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Updated: Dec 15, 2025
Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells
Published on: September 25, 2019
Efficient CRISPR-Cas9-Mediated Gene Ablation in Human Keratinocytes to Recapitulate Genodermatoses: Modeling of
Victoria Gálvez1,2,3, Esteban Chacón-Solano2,4,3, Jose Bonafont2,4,3
1Epithelial Biomedicine Division, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT), 28040 Madrid, Spain.
Abstract:
Current efforts to find specific genodermatoses treatments and define precise pathogenesis mechanisms require appropriate surrogate models with human cells. Although transgenic and gene knockout mouse models for several of these disorders exist, they often fail to faithfully replicate the clinical and histopathological features of the human skin condition. We have established a highly efficient method for precise deletion of critical gene sequences in primary human keratinocytes, based on CRISPR-Cas9-mediated gene editing. Using this methodology, in the present study we generated a model of Netherton syndrome by disruption of SPINK5. Gene-edited cells showed absence of LEKTI expression and were able to recapitulate a hyperkeratotic phenotype with most of the molecular hallmarks of Netherton syndrome, after grafting to immunodeficient mice and in organotypic cultures. To validate the model as a platform for therapeutic intervention, we tested an ex vivo gene therapy approach using a lentiviral vector expressing SPINK5. Re-expression of SPINK5 in an immortalized clone of SPINK5-knockout keratinocytes was capable of reverting from Netherton syndrome to a normal skin phenotype in vivo and in vitro. Our results demonstrate the feasibility of modeling genodermatoses, such as Netherton syndrome, by efficiently disrupting the causative gene to better understand its pathogenesis and to develop novel therapeutic approaches.
Insights
Researchers developed a CRISPR-Cas9 gene editing method to create a Netherton syndrome model using human keratinocytes. This model successfully replicated disease features and showed potential for gene therapy interventions.
Area of Science:
- Dermatology
- Molecular Biology
- Genetics
Background:
- Developing accurate human cell models for genodermatoses is crucial for understanding disease mechanisms and finding treatments.
- Existing mouse models often do not fully replicate human skin conditions.
Purpose of the Study:
- To establish a human cell-based model for Netherton syndrome using CRISPR-Cas9 gene editing.
- To validate this model for studying disease pathogenesis and testing therapeutic strategies.
Main Methods:
- Utilized CRISPR-Cas9 gene editing to disrupt the SPINK5 gene in primary human keratinocytes.
- Generated a Netherton syndrome model by observing LEKTI absence and hyperkeratosis.
- Validated the model through grafting onto immunodeficient mice and organotypic cultures.
- Tested an ex vivo gene therapy approach using lentiviral vectors to re-express SPINK5.
Main Results:
- Successfully generated human keratinocytes with disrupted SPINK5, leading to LEKTI absence and Netherton syndrome hallmarks.
- The gene-edited cells recapitulated hyperkeratotic phenotype in vivo and in vitro.
- Ex vivo gene therapy successfully restored SPINK5 expression and reverted the Netherton syndrome phenotype.
Conclusions:
- CRISPR-Cas9 mediated gene editing provides an efficient method for modeling genodermatoses like Netherton syndrome.
- This human cell-based model is valuable for understanding pathogenesis and developing novel therapeutic approaches.
