Development of SAGE, an AAV-based in vivo genetic screening platform, enables systematic identification of transcriptional and epigenetic regulators of alveolar repair, revealing that histone acetylation through the Kat8-NSL complex is essential for AT2-mediated regeneration, and that inflammatory signaling through NF-κB drives AT2 cells into a pathological basaloid-like transitional state rather than a reparative trajectory, linking inflammation to maladaptive epithelial remodeling in lung injury.

Effective alveolar repair after viral lung injury requires precise coordination of alveolar type 2 cell (AT2) proliferation and differentiation to restore lung function. To uncover causal regulators of this process in the native tissue environment, we developed SAGE (Stable Adeno-Associated Virus Genomic IntEgration), an engineered AAV system that enables high-throughput in vivo genetic interrogation. SAGE supports both bulk phenotypic screening (SAGE-Perturb) and single-cell transcriptomic profiling (SAGE-Perturb-seq). Using this approach, we identified lysine acetyltransferase 8 (Kat8) as essential for epithelial repair following viral infection through the Non-Specific-Lethal (NSL) complex, and generated a time-resolved, high-resolution functional map of transcription factor knockouts during alveolar repair, revealing transcription factor dependences for distinct alveolar epithelial repair trajectories. This map further defined two independent AT2-derived transitional states: a reparative state, and a pathological state that is transcriptionally similar to the basaloid population observed in human pulmonary fibrosis. Disruption of transcription factors in the NF-κB pathway prevented the emergence of the pathological transitional state, linking inflammation and maladaptive epithelial remodeling. SAGE represents a versatile platform for functional genomics in vivo, with applications extending across respiratory biology and disease.