Efficient genome editing in ASC-derived porcine lung organoids via GFP-guided enrichment

Adult stem cell-derived organoids are invaluable tools for disease modeling and translational medicine owing to their tissue-specific correspondence. However, their complex three-dimensional structure and low genome-editing efficiency have posed significant challenges for functional genetic studies. To address these limitations, this study aimed to establish porcine lung organoid (PLOs) and to develop a robust, high-efficiency genome-editing platform by leveraging the high physiological and anatomical similarity between pigs and humans. PLOs were derived from lung tissues and characterized by immunofluorescence and immunohistochemistry analysis. To enhance genome-editing efficiency using CRISPR/Cas9 and prime editing, a GFP-guided enrichment strategy was used by co-targeting a GFP transgene and endogenous loci, followed by the selection of GFP-negative organoids. Clonal expansion from single organoids was performed to generate genetically homogenous lines. Genetic modifications were validated through cDNA sequencing and western blot analysis. Structural integrity was assessed using AlphaFold2 modeling, and functional changes were evaluated using the forskolin-induced swelling assay. The established PLOs exhibited sustained self-renewal capacity and expression of surfactant protein C alongside both proximal epithelial and mesenchymal markers, reflecting a composite lung organoid identity. GFP-guided enrichment significantly enriched edited cells; the mutation ratio in the GFP-negative group was significantly higher than that in the GFP-positive group. This system successfully generated fully gene-edited clones with high genetic homogeneity, and effectively enriched for precisely edited organoids, even when applying complex modalities such as prime editing. The PLOs and genome-editing platform developed in this study provide a powerful resource for translational medicine. By enabling precise genetic manipulation in a model that closely mimics human lung physiology, this system offers an essential platform for recapitulating human lung pathologies, studying zoonotic infectious diseases, and validating the efficacy of gene therapies.