Journal article
Recombinant Adeno-Associated Virus-Mediated Editing of the G551D Cystic Fibrosis Transmembrane Conductance Regulator Mutation in Ferret Airway Basal Cells
Human Gene Therapy, Vol.33(19-20), pp.1023-1036
06/10/2022
DOI: 10.1089/hum.2022.036
PMCID: PMC9595624
PMID: 35686451
Abstract
Mutations in the cystic fibrosis transmembrane conductance regulator (
) gene cause cystic fibrosis (CF), a chronic disease that affects multiple organs including the lung. We developed a CF ferret model of a scarless G551→D substitution in
(
), enabling approaches to correct this gating mutation in CF airways via gene editing. Homology-directed repair (HDR) was tested in Cas9-expressing CF airway basal cells (Cas9-GKI) from this model, as well as reporter basal cells (Y66S-Cas9-GKI) that express an integrated non-fluorescent Y66S-EGFP mutant gene to facilitate rapid assessment of HDR by the restoration of fluorescence. rAAV vectors were used to deliver two DNA templates and sgRNAs for dual gene editing at the
and
genes, followed by fluorescence activated cell sorting (FACS) of
-corrected cells. When gene-edited airway basal cells were polarized at an air-liquid interface, unsorted and
-corrected sorted populations gave rise to 26.0% and 70.4% CFTR-mediated Cl- transport of that observed in non-CF cultures, respectively. The consequences of gene editing at the
locus by HDR and non-homology end joining (NHEJ) were assessed by targeted gene next generation sequencing (NGS) against a specific amplicon. NGS revealed HDR corrections of 3.1% of G551 sequences in the unsorted population of rAAV-infected cells, and 18.4% in the EGFPY66S-corrected cells. However, the largest proportion of sequences had indels surrounding the CRISPR cut site, demonstrating that NHEJ was the dominant repair pathway. This approach to simultaneously co-edit at two genomic loci using rAAV may have utility as a model system for optimizing gene editing efficiencies in proliferating airway basal cells through the modulation of DNA repair pathways in favor of HDR.
Details
- Title: Subtitle
- Recombinant Adeno-Associated Virus-Mediated Editing of the G551D Cystic Fibrosis Transmembrane Conductance Regulator Mutation in Ferret Airway Basal Cells
- Creators
- Ziying Yan - Roy J. and Lucille A. Carver College of MedicineKai Vorhies - Roy J. and Lucille A. Carver College of MedicineZehua Feng - Roy J. and Lucille A. Carver College of MedicineSoo-Yeun Park - Roy J. and Lucille A. Carver College of MedicineSoon H Choi - Roy J. and Lucille A. Carver College of MedicineYulong Zhang - Roy J. and Lucille A. Carver College of MedicineMicheal Winter - Roy J. and Lucille A. Carver College of MedicineXingshen Sun - Roy J. and Lucille A. Carver College of MedicineJohn F Engelhardt - Roy J. and Lucille A. Carver College of Medicine
- Resource Type
- Journal article
- Publication Details
- Human Gene Therapy, Vol.33(19-20), pp.1023-1036
- DOI
- 10.1089/hum.2022.036
- PMID
- 35686451
- PMCID
- PMC9595624
- ISSN
- 1043-0342
- eISSN
- 1557-7422
- Language
- English
- Date published
- 06/10/2022
- Academic Unit
- Roy J. Carver Department of Biomedical Engineering; Anatomy and Cell Biology; Radiation Oncology; Neuroscience and Pharmacology; Internal Medicine
- Record Identifier
- 9984288627602771
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