In vivo interrogation of transcriptional and epigenetic regulators of lung epithelial regeneration

Abstract 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. Competing Interest Statement F.C. is an academic founder of Curio Bioscience and Doppler Biosciences, and scientific advisor for Amber Bio. F.C's interests were reviewed and managed by the Broad Institute in accordance with their conflict-of-interest policies.