Efficient Dual Cas9 Nickase Correction of a Prevalent Pathogenic LAMB3 Variant for Junctional Epidermolysis Bullosa
Gene editing facilitated by homology-directed repair represents a promising strategy for precisely correcting pathogenic variants underlying monogenic disorders, including the life-threatening skin blistering condition junctional epidermolysis bullosa (JEB). Frequent reports of unintended off-target...
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Elsevier
2025-05-01
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| Series: | JID Innovations |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2667026724000912 |
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| author | Alex du Rand John Hunt Daniel Verdon Ben Buttle P. Rod Dunbar Diana Purvis Vaughan Feisst Hilary Sheppard |
| author_facet | Alex du Rand John Hunt Daniel Verdon Ben Buttle P. Rod Dunbar Diana Purvis Vaughan Feisst Hilary Sheppard |
| author_sort | Alex du Rand |
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| description | Gene editing facilitated by homology-directed repair represents a promising strategy for precisely correcting pathogenic variants underlying monogenic disorders, including the life-threatening skin blistering condition junctional epidermolysis bullosa (JEB). Frequent reports of unintended off-target genotoxicity associated with conventional Cas9 nuclease editing have increasingly led to the adoption of dual-Cas9 nickases (dual-Cas9n) owing to their improved safety profile. However, rates of precise repair obtained with such strategies remain low. In this study, we establish a dual-Cas9n approach targeting LAMB3, using electroporation to deliver Cas9-nickase ribonucleoproteins and modified single-stranded oligodeoxynucleotide repair templates into primary JEB keratinocytes. Targeting a hotspot pathogenic variant (c.1903C>T, p.R635∗), we report perfect correction efficiencies of up to 54% based on standard next-generation sequencing. Using a high-fidelity Cas9 nuclease, we also report perfect repair of up to 74% when using a small-molecule modulator of DNA repair. Dual-Cas9n–corrected JEB keratinocytes demonstrated restored laminin-332 expression and secretion in vitro, leading to improved cellular adhesion and accurate laminin-332 localization in engineered skin equivalents. This protocol represents a significant improvement in precision gene repair using Cas9 nickases for epidermolysis bullosa, with the potential to be applied to a large cohort of patients harboring this prevalent pathogenic variant. |
| format | Article |
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| institution | Kabale University |
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| language | English |
| publishDate | 2025-05-01 |
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| series | JID Innovations |
| spelling | doaj-art-d27057a7813240ca976949b81b0317f72025-01-30T05:15:09ZengElsevierJID Innovations2667-02672025-05-0153100343Efficient Dual Cas9 Nickase Correction of a Prevalent Pathogenic LAMB3 Variant for Junctional Epidermolysis BullosaAlex du Rand0John Hunt1Daniel Verdon2Ben Buttle3P. Rod Dunbar4Diana Purvis5Vaughan Feisst6Hilary Sheppard7School of Biological Sciences, The University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre, The University of Auckland, Auckland, New ZealandSchool of Biological Sciences, The University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre, The University of Auckland, Auckland, New ZealandSchool of Biological Sciences, The University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre, The University of Auckland, Auckland, New ZealandSchool of Biological Sciences, The University of Auckland, Auckland, New ZealandSchool of Biological Sciences, The University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre, The University of Auckland, Auckland, New ZealandTe Whatu Ora Health New Zealand, Te Toka Tumai, Auckland, New ZealandSchool of Biological Sciences, The University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre, The University of Auckland, Auckland, New ZealandSchool of Biological Sciences, The University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre, The University of Auckland, Auckland, New Zealand; Correspondence: Hilary Sheppard, School of Biological Sciences, The University of Auckland, 3a Symonds Street, Auckland 1010, New Zealand.Gene editing facilitated by homology-directed repair represents a promising strategy for precisely correcting pathogenic variants underlying monogenic disorders, including the life-threatening skin blistering condition junctional epidermolysis bullosa (JEB). Frequent reports of unintended off-target genotoxicity associated with conventional Cas9 nuclease editing have increasingly led to the adoption of dual-Cas9 nickases (dual-Cas9n) owing to their improved safety profile. However, rates of precise repair obtained with such strategies remain low. In this study, we establish a dual-Cas9n approach targeting LAMB3, using electroporation to deliver Cas9-nickase ribonucleoproteins and modified single-stranded oligodeoxynucleotide repair templates into primary JEB keratinocytes. Targeting a hotspot pathogenic variant (c.1903C>T, p.R635∗), we report perfect correction efficiencies of up to 54% based on standard next-generation sequencing. Using a high-fidelity Cas9 nuclease, we also report perfect repair of up to 74% when using a small-molecule modulator of DNA repair. Dual-Cas9n–corrected JEB keratinocytes demonstrated restored laminin-332 expression and secretion in vitro, leading to improved cellular adhesion and accurate laminin-332 localization in engineered skin equivalents. This protocol represents a significant improvement in precision gene repair using Cas9 nickases for epidermolysis bullosa, with the potential to be applied to a large cohort of patients harboring this prevalent pathogenic variant.http://www.sciencedirect.com/science/article/pii/S2667026724000912CRISPR/Cas9Gene correctionGene therapyHomology-directed repairSkin grafts |
| spellingShingle | Alex du Rand John Hunt Daniel Verdon Ben Buttle P. Rod Dunbar Diana Purvis Vaughan Feisst Hilary Sheppard Efficient Dual Cas9 Nickase Correction of a Prevalent Pathogenic LAMB3 Variant for Junctional Epidermolysis Bullosa JID Innovations CRISPR/Cas9 Gene correction Gene therapy Homology-directed repair Skin grafts |
| title | Efficient Dual Cas9 Nickase Correction of a Prevalent Pathogenic LAMB3 Variant for Junctional Epidermolysis Bullosa |
| title_full | Efficient Dual Cas9 Nickase Correction of a Prevalent Pathogenic LAMB3 Variant for Junctional Epidermolysis Bullosa |
| title_fullStr | Efficient Dual Cas9 Nickase Correction of a Prevalent Pathogenic LAMB3 Variant for Junctional Epidermolysis Bullosa |
| title_full_unstemmed | Efficient Dual Cas9 Nickase Correction of a Prevalent Pathogenic LAMB3 Variant for Junctional Epidermolysis Bullosa |
| title_short | Efficient Dual Cas9 Nickase Correction of a Prevalent Pathogenic LAMB3 Variant for Junctional Epidermolysis Bullosa |
| title_sort | efficient dual cas9 nickase correction of a prevalent pathogenic lamb3 variant for junctional epidermolysis bullosa |
| topic | CRISPR/Cas9 Gene correction Gene therapy Homology-directed repair Skin grafts |
| url | http://www.sciencedirect.com/science/article/pii/S2667026724000912 |
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