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."

中文翻译：

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CRISPR-Cas9 基因组编辑可在大鼠体内生成小鼠肺。


理由：最近在生物工程和胚胎干细胞 (ESC) 技术方面的努力使得在缺失关键形态发生基因的转基因小鼠中产生了源自 ESC 的小鼠肺组织。上皮细胞谱系是从 ESC 中有效产生的，但其他细胞类型是镶嵌的。供体 ESC 对肺组织的完全贡献从未实现过。小鼠肺从未在大鼠体内产生过。目的：我们试图在大鼠体内产生小鼠肺。方法：使用成簇规则间隔短回文重复序列 (CRISPR)-Cas9 基因组编辑来破坏大鼠单细胞受精卵中的 Nkx2-1 基因。通过将野生型小鼠 ESC 注射到 Nkx2-1 缺陷且肺发育不全的大鼠胚胎中，产生了种间小鼠-大鼠嵌合体。通过免疫染色、流式细胞术和单细胞 RNA 测序检查了小鼠 ESC 对肺组织的贡献。测量和主要结果：在 CRISPR-Cas9 介导的 Nkx2-1 基因破坏后，大鼠胚胎中周围肺组织和甲状腺组织消失。大鼠 Nkx2-1-/- 囊胚与小鼠 ESC 的互补恢复了小鼠-大鼠嵌合体中的肺和甲状腺结构，导致 ESC 对所有呼吸细胞谱系的贡献率接近 99%。 ESC 来源的肺中的上皮细胞、内皮细胞、造血细胞和基质细胞高度分化，并表现出与正常小鼠肺的呼吸细胞相似的谱系特异性基因特征。受体-配体相互作用的分析揭示了在大鼠中分化的小鼠ESC衍生的呼吸细胞之间的正常信号网络。 结论：采用CRISPR-Cas9基因组编辑和囊胚互补相结合的方法在大鼠体内产生小鼠肺，为下一代使用大型动物作为“生物反应器”的人类肺迈出了重要一步。"