title: Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa.
abstract: Genome editing with engineered site-specific endonucleases involves nonhomologous end-joining, leading to reading frame disruption. The approach is applicable to dominant negative disorders, which can be treated simply by knocking out the mutant allele, while leaving the normal allele intact. We applied this strategy to dominant dystrophic epidermolysis bullosa (DDEB), which is caused by a dominant negative mutation in the COL7A1 gene encoding type VII collagen (COL7). We performed genome editing with TALENs and CRISPR/Cas9 targeting the mutation, c.8068_8084delinsGA. We then cotransfected Cas9 and guide RNA expression vectors expressed with GFP and DsRed, respectively, into induced pluripotent stem cells (iPSCs) generated from DDEB fibroblasts. After sorting, 90% of the iPSCs were edited, and we selected four gene-edited iPSC lines for further study. These iPSCs were differentiated into keratinocytes and fibroblasts secreting COL7. RT-PCR and Western blot analyses revealed gene-edited COL7 with frameshift mutations degraded at the protein level. In addition, we confirmed that the gene-edited truncated COL7 could neither associate with normal COL7 nor undergo triple helix formation. Our data establish the feasibility of mutation site-specific genome editing in dominant negative disorders.
authors.affiliation: Department of Dermatology, Columbia University, New York, NY 10027; Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan;
authors.forename: Satoru
authors.initials: S
authors.lastname: Shinkuma
authors.affiliation: Department of Dermatology, Columbia University, New York, NY 10027;
authors.forename: Zongyou
authors.initials: Z
authors.lastname: Guo
authors.affiliation: Department of Dermatology, Columbia University, New York, NY 10027; Department of Genetics and Development, Columbia University, New York, NY 10027 amc65@columbia.edu.
authors.forename: Angela M
authors.initials: AM
authors.lastname: Christiano
mesh_terms: D000328:Adult; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D024143:Collagen Type VII / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D016108:Epidermolysis Bullosa Dystrophica / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism; D000072669:Gene Editing; D015870:Gene Expression; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000502:physiology* / Q000637:transplantation; D008297:Male; D008819:Mice, Nude; D013724:Teratoma / Q000473:pathology
chemical_list: C569053:COL7A1 protein, human; D024143:Collagen Type VII
keywords: CRISPR/Cas; TALENs; dominant negative effect; epidermolysis bullosa; gene editing