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essential for skin integrity. In this study, we developed a cell line suitable for gene expression studies of the JEB-associated COL17A1 encoding type XVII collagen (C17), a transmembrane protein involved in connecting basal keratinocytes to the underlying dermis of the skin. Using the CRISPR\/Cas9 system of Streptococcus pyogenes we fused the coding sequence of GFP to COL17A1 leading to the constitutive expression of GFP-C17 fusion proteins under the control of the endogenous promoter in human wild-type and JEB keratinocytes. We confirmed the accurate full-length expression and localization of GFP-C17 to the plasma membrane via fluorescence microscopy and Western blot analysis. As expected, the expression of GFP-C17mut fusion proteins in JEB keratinocytes generated no specific GFP signal. However, the CRISPR\/Cas9-mediated repair of a JEB-associated frameshift mutation in GFP-COL17A1mut-expressing JEB cells led to the restoration of GFP-C17, apparent in the full-length expression of the fusion protein, its accurate localization within the plasma membrane of keratinocyte monolayers as well as within the basement membrane zone of 3D-skin equivalents. Thus, this fluorescence-based JEB cell line provides the potential to serve as a platform to screen for personalized gene editing molecules and applications in vitro and in appropriate animal models in vivo.<\/jats:p>","DOI":"10.3390\/ijms24065197","type":"journal-article","created":{"date-parts":[[2023,3,9]],"date-time":"2023-03-09T02:33:16Z","timestamp":1678329196000},"page":"5197","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["A Novel Fluorescence-Based Screen of Gene Editing Molecules for Junctional Epidermolysis Bullosa"],"prefix":"10.3390","volume":"24","author":[{"given":"Janine","family":"Zwicklhuber","sequence":"first","affiliation":[{"name":"EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, 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Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anna","family":"Raninger","sequence":"additional","affiliation":[{"name":"Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University, 5020 Salzburg, Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Iris","family":"Gratz","sequence":"additional","affiliation":[{"name":"EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria"},{"name":"Department of Biosciences and Medical Biology, University of Salzburg, 5020 Salzburg, Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8705-3890","authenticated-orcid":false,"given":"Verena","family":"Wally","sequence":"additional","affiliation":[{"name":"EB House Austria, Research Program for 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