Rationale: Recent efforts in bioengineering and embryonic stem cell (ESC) technology allowed the generation of ESC-derived mouse lung tissues in transgenic mice that were missing critical morphogenetic genes. Epithelial cell lineages were efficiently generated from ESC, but other cell types were mosaic. A complete contribution of donor ESCs to lung tissue has never been achieved. The mouse lung has never been generated in a rat.

Objective: We sought to generate the mouse lung in a rat.

Methods: Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing was used to disrupt the Nkx2-1 gene in rat one-cell zygotes. Interspecies mouse–rat chimeras were produced by injection of wild-type mouse ESCs into Nkx2-1–deficient rat embryos with lung agenesis. The contribution of mouse ESCs to the lung tissue was examined by immunostaining, flow cytometry, and single-cell RNA sequencing.

Measurements and Main Results: Peripheral pulmonary and thyroid tissues were absent in rat embryos after CRISPR-Cas9–mediated disruption of the Nkx2-1 gene. Complementation of rat Nkx2-1^−/− blastocysts with mouse ESCs restored pulmonary and thyroid structures in mouse–rat chimeras, leading to a near-99% contribution of ESCs to all respiratory cell lineages. Epithelial, endothelial, hematopoietic, and stromal cells in ESC-derived lungs were highly differentiated and exhibited lineage-specific gene signatures similar to those of respiratory cells from the normal mouse lung. Analysis of receptor–ligand interactions revealed normal signaling networks between mouse ESC-derived respiratory cells differentiated in a rat.

Conclusions: A combination of CRISPR-Cas9 genome editing and blastocyst complementation was used to produce mouse lungs in rats, making an important step toward future generations of human lungs using large animals as “bioreactors.”