Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease
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 edi...
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Nature Portfolio
2025-08-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-62738-2 |
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| author | Jonas Holst Wolff Thomas Wisbech Skov Didde Haslund Sofie Rahbek Dorset Anne Louise S. Revenfeld Clotilde Aussel Sofie E. Jørgensen Mette Holm Martin K. Thomsen Sandra Ammann Toni Cathomen Trine H. Mogensen Bjarne Kuno Møller Rasmus O. Bak Jacob Giehm Mikkelsen |
| author_facet | Jonas Holst Wolff Thomas Wisbech Skov Didde Haslund Sofie Rahbek Dorset Anne Louise S. Revenfeld Clotilde Aussel Sofie E. Jørgensen Mette Holm Martin K. Thomsen Sandra Ammann Toni Cathomen Trine H. Mogensen Bjarne Kuno Møller Rasmus O. Bak Jacob Giehm Mikkelsen |
| author_sort | Jonas Holst Wolff |
| collection | DOAJ |
| description | 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+ 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. |
| format | Article |
| id | doaj-art-d2ec5e307af34c8fae6f46c031c304b9 |
| institution | DOAJ |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-d2ec5e307af34c8fae6f46c031c304b92025-08-20T03:05:10ZengNature PortfolioNature Communications2041-17232025-08-0116111610.1038/s41467-025-62738-2Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous diseaseJonas Holst Wolff0Thomas Wisbech Skov1Didde Haslund2Sofie Rahbek Dorset3Anne Louise S. Revenfeld4Clotilde Aussel5Sofie E. Jørgensen6Mette Holm7Martin K. Thomsen8Sandra Ammann9Toni Cathomen10Trine H. Mogensen11Bjarne Kuno Møller12Rasmus O. Bak13Jacob Giehm Mikkelsen14Department of Biomedicine, Aarhus UniversityDepartment of Biomedicine, Aarhus UniversityDepartment of Clinical Immunology, Aarhus University HospitalDepartment of Biomedicine, Aarhus UniversityDepartment of Clinical Immunology, Aarhus University HospitalInstitute for Transfusion Medicine and Gene Therapy, Medical Center – University of FreiburgDepartment of Infectious Diseases, Aarhus University HospitalDepartment of Paediatrics and Adolescent Medicine, Aarhus University HospitalDepartment of Biomedicine, Aarhus UniversityInstitute for Transfusion Medicine and Gene Therapy, Medical Center – University of FreiburgInstitute for Transfusion Medicine and Gene Therapy, Medical Center – University of FreiburgDepartment of Biomedicine, Aarhus UniversityDepartment of Clinical Immunology, Aarhus University HospitalDepartment of Biomedicine, Aarhus UniversityDepartment of Biomedicine, Aarhus UniversityAbstract 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+ 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.https://doi.org/10.1038/s41467-025-62738-2 |
| spellingShingle | Jonas Holst Wolff Thomas Wisbech Skov Didde Haslund Sofie Rahbek Dorset Anne Louise S. Revenfeld Clotilde Aussel Sofie E. Jørgensen Mette Holm Martin K. Thomsen Sandra Ammann Toni Cathomen Trine H. Mogensen Bjarne Kuno Møller Rasmus O. Bak Jacob Giehm Mikkelsen Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease Nature Communications |
| title | Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease |
| title_full | Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease |
| title_fullStr | Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease |
| title_full_unstemmed | Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease |
| title_short | Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease |
| title_sort | targeted gene editing and near universal cdna insertion of cyba and cybb as a treatment for chronic granulomatous disease |
| url | https://doi.org/10.1038/s41467-025-62738-2 |
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