bisulfite sequencing is considered the gold standard, however visualisation of massively parallel sequencing results remains a significant challenge. We created a program called Methpat that facilitates visualisation and interpretation of bisulfite sequencing data generated by massively parallel sequencing. To demonstrate this, we performed multiplex PCR that targeted 48 regions of interest across 95 human samples. The regions selected included known gene promoters associated with cancer, repetitive elements, known imprinted regions and mitochondrial genomic sequences. We interrogated a range of samples including human cell lines, primary tumours and primary tissue samples. Methpat generates two forms of output: a tab delimited text file for each sample that summarises DNA methylation patterns and their read counts for each amplicon and a HTML file that summarises this data visually. Methpat can be used with publicly available whole genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) datasets with sufficient read depths. Using Methpat, complex DNA methylation data derived from massively parallel sequencing can be summarised and visualised for biological interpretation. By accounting for allelic DNA methylation states and their abundance in a sample, Methpat can unmask the complexity of DNA methylation and reveal further biological insight in existing datasets. Multiplex bisulfite PCR and Next Generation sequencing of primary human samples and breast cancer cell lines.

2015-11-23 | E-GEOD-71804 | biostudies-arrayexpress

Multiplex base editing of BCL11A regulatory elements to treat sickle cell disease

Project description:Sickle cell disease (SCD) is a genetic anemia caused by the production of an abnormal adult hemoglobin. The clinical severity is lessened by elevated fetal hemoglobin (HbF) production in adulthood. A promising therapy is the transplantation of autologous, hematopoietic stem/progenitor cells (HSPCs) treated with CRISPR/Cas9 to downregulate the HbF repressor BCL11A via generation of double strand breaks (DSBs) in the +58-kb erythroid-specific enhancer. Here, to further enhance HbF production without increasing the mutagenic load, we targeted both +58-kb and +55-kb BCL11A erythroid-specific enhancers using base editors. We systematically dissected DNA motifs recognized by the key transcriptional activators within these regions and identified the critical nucleotides required for activator binding. Multiplex base editing of these residues was efficient and safe and generated no or little DSBs and genomic rearrangements. We observed substantial HbF reactivation, exceeding the levels achieved using the CRISPR/Cas9 nuclease-based strategy, thus efficiently rescuing the sickling phenotype. Multiplex base editing was efficient in long-term repopulating HSPCs and resulted in potent HbF reactivation in vivo. In summary, these results show that multiplex base editing of BCL11A erythroid-specific enhancers is a safe and potent strategy for treating sickle cell disease.

2025-07-28 | GSE291384 | GEO

Multiplex CRISPR/Cas9-Based Genome Editing of Mouse Hematopoietic Stem Cells Recapitulates Acute Erythroid Leukemia and Identifies Therapeutic Targets

Project description:This SuperSeries is composed of the SubSeries listed below.

2021-02-10 | GSE147344 | GEO

Multiplex genome-editing in Arabidopsis thaliana using Mb3Cas12a

Project description:The use of CRISPR/Cas proteins for the creation of multiplex genome-engineering represents an important avenue for crop improvement, and further improvements for creation of knock-in plant lines via CRISPR-based technologies may enable the high-throughput creation of designer alleles. To circumvent limitations of the commonly used CRISPR/Cas9 system for multiplex genome-engineering, we explored the use of Moraxella bovoculi 3 Cas12a (Mb3Cas12a) for multiplex genome-editing in Arabidopsis thaliana. We identified optimized promoter sequences for driving expression of single transcript multiplex crRNA arrays in A. thaliana, resulting in stable germline transmission of Mb3Cas12a-edited alleles at multiple target sites. By utilizing this system, we demonstrate single-transcript multiplexed genome-engineering using of up to 13 crRNA targets. We further show high target specificity of Mb3Cas12a-based genome-editing via whole-genome sequencing. Taken together, our method provides a simplified platform for efficient multiplex-genome-engineering in plant-based systems.

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Exemplary multiplex bisulfite amplicon data used to demonstrate the utility of Methpat.

2015-11-23 | GSE71804 | GEO

Dysregulated erythroid gene expression after TET2 loss is partially corrected by 5-azacytidine in TF-1 cells [Bisulfite-seq]

Project description:Using bisulfite padlock probes, mRNA sequencing, and hydroxymethylcytosine pull-down sequencing, we show that TET2 loss particularly influences DNA methylation (5mC) and hydroxymethylation (5hmC) patterns at erythroid gene enhancers and is associated with down-regulation of erythroid gene expression in the human erythroleukemia cell line TF-1. We show that 5-Aza disproportionately induces expression of these down regulated genes in TET2KO cells and that this effect may be related to 5mC changes at erythroid gene enhancers after 5-Aza exposure. This work highlights the role of 5mC and 5hmC changes at distal regulatory elements in regulating the expression of differentiation-associated gene signatures, and sheds light on how 5-Aza may be more effective in patients harboring TET2 mutations.

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