CRISPR/Cas9 Editing of Human Keratinocytes for Organotypic Modeling of Genetic Epidermal Disorders.

CRISPR/Cas9-based DNA editing has revolutionized biomedical research by enabling investigators to precisely and permanently inactivate genes in human cells to provide greater insight into the mechanisms that support normal physiology and drive disease pathology. To leverage this technology for cutaneous biology research, we optimized a strategy for efficient gene editing in human keratinocytes, which can be grown into a differentiated organotypic epidermis to extend studies of gene function to a mature 3D tissue model. Here, we describe step-by-step methods for deploying CRISPR/Cas9 to engineer a TERT-immortalized human epidermal keratinocyte line (N/TERT-2G) to generate monoclonal heterozygous and homozygous knockout cell lines. Additionally, we provide a detailed protocol for growing gene-edited keratinocytes into organotypic epidermis, which we have used to model the pathology of monogenic dermatologic disorders that were not replicated in traditional knockout mice.