Direct correction of haemoglobin E / beta-thalassaemia using base editors
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA849199 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR19650827_1.fastq.gz | Fastqsanger.gz | |||
| SRR19650827_2.fastq.gz | Fastqsanger.gz | |||
| SRR19650828_1.fastq.gz | Fastqsanger.gz | |||
| SRR19650828_2.fastq.gz | Fastqsanger.gz | |||
| SRR19650829_1.fastq.gz | Fastqsanger.gz |
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Project description:Haemoglobin E (HbE)/β-thalassaemia causes approximately 50% of all severe thalassaemia worldwide; equating to around 30,000 births per year. HbE/β-thalassaemia is due to a point mutation in codon 26 of the human HBB gene on one allele (GAG; glutamate → GAA; lysine, E26K), and any mutation causing severe β-thalassaemia on the other. When inherited together in compound heterozygosity these mutations can cause a severe thalassaemic phenotype. However, if only one allele is mutated individuals are carriers for the respective mutation and have an asymptomatic phenotype (β-thalassaemia trait). Here we describe a base editing strategy which corrects the HbE mutation either to wildtype (WT) or a normal variant haemoglobin (E26G) known as Hb Aubenas and thereby recreates the asymptomatic trait phenotype. We have achieved editing efficiencies in excess of 85% in primary human CD34+ cells. We demonstrate editing of long-term repopulating haematopoietic stem cells (LT-HSCs) using serial xenotransplantation in NSG mice. We have profiled the off-target effects using a combination of CIRCLE-seq and deep targeted capture and have developed machine-learning based methods to predict functional effects of candidate off-target mutations.
2023-01-25 | GSE206098 | GEO
Project description:Haemoglobin E (HbE) β-thalassaemia causes approximately 50% of all severe thalassaemia worldwide; equating to around 30,000 births per year. HbE β-thalassaemia is due to a point mutation in codon 26 of the human HBB gene on one allele (GAG; glutamatic acid → AAG; lysine, E26K), and any mutation causing severe β-thalassaemia on the other. When inherited together in compound heterozygosity these mutations can cause a severe thalassaemic phenotype. However, if only one allele is mutated individuals are carriers for the respective mutation and have an asymptomatic phenotype (β-thalassaemia trait). Here we describe a base editing strategy which corrects the HbE mutation either to wildtype (WT) or a normal variant haemoglobin (E26G) known as Hb Aubenas and thereby recreates the asymptomatic trait phenotype. We have achieved editing efficiencies in excess of 90% in primary human CD34 + cells. We demonstrate editing of long-term repopulating haematopoietic stem cells (LT-HSCs) using serial xenotransplantation in NSG mice. We have profiled the off-target effects using a combination of circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) and deep targeted capture and have developed machine-learning based methods to predict functional effects of candidate off-target mutations.
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Project description:A variety of base editors have been developed to achieve C-to-T editing in different genomic contexts. Here, we compare a panel of five base editors on their C-to-T editing efficiencies and product purity at commonly-editable sites, including some human pathogenic C-to-T mutations. We further profile the accessibilities of twenty base editors to all possible pathogenic mutations in silico. Finally, we build the BEable-GPS (Base Editable prediction of Global Pathogenic SNVs) database for users to select proper base editors to model or correct disease-related mutations. This in-vivo comparison and in-silico profiling catalogs the availability of base editors and their broad applications in biomedical studies.
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Project description:CRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described “enhanced” A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.
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Project description:CRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described “enhanced” A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.
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Project description:The therapeutic application of base editors is currently limited by their large sizes, which are often beyond the packaging capabilities of adeno-associated viral (AAV) vectors. Despite recent progress in mega genome mining that has identified a diverse array of compact CRISPR proteins (e.g. Cas12f, TnpB, and IscB), the resulting miniature base editors are often exhibited reduced activities and a limited targeting scope, leaving a broad spectrum of disease-relevant genetic variants inaccessible. In this study, we have developed a platform, designated as Zinc Finger Proteins (ZFP)-enhanced miniature base editor (zmBE), which integrates programmable DNA-binding domains to enhance the efficiency and expand targeting scope of miniature base editors, including those based on Un1Cas12f1 and OgeuIscB. Utilizing protein language model (PLM) for the design of ZFPs further simplified development and optimization of zmBEs tailored to a specific target. Leveraging these methodologies, we engineered a zmBE that effectively induced the SMN2 exon 7 A6>G conversion and restored SMN2 exon 7 inclusion. Our study thus provides a versatile platform for developing miniature base editors for in vivo therapeutic applications.
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Project description:Techniques for exclusion of exons from mature transcripts have been applied as gene therapies for treating many different diseases. Since exon skipping has been traditionally accomplished using technologies that have a transient effect, it is particularly important to develop new techniques that enable permanent exon skipping. We have recently demonstrated that this can be accomplished using cytidine base editors for permanently disabling the splice acceptor of target exons. We now demonstrate the application of adenine-deaminase base editors to disrupt the conserved adenosine within splice acceptor sites for programmable exon skipping. We also demonstrate that by altering the amino acid sequence of the linker between the adenosine deaminase domain and the Cas9 nickase or by coupling the adenine base editor with a uracil glycosylase inhibitor, the DNA editing efficiency and exon skipping rates improve significantly. Finally, we developed a split base editor architecture compatible with adeno-associated viral packaging. Collectively, these results represent significant progress towards permanent in vivo exon skipping through base editing and, ultimately, a new modality of gene therapy for the treatment of genetic diseases.
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Project description:CRISPR gene editing has revolutionized biomedicine and biotechnology by providing a simple means to engineer genes in vivo by introducing mutations at target sites in the genomic DNA of living cells. However, given the stochasticity of cellular DNA repair mechanisms and the potential for introducing mutations at off-target sites, technologies capable of introducing targeted changes with increased precision, such as cytidine deaminase single-base editors, are preferred. We here present a versatile method termed CRISPR-SKIP that utilizes cytidine deaminase single-base editors to program de-novo exon skipping by mutating target DNA bases within splice acceptor sites. Given its simplicity and precision, CRISPR-SKIP will be broadly applicable in gene therapy and synthetic biology.
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Project description:CRISPR gene editing has revolutionized biomedicine and biotechnology by providing a simple means to engineer genes in vivo by introducing mutations at target sites in the genomic DNA of living cells. However, given the stochasticity of cellular DNA repair mechanisms and the potential for introducing mutations at off-target sites, technologies capable of introducing targeted changes with increased precision, such as cytidine deaminase single-base editors, are preferred. We here present a versatile method termed CRISPR-SKIP that utilizes cytidine deaminase single-base editors to program de-novo exon skipping by mutating target DNA bases within splice acceptor sites. Given its simplicity and precision, CRISPR-SKIP will be broadly applicable in gene therapy and synthetic biology.
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Project description:CRISPR gene editing has revolutionized biomedicine and biotechnology by providing a simple means to engineer genes in vivo by introducing mutations at target sites in the genomic DNA of living cells. However, given the stochasticity of cellular DNA repair mechanisms and the potential for introducing mutations at off-target sites, technologies capable of introducing targeted changes with increased precision, such as cytidine deaminase single-base editors, are preferred. We here present a versatile method termed CRISPR-SKIP that utilizes cytidine deaminase single-base editors to program de-novo exon skipping by mutating target DNA bases within splice acceptor sites. Given its simplicity and precision, CRISPR-SKIP will be broadly applicable in gene therapy and synthetic biology.
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