Investigations into the FLG Null Phenotype: Showcasing the Methodology for CRISPR/Cas9 Editing of Human Keratinocytes

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 co...

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Published in:	Journal of investigative dermatology Vol. 143; no. 8; pp. 1520 - 1528.e5
Main Authors:	Smits, Jos P.H., van den Brink, Noa J.M., Meesters, Luca D., Hamdaoui, Hadia, Niehues, Hanna, Jansen, Patrick A.M., van Vlijmen-Willems, Ivonne M.J.J., Rodijk-Olthuis, Diana, Evrard, Céline, Poumay, Yves, van Geel, Michel, Hendriks, Wiljan J.A.J., Schalkwijk, Joost, Zeeuwen, Patrick L.J.M., van den Bogaard, Ellen H.
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 01-08-2023
Subjects:	CRISPR-Cas Systems Filaggrin Proteins Humans Intermediate Filament Proteins - metabolism Keratinocytes - metabolism Phenotype AD sgRNA NMF K2 KC HEE HRNR
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Summary:	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. [Display omitted]
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0022-202X 1523-1747
DOI:	10.1016/j.jid.2023.02.021