Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease

Chronic granulomatous disease (CGD) is a severe inborn error of immunity caused by NADPH oxidase defects. Here, we develop CRISPR/Cas9-based gene editing strategies for correction of variants in the CYBA and CYBB genes causing CGD. For X-linked CGD, we also develop a near-universal gene editing strategy by targeted integration of a truncated CYBB cDNA in CD34<sup>+</sup> hematopoietic stem and progenitor cells (HSPCs). Throughout, off-target editing and chromosomal translocations are evident, which negatively impact the ability of gene-edited HSPCs to engraft in immunodeficient mice. However, by employing a high-fidelity Cas9 to minimize off-target editing, we demonstrate restoration of the multilineage engraftment potential of gene-edited HSPCs. Moreover, to further improve safety, we develop a D10A Cas9n editing approach with no detectable off-target activity or chromosomal translocations. Collectively, through risk assessments of different gene editing approaches, we present a D10A Cas9n-based strategy with improved safety, offering a potentially curative treatment for CGD patients.

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Publication Details

Journal
Nature Communications
Published
2025-08-12
DOI
https://doi.org/10.1038/s41467-025-62738-2
Primary Topic
CRISPR and Genetic Engineering
Type
article
Field-Weighted Citation Impact
0.00

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article

Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease

Thomas Wisbech Skov, Sofie E. Jørgensen, Rasmus Otkjær Bak, Didde Haslund et al.
Nature Communications
CRISPR and Genetic Engineering
article

Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease

Thomas Wisbech Skov, Sofie E. Jørgensen, Rasmus Otkjær Bak, Didde Haslund, Martin Kristian Thomsen, Anne Louise Revenfeld, Bjarne Kuno Møller, Trine Hyrup Mogensen, Jonas Holst Wolff, Mette Holm, Sandra Ammann, Jacob Giehm Mikkelsen, Toni Cathomen, Sofie Rahbek Dorset, Clotilde Aussel
article en

Abstract

Chronic granulomatous disease (CGD) is a severe inborn error of immunity caused by NADPH oxidase defects. Here, we develop CRISPR/Cas9-based gene editing strategies for correction of variants in the CYBA and CYBB genes causing CGD. For X-linked CGD, we also develop a near-universal gene editing strategy by targeted integration of a truncated CYBB cDNA in CD34<sup>+</sup> hematopoietic stem and progenitor cells (HSPCs). Throughout, off-target editing and chromosomal translocations are evident, which negatively impact the ability of gene-edited HSPCs to engraft in immunodeficient mice. However, by employing a high-fidelity Cas9 to minimize off-target editing, we demonstrate restoration of the multilineage engraftment potential of gene-edited HSPCs. Moreover, to further improve safety, we develop a D10A Cas9n editing approach with no detectable off-target activity or chromosomal translocations. Collectively, through risk assessments of different gene editing approaches, we present a D10A Cas9n-based strategy with improved safety, offering a potentially curative treatment for CGD patients.

Nature CommunicationsVol. 16(1)
University of Freiburg (DE), Aarhus University (DK), Aarhus University Hospital (DK)
Aarhus Universitet, Innovationsfonden, Deutsche Forschungsgemeinschaft, Carlsbergfondet, Novo Nordisk, Novo Nordisk Fonden
Good health and well-being
Openalex Percentile: Top 82%
CRISPR and Genetic Engineering
DOIOpen Access PDFOpenAlex
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