Generation and Characterization of a CRISPR/Cas9-Mediated SNAP29 Knockout in Human Fibroblasts |
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| Authors: | Marie Christine Martens,Janin Edelkamp,Christina Seebode,Mirijam Sch fer,Susanne St hlke,Saskia Krohn,Ole Jung,Hugo Murua Escobar,Steffen Emmert,Lars Boeckmann |
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| Affiliation: | 1.Clinic and Policlinic for Dermatology and Venerology, University Medical Center Rostock, 18057 Rostock, Germany; (M.C.M.); (J.E.); (C.S.); (M.S.); (O.J.); (S.E.);2.Department of Cell Biology, University Medical Center Rostock, 18057 Rostock, Germany;3.Clinic for Hematology, Oncology and Palliative Care, University Medical Center Rostock, 18057 Rostock, Germany; (S.K.); (H.M.E.) |
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| Abstract: | Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) lead to the rare autosomal recessive neurocutaneous cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. SNAP29 is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. So far, it has been shown to be involved in membrane fusion, epidermal differentiation, formation of primary cilia, and autophagy. Recently, we reported the successful generation of two mouse models for the human CEDNIK syndrome. The aim of this investigation was the generation of a CRISPR/Cas9-mediated SNAP29 knockout (KO) in an immortalized human cell line to further investigate the role of SNAP29 in cellular homeostasis and signaling in humans independently of animal models. Comparison of different methods of delivery for CRISPR/Cas9 plasmids into the cell revealed that lentiviral transduction is more efficient than transfection methods. Here, we reported to the best of our knowledge the first successful generation of a CRISPR/Cas9-mediated SNAP29 KO in immortalized human MRC5Vi fibroblasts (c.169_196delinsTTCGT) via lentiviral transduction. |
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| Keywords: | CEDNIK syndrome SNAP29 ichthyosis CRISPR/Cas9 lentiviral transduction |
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