CRISPR/Cas9 Genome Editing Allows Generation of the Mouse Lung in a Rat.

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

OBJECTIVE: To generate the mouse lung in a rat.

METHODS: CRISPR/Cas9 genome editing was used to disrupt the Nkx2-1 gene in rat 1-cell zygotes. Interspecies mouse-rat chimeras were produced by injection of wild-type mouse ESC into Nkx2-1 -deficient rat embryos with lung agenesis. The contribution of mouse ESC 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 ESC restored pulmonary and thyroid structures in mouse-rat chimeras leading to a near 99% contribution of ESC 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 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".