Deficiency of basement membrane heterotrimeric laminin 332 component, coded by LAMA3, LAMB3, and LAMC2genes, causes junctional epidermolysis bullosa (JEB), a severe skin adhesion defect. Herein, we report the first application of CRISPR/Cas9-mediated homology direct repair (HDR) to in siturestore LAMB3 expression in JEB keratinocytes in vitroand in immunodeficient mice transplanted with genetically corrected skin equivalents. We packaged an adenovector carrying Cas9/guide RNA (gRNA) tailored to the intron 2 of LAMB3gene and an integration defective lentiviral vector bearing a promoterless quasi-complete LAMB3cDNA downstream a splice acceptor site and flanked by homology arms. Upon genuine HDR, we exploited the in vitroadhesion advantage of laminin 332 production to positively select LAMB3-expressing keratinocytes. HDR and restored laminin 332 expression were evaluated at single-cell level. Notably, monoallelic-targeted integration of LAMB3cDNA was sufficient to in vitrorecapitulate the adhesive property, the colony formation typical of normal keratinocytes, as well as their cell growth. Grafting of genetically corrected skin equivalents onto immunodeficient mice showed a completely restored dermal-epidermal junction. This study provides evidence for efficient CRISPR/Cas9-mediated in siturestoration of LAMB3 expression, paving the way for ex vivoclinical application of this strategy to laminin 332 deficiency.