CRISPR/Cas9-mediated knockouts in reconstructed human epidermis to profile skin microbiome interactions

Duke University, Durham NC

Investigators

Abstract

PROJECT SUMMARY / ABSTRACT Our goal is to enable the investigation of interactions between the skin microbiome and skin cell populations using genetically modified 3D in vitro models. The human skin microbiome – encompassing hundreds of bacterial and fungal species – has essential roles in maintaining skin health. Skin microbiome dysfunction or pathobiont expansion can contribute to diverse skin infections, inflammatory disorders, and skin cancer. It is important to identify the interactions that could go awry in skin disease and to evaluate the impact of potential therapeutic approaches for skin diseases. However, a detailed mechanistic understanding of how these various microbes interact with host cells to maintain skin health or promote skin disease is lacking. This knowledge gap is exacerbated due to an inability to examine host-microbe interactions at the scale needed to probe potentially hundreds of microbes and host genes of interest involved in these mechanisms. Here, we will leverage 3D human reconstructed epidermis (RHE) cultures built from keratinocytes and fibroblasts in an air- liquid interface (ALI) to develop a platform that allows us to knock out human gene function and colonize with diverse skin microbiota. We have previously used the RHE model to examine the effects of colonization of 180 genetically diverse skin staphylococci on skin barrier and innate immunity. Using single-cell RNA-sequencing (scRNA-seq) and spatial transcriptomics (ST), we were able to pinpoint cell-type-specific interactions in our model. However, a major gap was the ability to interrogate host mechanisms, most effectively by gene knockout or knockdown. Recently, we built 3D ALI cultures with CRISPR/Cas9 KOs in primary bronchial epithelial cells to investigate the innate immune response to microbial colonization. We propose using a similar approach to build KO keratinocytes and fibroblasts to generate multi-cell type 3D KO RHEs that will enable us to investigate skin's host-microbiome interactions. In Aim 1, we will create 3D RHE ALI cultures with CRISPR/Cas9 knockouts (KO RHE) in a panel of skin barrier and innate immunity genes in immortalized N/TERT keratinocytes, which we will then colonize with commensal and pathogenic skin microbes. In Aim 2, we will add an immortalized hTERT fibroblast layer to the KO RHE model (F-RHE) and similarly colonize with the microbial panel. We will use immunohistochemistry, RNA-seq, scRNA-seq, and ST to characterize KO RHEs' effect on fibroblasts to pinpoint microbial and keratinocyte-fibroblast interactions that change with KO of important skin barrier and immune components. By developing a 3D skin KO model coupled with a cutting-edge `omics approach, this proof-of- principle study will enable the construction of large-scale, high-resolution spatial and functional maps of skin microbiome-host cell interactions. Given the extensive biodiversity of the human skin microbiome, this platform will be valuable for deconstructing and pinpointing interactions that could be disrupted in skin disease and for evaluating the impact of potential therapeutic approaches for skin diseases.