A Novel Fluorescence-Based Screen of Gene Editing Molecules for Junctional Epidermolysis Bullosa

Janine Zwicklhuber, Thomas Kocher, Bernadette Liemberger, Stefan Hainzl, Johannes Bischof, Dirk Strunk, Anna M Raninger, Iris Gratz, Verena Wally, Christina Guttmann-Gruber, Josefina Piñón Hofbauer, Johann W Bauer, Ulrich Koller

Research output: Contribution to journalArticlepeer-review

Abstract

Junctional epidermolysis bullosa (JEB) is a severe blistering skin disease caused by mutations in genes encoding structural proteins 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.

Original languageEnglish
JournalInternational Journal of Molecular Sciences
Volume24
Issue number6
DOIs
Publication statusPublished - 8 Mar 2023

Keywords

  • Animals
  • Humans
  • Epidermolysis Bullosa, Junctional/genetics
  • Gene Editing
  • Skin
  • Mutation
  • Keratinocytes
  • Epidermolysis Bullosa/genetics

Fields of Science and Technology Classification 2012

  • 106 Biology
  • 304 Medical Biotechnology

Cite this