Allele-Specific gene editing approach for a dominant form of Epidermolysis Bullosa Simplex I

Epidermolysis Bullosa Simplex (EBS) is the most common form of Epidermolysis Bullosa (EB) and it is mainly inherited in an autosomal dominant manner (prevalence 1/30000 – 1/50000). Several clinical variants have been described based on the mutated gene, the site of blister formation and the anatomical distribution, but the vast majority of the patients display dominant mutations in genes encoding keratin 5 (KRT5) and keratin 14 (KRT14). The lack of functional keratin intermediate filaments causes basal keratinocytes to exhibit a dramatic cytoplasmatic softening and rupture, when subjected to minor mechanical traction, leading to the distinctive EBS patients intraepidermal blisters formation. Whilst viral mediated addition of a corrected copy of the altered gene is the ascertained approach to tackle recessively inherited EB (such as Junctional and Dystrophic EB), a potential successful combined cell and gene therapy for EBS dominant forms requires the editing of the mutated gene. In this case study, we outlined an allele specific CRISPR/Cas9 gene editing approach able to specifically detect and disrupt a de novo monoallelic c.475/495del21 mutation within exon 1 of KRT14. Taking advantage of the tailored CRISPR/Cas9 system to induce a NHEJ mediated frameshift mutations introduction, we attained a remarkable mutant allele knock-out efficiency. Following KRT14 mutant allele specific gene editing, patient derived primary keratinocytes (EBS01) restored a normal intermediate filament network and mechanical stress resilience. Overall design: Single cell-RNA seq of in vitro cultivated human primary keratinocytes derived from patient affected by Epidermolysis Bullosa Symplex, untreated or treated with KRT14-specific editing strategy