Conditional knockout (cKO) mice have contributed greatly to understanding the tissue- or stage-specific functions of genes in vivo. However, the current cKO method requires considerable time and effort because of the need to generate two gene-modified mouse strains (Cre transgenic and loxP knockin) for crossing. Here, we examined whether we could analyze the germ cell-related functions of embryonic lethal genes in F0 chimeric mice by restricting the origin of germ cells to mutant embryonic stem cells (ESCs). We confirmed that the full ESC origin of spermatozoa in fertile chimeric mice was achieved by the CRISPR/Cas9 system using three guide RNAs targeting Nanos3, which induced germ cell depletion in the host blastocyst-derived tissues. Among these fertile chimeric mice, those from male ESCs with a Dnmt3b mutation, which normally causes embryo death, also produced F1 mice derived exclusively from the mutant ESCs. Thus, our new chimeric strategy readily revealed that Dnmt3b is dispensable for male germ cell development, in agreement with a previous cKO study. Our new approach enables us to analyze the germ cell functions of embryonic lethal genes in the F0 generation without using the current cKO method.

中文翻译：

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使用 Nanos3† 的三靶 CRISPR 方法生成具有完全源自胚胎干细胞的精子的嵌合小鼠。

条件敲除 (cKO) 小鼠对理解体内基因的组织或阶段特异性功能做出了巨大贡献。然而，目前的 cKO 方法需要大量的时间和精力，因为需要生成两个基因修饰的小鼠品系（Cre 转基因和 loxP 敲入）进行杂交。在这里，我们检查了是否可以通过将生殖细胞的起源限制为突变胚胎干细胞 (ESC) 来分析 F0 嵌合小鼠胚胎致死基因的生殖细胞相关功能。我们证实，可育嵌合小鼠中精子的完整 ESC 起源是通过 CRISPR/Cas9 系统使用三种靶向Nanos3 的引导 RNA实现的，这会诱导宿主胚泡衍生组织中的生殖细胞消耗。在这些可育的嵌合小鼠中，来自雄性 ESC 的小鼠具有通常会导致胚胎死亡的Dnmt3b突变也产生了完全源自突变 ESC 的 F1 小鼠。因此，我们的新嵌合策略很容易揭示Dnmt3b对于雄性生殖细胞发育是可有可无的，这与之前的 cKO 研究一致。我们的新方法使我们能够在不使用当前 cKO 方法的情况下分析 F0 代胚胎致死基因的生殖细胞功能。