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Because HDR is restricted to dividing cells, whereas NHEJ functions in both dividing and non-dividing cells, NHEJ-based approaches are better suited for in vivo gene editing in the largely post-mitotic airway epithelium. Homology-independent targeted insertion (HITI), an NHEJ-based method, offers a promising strategy for cystic fibrosis (CF) gene therapy. Here, we applied HITI to drive the expression of a promoterless reporter through an exon trap strategy in both proliferating airway basal cells and well-differentiated primary airway epithelial cultures derived from transgenic ROSAmTmG ferrets. We also established a versatile human gene editing reporter (GER) airway basal cell line capable of multipotent differentiation, enabling real-time visualization of editing outcomes and the quantitative assessment of HDR- and NHEJ-based editing efficiencies. Together, these platforms provide easily accessible tools for optimizing genome editing strategies in the respiratory epithelium and advancing clinically relevant delivery strategies for CF gene therapy.<\/jats:p>","DOI":"10.3390\/v17060821","type":"journal-article","created":{"date-parts":[[2025,6,6]],"date-time":"2025-06-06T03:52:28Z","timestamp":1749181948000},"page":"821","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["A Versatile Reporter Platform for Evaluating HDR- and NHEJ-Based Genome Editing in Airway Epithelial Cell Cultures Using an rAAV Vector"],"prefix":"10.3390","volume":"17","author":[{"given":"Soo Yeun","family":"Park","sequence":"first","affiliation":[{"name":"Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA"},{"name":"Division of Pulmonary, Allergy, Critical Care & Sleep Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zehua","family":"Feng","sequence":"additional","affiliation":[{"name":"Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-4268-4171","authenticated-orcid":false,"given":"Xiujuan","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KS 66160, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4532-7598","authenticated-orcid":false,"given":"Yinghua","family":"Tang","sequence":"additional","affiliation":[{"name":"Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA"},{"name":"Division of Pulmonary, Allergy, Critical Care & Sleep Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Donovan","family":"Richart","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KS 66160, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8673-0703","authenticated-orcid":false,"given":"Kai E.","family":"Vorhies","sequence":"additional","affiliation":[{"name":"Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9850-1695","authenticated-orcid":false,"given":"Jianming","family":"Qiu","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KS 66160, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2389-9277","authenticated-orcid":false,"given":"John F.","family":"Engelhardt","sequence":"additional","affiliation":[{"name":"Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA"},{"name":"Division of Pulmonary, Allergy, Critical Care & Sleep Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8210-5567","authenticated-orcid":false,"given":"Ziying","family":"Yan","sequence":"additional","affiliation":[{"name":"Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA"},{"name":"Division of Pulmonary, Allergy, Critical Care & Sleep Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1066","DOI":"10.1126\/science.2475911","article-title":"Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA","volume":"245","author":"Riordan","year":"1989","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1126\/science.2772657","article-title":"Identification of the cystic fibrosis gene: Chromosome walking and jumping","volume":"245","author":"Rommens","year":"1989","journal-title":"Science"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1574","DOI":"10.1056\/NEJMra1300109","article-title":"Origins of cystic fibrosis lung disease","volume":"372","author":"Stoltz","year":"2015","journal-title":"N. 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