Generation of Alpha-1 Antitrypsin Knockout and PI*ZZ Ferrets Using Crispr/Cas9. A Genetic Model of Emphysema

Rationale: The most prevalent genetic cause of chronic obstructive pulmonary disease is alpha-1 antitrypsin (A1AT) deficiency, a disorder that has yet to be widely modeled in animals because of species-specific differences between rodents and humans.Objectives: To address these challenges, we engineered two A1AT ferret models using zygote gene editing to test the hypothesis that unopposed protease activity within the lung leads to emphysema and bronchitis.Methods: Guide RNAs targeting exon 2 (for knockout) and exon 5 (for Z-allele mutation, Pi*Z) of the ferret A1AT gene were injected into ferret zygotes with Cas9 mRNA. For PI*Z targeting, a short oligonucleotide carrying the mutation was included. Offspring were genotyped and plasma levels of A1AT determined by Western blot before entry into a longitudinal study. Adult A1AT ferrets underwent bronchoscopy and FlexiVent over time to characterize lung disease. Control animals were wild-type (WT) and age, sex, and size matched.Results: A1AT-deficient ferrets ...