Self-Assembled Skin Equivalents with Monoclonal CRISPR/Cas9-Modified N/TERT-1 Keratinocytes: A Cutting-Edge Model for Human Skin and its Diseases.

Human skin is a complex organ consisting of multiple cell types and serves as an essential barrier against environmental stressors. Due to ethical considerations and interspecies differences, in vitro human skin equivalents (SEs) are increasingly used to complement or replace animal models in mechanistic, pharmacological, and disease-modeling studies. Scaffold-free full-thickness SEs, in which fibroblasts generate their own extracellular matrix, are particularly attractive because they provide high structural stability even during extended culture. However, the use of genetically defined keratinocyte populations in these SEs has remained limited. Here, scaffold-free full-thickness SEs incorporating wild-type, polyclonal or monoclonal CRISPR/Cas9-modified N/TERT-1 keratinocytes, generated via electroporation, are established. Monoclonal N/TERT-1 keratinocytes with targeted knockout (KO) of the crucial inflammasome component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) form a differentiated epidermis but fail to secrete the proinflammatory cytokines interleukin (IL)-1β and IL-18 upon inflammasome activation, indicating complete functional ablation of inflammasome signaling in the 3D model. Moreover, SEs generated with gasdermin A (GSDMA)-KO N/TERT-1 keratinocytes illustrate the feasibility of analyzing genes induced during keratinocyte differentiation under physiological conditions. These results establish scaffold-free full-thickness SEs with monoclonal genetically modified N/TERT-1 keratinocytes as a robust and reproducible human skin model for mechanistic studies and future disease-modeling applications.