title: CRISPR-Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18.
abstract: Targeted knockout of genes in primary human cells using CRISPR-Cas9-mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a proinflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9-mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a proinflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli.
authors.affiliation: Department of Medicine, National Jewish Health, Denver, CO, USA.
authors.forename: H W
authors.initials: HW
authors.lastname: Chu
authors.affiliation: Integrated Center for Genes, Environment, and Health, National Jewish Health, Denver, CO, USA.
authors.forename: C
authors.initials: C
authors.lastname: Rios
authors.affiliation: Department of Medicine, National Jewish Health, Denver, CO, USA.
authors.forename: C
authors.initials: C
authors.lastname: Huang
authors.affiliation: Integrated Center for Genes, Environment, and Health, National Jewish Health, Denver, CO, USA.
authors.forename: A
authors.initials: A
authors.lastname: Wesolowska-Andersen
authors.affiliation: Department of Medicine, University of California-San Francisco, San Francisco, CA, USA.|Department of Bioengineering & Therapeutic Sciences, University of California-San Francisco, San Francisco, CA, USA.
authors.forename: E G
authors.initials: EG
authors.lastname: Burchard
authors.affiliation: Integrated Center for Genes, Environment, and Health, National Jewish Health, Denver, CO, USA.
authors.forename: B P
authors.initials: BP
authors.lastname: O'Connor
authors.affiliation: Integrated Center for Genes, Environment, and Health, National Jewish Health, Denver, CO, USA.
authors.forename: T E
authors.initials: TE
authors.lastname: Fingerlin
authors.affiliation: Department of Pediatrics, National Jewish Health, Denver, CO, USA.
authors.forename: D
authors.initials: D
authors.lastname: Nichols
authors.affiliation: Department of Pediatrics, National Jewish Health, Denver, CO, USA.
authors.forename: S D
authors.initials: SD
authors.lastname: Reynolds
authors.affiliation: Integrated Center for Genes, Environment, and Health, National Jewish Health, Denver, CO, USA.|Department of Pediatrics, National Jewish Health, Denver, CO, USA.|Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado Denver, Denver, CO, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Seibold
mesh_terms: D051929:CD146 Antigen / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D015870:Gene Expression; D055786:Gene Knockout Techniques; D005822:Genetic Vectors; D006801:Humans; D007249:Inflammation / Q000235:genetics; D016086:Lentivirus; D061251:Primary Cell Culture; D020545:Respiratory Mucosa / Q000276:immunology / Q000378:metabolism*
chemical_list: D051929:CD146 Antigen; C497902:MCAM protein, human