LAUSR

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) 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 CFTR (CFTRG551D-KI), 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 EGFP and CFTR genes, followed by fluorescence activated cell sorting (FACS) of EGFPY66S-corrected cells. When gene-edited airway basal cells were polarized at an air-liquid interface, unsorted and EGFPY66S-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 CFTRG551D 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.