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Potent and uniform fetal hemoglobin induction via base editing [Cut & Run]

ABSTRACT: Potent and uniform fetal hemoglobin induction via base editing [Cut & Run]

PROVIDER: PRJNA951690 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR24049332_1.fastq.gz	Fastqsanger.gz
	SRR24049332_2.fastq.gz	Fastqsanger.gz
	SRR24049333_1.fastq.gz	Fastqsanger.gz
	SRR24049333_2.fastq.gz	Fastqsanger.gz
	SRR24049334_1.fastq.gz	Fastqsanger.gz

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Potent and uniform fetal hemoglobin induction via base editing

Project description:Potent and uniform fetal hemoglobin induction via base editing

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Potent and uniform fetal hemoglobin induction via base editing [Cut & Run]

Project description:Genetic manipulations to increase fetal hemoglobin (HbF, α2γ2) in postnatal red blood cells (RBCs) can alleviate β-thalassemia and sickle cell disease. We compared five strategies in CD34+ hematopoietic stem and progenitor cells, using either Cas9 nuclease, which creates uncontrolled indel mixtures, or adenine base editors (ABE), which generate more precise nucleotide changes. The most potent modification was ABE generation of γ-globin (HBG1/HBG2) −175 A>G, which creates a promoter binding motif for the transcriptional activator TAL1. In erythroid colonies with >87.5% on-target edits, those with −175 A>G expressed 81 ± 7% HbF, versus 17 ± 11% in unedited controls. In comparison, HbF levels were lower and more variable in erythroid cells modified with either of two Cas9 nuclease strategies with similar editing efficiencies, being 32 ± 19% when a BCL11A repressor binding motif in the γ-globin promoter was targeted and 52 ± 13% when the +58 BCL11A erythroid enhancer was targeted. Contrary to currently accepted models of γ-globin regulation, HbF levels varied significantly with different Cas9 indels that disrupted the γ-globin promoter BCL11A binding motif. The −175 A>G base edit also induced HbF more potently than did the Cas9 nuclease approaches in RBCs generated after transplantation of modified normal or SCD patient CD34+ cells into mice. Our data suggest a strategy for potent, uniform induction of HbF and provide insights into γ-globin gene regulation. More generally, we demonstrate that diverse indels generated by Cas9 nuclease can cause unexpected variations in biological outcomes that can be circumvented by base editing, with important implications for therapeutic gene editing efforts.

2023-04-28 | GSE228819 | GEO

Potent and uniform fetal hemoglobin induction via base editing.

Project description:Inducing fetal hemoglobin (HbF) in red blood cells can alleviate β-thalassemia and sickle cell disease. We compared five strategies in CD34+ hematopoietic stem and progenitor cells, using either Cas9 nuclease or adenine base editors. The most potent modification was adenine base editor generation of γ-globin -175A>G. Homozygous -175A>G edited erythroid colonies expressed 81 ± 7% HbF versus 17 ± 11% in unedited controls, whereas HbF levels were lower and more variable for two Cas9 strategies targeting a BCL11A binding motif in the γ-globin promoter or a BCL11A erythroid enhancer. The -175A>G base edit also induced HbF more potently than a Cas9 approach in red blood cells generated after transplantation of CD34+ hematopoietic stem and progenitor cells into mice. Our data suggest a strategy for potent, uniform induction of HbF and provide insights into γ-globin gene regulation. More generally, we demonstrate that diverse indels generated by Cas9 can cause unexpected phenotypic variation that can be circumvented by base editing.

| S-EPMC10722557 | biostudies-literature

Potent and uniform fetal hemoglobin induction via base editing

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

2023-04-28 | GSE228822 | GEO

Base editing improvement

Project description:Base editing improvement

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CUT&RUN of erythroblasts upon perturbation of gamma-hemoglobin repressors (part II)

Project description:Through high throughput compound screening, we've identified compounds that induce the expression of fetal hemoglobin. This study contains CUT&RUN data of a novel target, WIZ.

2024-06-19 | E-MTAB-13890 | biostudies-arrayexpress

Base editing improvement

Project description:We examined the off-targets of new base editing tools at RNA level

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Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

Project description:CRISPR-Cas base editors are preferred tool for genome editing as they generate desired editing without any double strand break in the genome, as double stand break is detrimental to the cells. In our study we have demonstrated the significance of base editors in editing the highly homologous HBG promoter (HBG1 and HBG2) region to introduce novel HPFH-like mutation to elevate HbF for therapeutical applications. Previous studies revealed that the base editors can cause unintended Cas-independent edits at transcriptome level. To validate off-target at RNA level, we performed a transcriptome wide analysis. The frequency of unintended edits in the HUDEP-2 stable cell lines expressing the base editors with the gRNA were not significant compared to the control. We determined the ABE mediated A to I conversion and CBE mediated C to U conversion across the base edited samples. The RNA off-target analysis was carried out with the help of REDItools v 2 tool. This data suggests that despite high on-target editing in DNA, the Cas-independent RNA off-target were not at detectable range compared to control. The differential expression of 34 selected genes which necessitate globin regulation were compared between the unedited HUDEP WT, CBE control, ABE control, and edited ABE (with gRNA 2/11) and edited CBE (with gRNA 2/11). We observed that there is no significant differential gene expression between the edited and control cells except the gamma and delta globin genes. These results suggest that base editors are preferred tools to edit highly homologous HBG promoter region to created HPFH-like mutations inducing HbF levels without causing double strand breaks, larger deletions and no significant RNA off-targets which are detrimental to the gene edited cells.

2022-01-02 | GSE192801 | GEO

RNA-seq and CUT&RUN of erythroblasts upon perturbation of gamma-hemoglobin repressors

Project description:Through high throughput compound screening, we've identified compounds that induce the expression of fetal hemoglobin. This study contains RNA-seq and CUT&RUN data of a novel target, WIZ.

2024-06-19 | GSE247096 | GEO

Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

Project description:Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

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