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.
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The sequencing data used in this study have been deposited and are available in GEO (GSE181357).
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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.
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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.
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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