Journal Article Epublish 1 citation
PubMed: 39944129 DOI: 10.1016/j.xjidi.2024.100343
Efficient Dual Cas9 Nickase Correction of a Prevalent Pathogenic Variant for Junctional Epidermolysis Bullosa.
JID Innov 5(3):100343
Authors: Alex du Rand, John Hunt, Daniel Verdon, Ben Buttle, P Rod Dunbar, Diana Purvis, Vaughan Feisst, Hilary Sheppard
Affiliations
- School of Biological Sciences, The University of Auckland, Auckland, New Zealand.
- Maurice Wilkins Centre, The University of Auckland, Auckland, New Zealand.
- Te Whatu Ora Health New Zealand, Te Toka Tumai, Auckland, New Zealand.
Abstract
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 , 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 , 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.
MeSH
Skin, Genes, Epidermolysis Bullosa, Junctional, Epidermolysis Bullosa, Keratinocytes, Laminin