title: Efficient in vivo gene editing using ribonucleoproteins in skin stem cells of recessive dystrophic epidermolysis bullosa mouse model.
abstract: The prokaryotic CRISPR/Cas9 system has recently emerged as a powerful tool for genome editing in mammalian cells with the potential to bring curative therapies to patients with genetic diseases. However, efficient in vivo delivery of this genome editing machinery and indeed the very feasibility of using these techniques in vivo remain challenging for most tissue types. Here, we show that nonreplicable Cas9/sgRNA ribonucleoproteins can be used to correct genetic defects in skin stem cells of postnatal recessive dystrophic epidermolysis bullosa (RDEB) mice. We developed a method to locally deliver Cas9/sgRNA ribonucleoproteins into the skin of postnatal mice. This method results in rapid gene editing in epidermal stem cells. Using this method, we show that Cas9/sgRNA ribonucleoproteins efficiently excise exon80, which covers the point mutation in our RDEB mouse model, and thus restores the correct localization of the collagen VII protein in vivo. The skin blistering phenotype is also significantly ameliorated after treatment. This study provides an in vivo gene correction strategy using ribonucleoproteins as curative treatment for genetic diseases in skin and potentially in other somatic tissues.
authors.affiliation: Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program, School of Life Sciences, Peking University, Beijing 100871, China.|National Institute of Biological Sciences, Beijing 102206, China.
authors.forename: Wenbo
authors.initials: W
authors.lastname: Wu
authors.affiliation: National Institute of Biological Sciences, Beijing 102206, China.
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Lu
authors.affiliation: National Institute of Biological Sciences, Beijing 102206, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Li
authors.affiliation: National Institute of Biological Sciences, Beijing 102206, China.
authors.forename: Wenjie
authors.initials: W
authors.lastname: Wang
authors.affiliation: National Institute of Biological Sciences, Beijing 102206, China.
authors.forename: Nannan
authors.initials: N
authors.lastname: Qian
authors.affiliation: National Institute of Biological Sciences, Beijing 102206, China.
authors.forename: Jinzhi
authors.initials: J
authors.lastname: Duan
authors.affiliation: National Institute of Biological Sciences, Beijing 102206, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: National Institute of Biological Sciences, Beijing 102206, China.
authors.forename: Fengchao
authors.initials: F
authors.lastname: Wang
authors.affiliation: National Institute of Biological Sciences, Beijing 102206, China chenting@nibs.ac.cn.
authors.forename: Ting
authors.initials: T
authors.lastname: Chen
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics; D024143:Collagen Type VII / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D016108:Epidermolysis Bullosa Dystrophica / Q000235:genetics / Q000628:therapy*; D000072669:Gene Editing / Q000379:methods*; D005808:Genes, Recessive; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009154:Mutation; D015203:Reproducibility of Results; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism; D012867:Skin / Q000378:metabolism / Q000473:pathology; D013234:Stem Cells / Q000378:metabolism*
chemical_list: C583886:Col7a1 protein, mouse; D024143:Collagen Type VII; D012261:Ribonucleoproteins
keywords: Cas9/sgRNA ribonucleoproteins; RDEB; electroporation; in vivo gene editing; skin stem cell