Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13-induced Mucostasis
| العنوان: | Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13-induced Mucostasis |
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| المؤلفون: | David J. Erle, Lorna Zlock, Dan Cheng, Kyung Duk Koh, Walter E. Finkbeiner, Dingyuan I. Sun, Sana Siddiqui, Luke R. Bonser, Prescott G. Woodruff |
| المصدر: | American Journal of Respiratory Cell and Molecular Biology American journal of respiratory cell and molecular biology, vol 62, iss 3 |
| سنة النشر: | 2019 |
| مصطلحات موضوعية: | 0301 basic medicine, Respiratory System, Clinical Biochemistry, Cardiorespiratory Medicine and Haematology, Mucin 5AC, goblet, 0302 clinical medicine, Gene expression, 2.1 Biological and endogenous factors, CRISPR, SPDEF, Kinetoplastida, Aetiology, Lung, Cells, Cultured, 0303 health sciences, Cultured, Interleukin-13, Electroporation, Gene targeting, respiratory system, 3. Good health, Cell biology, medicine.anatomical_structure, Ribonucleoproteins, Mucociliary Clearance, 030220 oncology & carcinogenesis, Interleukin 13, Gene Targeting, Respiratory, Goblet Cells, Biotechnology, RNA, Guide, Kinetoplastida, Pulmonary and Respiratory Medicine, Cells, Primary Cell Culture, Down-Regulation, Bronchi, Biology, 03 medical and health sciences, Mediator, human bronchial epithelial cells, medicine, Genetics, Humans, Molecular Biology, Transcription factor, Gene, 030304 developmental biology, Goblet cell, Metaplasia, Proto-Oncogene Proteins c-ets, Editorials, Epithelial Cells, Cell Biology, MUC5AC, Asthma, Good Health and Well Being, 030104 developmental biology, 030228 respiratory system, Gene Expression Regulation, RNA, CRISPR-Cas Systems, Transcriptome, Guide |
| الوصف: | 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 recombinant Cas9 (rCas9) 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 MUC5AC 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. |
| وصف الملف: | application/pdf |
| تدمد: | 1535-4989 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::451c6bbeb12c16a9b1e48ad8c4d00999 https://pubmed.ncbi.nlm.nih.gov/31633992 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....451c6bbeb12c16a9b1e48ad8c4d00999 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15354989 |
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