{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T06:59:36Z","timestamp":1792652376586,"version":"4.5.0"},"reference-count":34,"publisher":"Oxford University Press (OUP)","issue":"3","license":[{"start":{"date-parts":[[2020,3,1]],"date-time":"2020-03-01T00:00:00Z","timestamp":1583020800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/pages\/standard-publication-reuse-rights"}],"content-domain":{"domain":["www.atsjournals.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2020,3,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>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 MUC5AC expression in HBECs stimulated with IL-13, a central mediator of allergic asthma. Near-complete targeting of SPDEF abolished IL-13\u2013induced MUC5AC expression and goblet cell differentiation. In addition, targeting of SPDEF prevented IL-13\u2013induced 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.<\/jats:p>","DOI":"10.1165\/rcmb.2019-0266oc","type":"journal-article","created":{"date-parts":[[2019,10,9]],"date-time":"2019-10-09T16:44:27Z","timestamp":1570639467000},"page":"373-381","update-policy":"https:\/\/doi.org\/10.1164\/crossmarkpolicies","source":"Crossref","is-referenced-by-count":43,"title":["Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13\u2013induced Mucostasis"],"prefix":"10.1093","volume":"62","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3326-8217","authenticated-orcid":false,"given":"Kyung Duk","family":"Koh","sequence":"first","affiliation":[{"name":"Lung Biology Center"},{"name":"Cardiovascular Research 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and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lorna T.","family":"Zlock","sequence":"additional","affiliation":[{"name":"Department of Pathology, University of California San Francisco, San Francisco, California; and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Walter E.","family":"Finkbeiner","sequence":"additional","affiliation":[{"name":"Department of Pathology, University of California San Francisco, San Francisco, California; and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Prescott G.","family":"Woodruff","sequence":"additional","affiliation":[{"name":"Cardiovascular Research Institute"},{"name":"Division of Pulmonary, Critical Care, Sleep, and Allergy, and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2171-0648","authenticated-orcid":false,"given":"David J.","family":"Erle","sequence":"additional","affiliation":[{"name":"Lung Biology Center"},{"name":"Cardiovascular 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