Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13-induced Mucostasis
Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13-induced Mucostasis
复制标题
高效 RNP 导向的人类基因靶向揭示 SPDEF 是 IL-13 诱导的粘膜停滞所必需的
DOI：
10.1165/rcmb.2019-0266oc
复制
发表时间：
2020-03-01
影响因子：
6.4
通讯作者：
Erle, David J.
中科院分区：
文献类型：
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作者：
Koh, Kyung Duk;Siddiqui, Sana;Erle, David J.
原代人支气管上皮细胞（HBEC）培养是研究肺部健康和主要气道疾病的有用模型。然而，机制研究受到我们选择性破坏这些细胞中特定基因的能力的限制。在这里，我们优化了HBECs基因靶向的方法，即通过电穿孔直接递送单导RNA （sgRNA）和rCas9（重组Cas9）复合物，而不需要质粒、病毒或抗生素选择。递送方法、sgRNA和rCas9浓度以及sgRNA序列的变化都对靶向效率有影响，从而可以对基因靶向程度进行可预测的控制，并几乎完全破坏基因表达。为了证明该系统的价值，我们将SPDEF作为目标，SPDEF编码一种转录因子，该转录因子先前被证明对哮喘小鼠模型中产生muc5ac的杯状细胞的分化至关重要。靶向SPDEF导致IL-13刺激的HBECs中MUCSAC表达成比例降低，IL-13是过敏性哮喘的中枢介质。几乎完全靶向SPDEF可消除il -13诱导的MUC5AC表达和杯状细胞分化。此外，靶向SPDEF可防止il -13诱导的粘膜纤毛清除损伤，这可能是哮喘气道阻塞、发病率和死亡率的重要因素。我们得出结论，sgRNA和rCas9复合物的直接递送允许可预测和有效的基因靶向，并使原发性HBECs疾病相关途径的机制研究成为可能。
Primary human bronchial epithelial cell (HBEC) cultures are a useful model for studies of lung health and major airway diseases. However, mechanistic studies have been limited by our ability to selectively disrupt specific genes in these cells. Here we optimize methods for gene targeting in HBECs by direct delivery of single guide RNA (sgRNA) and rCas9 (recombinant Cas9) complexes by electroporation, without a requirement for plasmids, viruses, or antibiotic selection. Variations in the method of delivery, sgRNA and rCas9 concentrations, and sgRNA sequences all had effects on targeting efficiency, allowing for predictable control of the extent of gene targeting and for near-complete disruption of gene expression. To demonstrate the value of this system, we targeted SPDEF, which encodes a transcription factor previously shown to be essential for the differentiation of MUC5AC-producing goblet cells in mouse models of asthma. Targeting SPDEF led to proportional decreases in MUCSAC expression in HBECs stimulated with IL-13, a central mediator of allergic asthma. Near-complete targeting of SPDEF abolished IL-13-induced MUC5AC expression and goblet cell differentiation. In addition, targeting of SPDEF prevented IL-13-induced impairment of mucociliary clearance, which is likely to be an important contributor to airway obstruction, morbidity, and mortality in asthma. We conclude that direct delivery of sgRNA and rCas9 complexes allows for predictable and efficient gene targeting and enables mechanistic studies of disease-relevant pathways in primary HBECs.