Recessive dystrophic epidermolysis bullosa (RDEB) is an intractable genetic disease of the skin caused by mutations in the COL7A1 gene. The majority of patients with RDEB harbor compound heterozygous mutations—two distinct mutations on each chromosome—without any apparent hotspots in the COL7A1 mutation pattern. This situation has made it challenging to establish a reliable RDEB mouse model with mutations that accurately mimic the genomic background of patients. Here, we established an RDEB mouse model harboring patient-type mutations in a compound heterozygous manner, using the CRISPR-based genome-editing technology i-GONAD. We selected two mutations, c.5818delC and E2857X, that have frequently been identified in cohorts of Japanese patients with RDEB. These mutations were introduced into the mouse genome at locations corresponding to those identified in patients. Mice homozygous for the 5818delC mutation developed severe RDEB-like phenotypes and died immediately after birth, whereas E2857X homozygous mice did not have a shortened lifespan compared to wild-type mice. Adult E2857X homozygous mice showed hair abnormalities, syndactyly, and nail dystrophy; these findings indicate that E2857X is indeed pathogenic in mice. Mice with the c.5818delC/E2857X compound heterozygous mutation presented an intermediate phenotype between the c.5818delC and E2857X homozygous mice. Single-cell RNA sequencing further clarified that the intrafollicular keratinocytes in c.5818delC/E2857X compound heterozygous mice exhibited abnormalities in cell cycle regulation. The proposed strategy to produce compound heterozygous mice, in addition to the established mouse line, will facilitate research on RDEB pathogenesis to develop a cure for this devastating disease.
This is a preview of subscription content
Subscribe to Journal
Get full journal access for 1 year
We are sorry, but there is no personal subscription option available for your country.
Get time limited or full article access on ReadCube.
All prices are NET prices.
The sequencing data used in this study have been deposited and are available in GEO (GSE181357).
Bardhan, A. et al. Epidermolysis bullosa. Nat. Rev. Dis. Primers 6, 78 (2020).
Has, C. et al. Consensus reclassification of inherited epidermolysis bullosa and other disorders with skin fragility. Br. J. Dermatol. 183, 614–627 (2020).
Christiano, A. M. et al. Structural organization of the human type VII collagen gene (COL7A1), composed of more exons than any previously characterized gene. Genomics 21, 169–179 (1994).
Varki, R., Sadowski, S., Uitto, J. & Pfendner, E. Epidermolysis bullosa. II. Type VII collagen mutations and phenotype-genotype correlations in the dystrophic subtypes. J. Med. Genet. 44, 181–192 (2007).
Nystrom, A. & Bruckner-Tuderman, L. Injury- and inflammation-driven skin fibrosis: the paradigm of epidermolysis bullosa. Matrix Biol. 68-69, 547–560 (2018).
Fine, J. D., Johnson, L. B., Weiner, M., Li, K. P. & Suchindran, C. Epidermolysis bullosa and the risk of life-threatening cancers: the National EB Registry experience, 1986-2006. J. Am. Acad. Dermatol. 60, 203–211 (2009).
Guerra, L., Odorisio, T., Zambruno, G. & Castiglia, D. Stromal microenvironment in type VII collagen-deficient skin: the ground for squamous cell carcinoma development. Matrix Biol. 63, 1–10 (2017).
Has, C., South, A. & Uitto, J. Molecular therapeutics in development for epidermolysis bullosa: update 2020. Mol. Diagn. Ther. 24, 299–309 (2020).
Vandamme, T. F. Use of rodents as models of human diseases. J. Pharm. Bioallied. Sci. 6, 2–9 (2014).
Heinonen, S. et al. Targeted inactivation of the type VII collagen gene (Col7a1) in mice results in severe blistering phenotype: a model for recessive dystrophic epidermolysis bullosa. J. Cell Sci. 112(Pt 21), 3641–3648 (1999).
Fritsch, A. et al. A hypomorphic mouse model of dystrophic epidermolysis bullosa reveals mechanisms of disease and response to fibroblast therapy. J. Clin. Invest. 118, 1669–1679 (2008).
Landrum, M. J. et al. ClinVar: improving access to variant interpretations and supporting evidence. Nucleic Acids Res. 46, D1062–D1067 (2018).
Shalem, O., Sanjana, N. E. & Zhang, F. High-throughput functional genomics using CRISPR-Cas9. Nat. Rev. Genet 16, 299–311 (2015).
Huijbers, I. J. Generating genetically modified mice: a decision guide. Methods Mol. Biol. 1642, 1–19 (2017).
Ohtsuka, M. et al. i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases. Genome Biol. 19, 25 (2018).
Tamai, K. et al. Recurrent COL7A1 mutations in Japanese patients with dystrophic epidermolysis bullosa: positional effects of premature termination codon mutations on clinical severity. Japanese Collaborative Study Group on Epidermolysis Bullosa. J. Invest. Dermatol. 112, 991–993 (1999).
Sawamura, D. et al. Genetic studies of 20 Japanese families of dystrophic epidermolysis bullosa. J. Hum. Genet 50, 543–546 (2005).
Dang, N. & Murrell, D. F. Mutation analysis and characterization of COL7A1 mutations in dystrophic epidermolysis bullosa. Exp. Dermatol. 17, 553–568 (2008).
Koshida, S. et al. Hallopeau-Siemens dystrophic epidermolysis bullosa due to homozygous 5818delC mutation in the COL7A gene. Pediatr. Int. 55, 234–237 (2013).
Saito, M., Masunaga, T., Teraki, Y., Takamori, K. & Ishiko, A. Genotype-phenotype correlations in six Japanese patients with recessive dystrophic epidermolysis bullosa with the recurrent p.Glu2857X mutation. J. Dermatol. Sci. 52, 13–20 (2008).
Gurumurthy, C. B. et al. Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD. Nat. Protoc. 14, 2452–2482 (2019).
Aoto, K. et al. ATP6V0A1 encoding the a1-subunit of the V0 domain of vacuolar H+-ATPases is essential for brain development in humans and mice. Nat. Commun. 12, 2107 (2021).
Wasylishen, A. R. et al. Daxx maintains endogenous retroviral silencing and restricts cellular plasticity in vivo. Sci. Adv. 6, eaba8415 (2020).
Ho, Y. T. et al. Longitudinal single-cell transcriptomics reveals a role for Serpina3n-mediated resolution of inflammation in a mouse colitis model. Cell Mol. Gastroenterol. Hepatol. 12, 547–566 (2021).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411–420 (2018).
Wolf, F. A., Angerer, P. & Theis, F. J. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 19, 15 (2018).
Finak, G. et al. MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data. Genome Biol. 16, 278 (2015).
Kuleshov, M. V. et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 44, W90–W97 (2016).
Joost, S. et al. Single-cell transcriptomics reveals that differentiation and spatial signatures shape epidermal and hair follicle heterogeneity. Cell Syst. 3, 221–237 e229 (2016).
Joost, S. et al. The molecular anatomy of mouse skin during hair growth and rest. Cell Stem Cell 26, 441–457.e447 (2020).
Chacon-Solano, E. et al. Fibroblast activation and abnormal extracellular matrix remodelling as common hallmarks in three cancer-prone genodermatoses. Br. J. Dermatol. 181, 512–522 (2019).
Capecchi, M. R. Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat. Rev. Genet 6, 507–512 (2005).
We would like to thank Editage [http://www.editage.com] for editing and reviewing this manuscript for English language.
This study was supported by JSPS KAKENHI Grant Numbers JP21K08324 (T.S.) and JP19H03682 (K.T.) and a research fund from StemRIM Inc.
K.T. is a scientific founder of and received research funding from StemRIM. K.T. and T.S. are StemRIM stockholders. S.T., K.I., T.K., Y.Y., and S.Y. are employees of StemRIM.
Ethics approval and consent to participate
All animals were handled in accordance with the guidelines of the Animal Committee of Osaka University Graduate School of Medicine that approved the experimental protocol.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Takaki, S., Shimbo, T., Ikegami, K. et al. Generation of a recessive dystrophic epidermolysis bullosa mouse model with patient-derived compound heterozygous mutations. Lab Invest 102, 574–580 (2022). https://doi.org/10.1038/s41374-022-00735-5