Investigations into the FLG Null Phenotype: showcasing the methodology for CRISPR/Cas9 editing of human keratinocytes

Jos P H Smits, Noa J M van den Brink, Luca D Meesters, Hadia Hamdaoui, Hanna Niehues, Patrick A M Jansen, Ivonne M J J van Vlijmen-Willems, Diana Rodijk-Olthuis, Céline Evrard, Yves Poumay, Michel van Geel, Wiljan J A J Hendriks, Joost Schalkwijk, Patrick L J M Zeeuwen, Ellen H van den Bogaard

Research output: Contribution to journalArticlepeer-review


Ever since the association between FLG loss-of-function variants and ichthyosis vulgaris and atopic dermatitis disease onset was identified, FLGs function has been under investigation. Intraindividual genomic predisposition, immunological confounders, and environmental interactions complicate the comparison between FLG genotypes and related causal effects. Using CRISPR/Cas9, we generated human FLG-knockout (ΔFLG) N/TERT-2G keratinocytes. FLG deficiency was shown by immunohistochemistry of human epidermal equivalent cultures. Next to (partial) loss of structural proteins (involucrin, hornerin, keratin 2, and transglutaminase 1), the stratum corneum was denser and lacked the typical basket weave appearance. In addition, electrical impedance spectroscopy and transepidermal water loss analyses highlighted a compromised epidermal barrier in ΔFLG human epidermal equivalents. Correction of FLG reinstated the presence of keratohyalin granules in the stratum granulosum, FLG protein expression, and expression of the proteins mentioned earlier. The beneficial effects on stratum corneum formation were reflected by the normalization of electrical impedance spectroscopy and transepidermal water loss. This study shows the causal phenotypical and functional consequences of FLG deficiency, indicating that FLG is not only central in epidermal barrier function but also vital for epidermal differentiation by orchestrating the expression of other important epidermal proteins. These observations pave the way to fundamental investigations into the exact role of FLG in skin biology and disease.

Original languageEnglish
Pages (from-to)1520-1528.e5
JournalThe journal of investigative dermatology
Issue number8
Early online date7 Mar 2023
Publication statusPublished - 1 Aug 2023


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