Methods to create genetically engineered mice involve three major steps: harvesting embryos from one set of females, microinjection of reagents into embryos ex vivo and their surgical transfer to another set of females. Although tedious, these methods have been used for more than three decades to create mouse models. We recently developed a method named GONAD (genome editing via oviductal nucleic acids delivery), which bypasses these steps. GONAD involves injection of CRISPR components (Cas9 mRNA and guide RNA (gRNA)) into the oviducts of pregnant females 1.5 d post conception, followed by in vivo electroporation to deliver the components into the zygotes in situ. Using GONAD, we demonstrated that target genes can be disrupted and analyzed at different stages of mouse embryonic development. Subsequently, we developed improved GONAD (i-GONAD) by delivering CRISPR ribonucleoproteins (RNPs; Cas9 protein or Cpf1 protein and gRNA) into day-0.7 pregnant mice, which made it suitable for routine generation of knockout and large-deletion mouse models. i-GONAD can also generate knock-in models containing up to 1-kb inserts when single-stranded DNA (ssDNA) repair templates are supplied. i-GONAD offers other advantages: it does not require vasectomized males and pseudo-pregnant females, the females used for i-GONAD are not sacrificed and can be used for other experiments, it can be easily adopted in laboratories lacking sophisticated microinjection equipment, and can be implemented by researchers skilled in small-animal surgery but lacking embryo-handling skills. Here, we provide a step-by-step protocol for establishing the i-GONAD method. The protocol takes ∼6 weeks to generate the founder mice.

创建基因工程小鼠的方法包括三个主要步骤：从一组雌性中获取胚胎、将试剂显微注射到体外胚胎中以及将它们通过手术转移到另一组雌性中。尽管很乏味，但这些方法已被用于创建小鼠模型超过三年。我们最近开发了一种名为 GONAD（通过输卵管核酸递送进行基因组编辑）的方法，它绕过了这些步骤。GONAD 涉及在受孕后 1.5 天将 CRISPR 成分（Cas9 mRNA 和引导 RNA (gRNA)）注射到怀孕女性的输卵管中，然后进行体内电穿孔以将成分原位输送到受精卵中。使用 GONAD，我们证明了可以在小鼠胚胎发育的不同阶段破坏和分析目标基因。随后，我们开发了改进的 GONAD（i- GONAD) 通过将 CRISPR 核糖核蛋白（RNP；Cas9 蛋白或 Cpf1 蛋白和 gRNA）输送到第 0.7 天的怀孕小鼠体内，使其适用于敲除和大缺失小鼠模型的常规生成。当提供单链 DNA (ssDNA) 修复模板时，i- GONAD 还可以生成包含多达 1-kb 插入片段的敲入模型。i- GONAD 提供其他优点：它不需要输精管切除术的男性和假孕女性，女性用于i-GONAD 不会被牺牲并可用于其他实验，它可以在缺乏复杂显微注射设备的实验室中轻松采用，并且可以由擅长小动物手术但缺乏胚胎处理技能的研究人员实施。在这里，我们提供了用于建立i- GONAD 方法的分步协议。该协议需要大约6 周的时间来生成创始人小鼠。