Martens Marie Christine, Edelkamp Janin, Seebode Christina, Schäfer Mirijam, Stählke Susanne, Krohn Saskia, Jung Ole, Murua Escobar Hugo, Emmert Steffen, Boeckmann Lars
Clinic and Policlinic for Dermatology and Venerology, University Medical Center Rostock, 18057 Rostock, Germany.
Department of Cell Biology, University Medical Center Rostock, 18057 Rostock, Germany.
Int J Mol Sci. 2021 May 18;22(10):5293. doi: 10.3390/ijms22105293.
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 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 KO in immortalized human MRC5Vi fibroblasts (c.169_196delinsTTCGT) via lentiviral transduction.
突触小体相关蛋白29（SNAP29）的功能丧失突变会导致罕见的常染色体隐性神经皮肤脑发育不全、神经病变、鱼鳞病和角皮症（CEDNIK）综合征。SNAP29是一种可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白。到目前为止，已表明它参与膜融合、表皮分化、初级纤毛形成和自噬。最近，我们报道了成功构建出两种人类CEDNIK综合征的小鼠模型。本研究的目的是在永生化人类细胞系中通过CRISPR/Cas9介导产生基因敲除（KO），以独立于动物模型进一步研究SNAP29在人类细胞内稳态和信号传导中的作用。将CRISPR/Cas9质粒导入细胞的不同方法的比较显示，慢病毒转导比转染方法更有效。在此，据我们所知，首次通过慢病毒转导在永生化人类MRC5Vi成纤维细胞（c.169_196delinsTTCGT）中成功构建出CRISPR/Cas9介导的基因敲除。
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