Genetic disruption of cutaneous de novo glucocorticoid synthesis: a CRISPR-Cas9 CYP11B1-knockout human epidermal model

Abstract

Human skin functions as both a physical barrier and a neuroendocrine and steroidogenic organ. Glucocorticoids (GC) synthesized in the skin help to maintain skin immune homeostasis and to mediate responses to environmental stressors. Cutaneous GC synthesis has so far been disrupted only pharmacologically, with inhibitors that do not discriminate CYP11B1 from its close paralogue CYP11B2, or genetically in mouse keratinocytes, so the consequences of blocking this pathway in human keratinocytes remain untested. Here, we disrupted de novo GC synthesis by knocking out the CYP11B1 gene using CRISPR-Cas9, thereby interrupting the terminal enzymatic process of converting 11-deoxycortisol to cortisol in mitochondria. Two independent knockout clones were established, one carrying a 28 bp deletion spanning the start codon and one carrying a multi-exon deletion. Both clones reconstituted stratified 3D epidermal models as in wild-type models, indicating that loss of CYP11B1 does not by itself impair epidermal differentiation. Wild-type models responded with increased CYP11B1 transcript abundance and immunofluorescence signal, whereas both knockout clones retained a UVB-inducible transcript signal but showed no detectable CYP11B1 protein, confirming loss of the protein product at the tissue level. This work provides, to our knowledge, the first human keratinocyte model carrying a genomically defined block in de novo GC synthesis, and a tissue-level platform for resolving the role of keratinocyte-derived GC in epidermal homeostasis and environmental stress responses.