The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.

中文翻译：

---

通过CRISPR / Cas9介导的具有多个sgRNA的基因编辑一步一步生成完整的基因敲除小鼠和猴子。

CRISPR / Cas9系统是一种有效的基因编辑方法，但是大多数经过基因编辑的动物均表现出镶嵌性，仅在部分细胞中进行了编辑。在这里我们显示，通过合子注射Cas9 mRNA和多个相邻的单向导RNA（间隔10-200 bp）（仅针对每个基因的一个关键外显子），可以在小鼠和猴胚胎中完全敲除单个基因或多个基因。 。对F0小鼠的表型分析分别针对Y染色体上的八个基因进行有针对性的删除后，证明了这种方法在产生基因敲除小鼠中的鲁棒性。重要的是，这种方法可在猴胚胎中以高效率（在Arnt1上为100％，在Prrt2上为91％）提供完整的基因敲除。最后，我们可以一步完成一个完整的Prrt2基因敲除猴子，