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Deficiency of basement membrane heterotrimeric laminin 332 component, coded by LAMA3, LAMB3, and LAMC2 genes, 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 situ restore LAMB3 expression in JEB keratinocytes in vitro and in immunodeficient mice transplanted with genetically corrected skin equivalents. We packaged an adenovector carrying Cas9/guide RNA (gRNA) tailored to the intron 2 of LAMB3 gene and an integration defective lentiviral vector bearing a promoterless quasi-complete LAMB3 cDNA downstream a splice acceptor site and flanked by homology arms. Upon genuine HDR, we exploited the in vitro adhesion 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 LAMB3 cDNA was sufficient to in vitro recapitulate 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 situ restoration of LAMB3 expression, paving the way for ex vivo clinical application of this strategy to laminin 332 deficiency.