137 Filaggrin knock-out in keratinocytes indicates a functional role in skin barrier formation

Abstract

Skin barrier function is the result of orchestrated terminal differentiation of keratinocytes, forming the lipid-surrounded hydrophobic cornified envelope of the stratum corneum. CRISPR-Cas9 has the potential to meticulously dissect the functional and structural components of the stratum corneum by precisely editing any gene of interest. We here illustrate the complementary possibilities of introducing CRISPR-Cas9 machinery in immortalized N/TERT keratinocytes to generate Filaggrin knockout (ΔFLG) isogenic cell lines and organotypic human ΔFLG epidermal equivalents (ΔFLG- HEE). FLG deficiency was accompanied by (partial) loss of other structural and functional proteins, such as involucrin, hornerin, and transglutaminases. Consequently, transepithelial electrical resistance (TEER) indicated a decreased barrier function in ΔFLG-HEEs. Homology directed repair of the ΔFLG clonal lines reinstated FLG protein expression and the concomitant expression of the aforementioned epidermal differentiation proteins in FLG-restored HEEs. The phenotypical consequences of FLG deficiency in cell lines with an identical genetic background and in absence of predicted CRISPR-Cas9 off-target effects indicate a functional role for FLG – not only in epidermal barrier function – but also in epidermal barrier development which provides new insights into the disease pathogenesis of atopic dermatitis and ichthyosis vulgaris.