Genome editing has had profound effects on biological experimentation and can now be applied to many organisms, including non-conventional models. However, the introduction of genome editing components is time- and labor-consuming and sometimes requires special skills for microinjection. In this study, we developed a technique to deliver exogenous proteins into eggs by injection into the mother’s ovary (IMO), which leads to the delivery of CRISPR/Cas9 into the eggs of oviparous animals, including fish. To test this technique, we examined whether exogenous proteins tagged with GFP or luciferase (Luc), and fluorescent-labeled RNP (Cas9 and sgRNA complex), can be delivered into eggs by IMO. When GFP-Luc or Cas9-Luc was delivered by IMO, their incorporation into fertilized eggs was confirmed by GFP fluorescence or luciferase activity; proteins were accumulated in the yolk. Cas9-RNP (targeting tyrosinase) was also incorporated into the eggs. However, genome editing of the target gene, tyrosinase, was not observed yet. This is presumably because the RNP delivered by IMO was packed in the yolk granules and did not reach into the embryonic nuclei. Thus, this report shows that exogenous molecules including Cas9-RNP were successfully delivered into fertilized eggs by IMO. Transferring the delivered RNP into nuclei will be critical for successful genome editing via the IMO delivery system.

基因组编辑对生物实验产生了深远的影响，现在可以应用于许多生物体，包括非常规模型。然而，基因组编辑组件的引入既费时又费力，有时需要特殊的显微注射技能。在这项研究中，我们开发了一种技术，通过注射到母体卵巢 (IMO) 将外源蛋白质输送到卵子中，从而将 CRISPR/Cas9 输送到卵生动物（包括鱼）的卵子中。为了测试这项技术，我们检查了用 GFP 或荧光素酶 (Luc) 标记的外源蛋白质和荧光标记的 RNP（Cas9 和 sgRNA 复合物）是否可以通过 IMO 输送到鸡蛋中。当 IMO 递送 GFP-Luc 或 Cas9-Luc 时，通过 GFP 荧光或荧光素酶活性证实它们掺入受精卵；蛋白质在蛋黄中积累。Cas9-RNP（靶向酪氨酸酶）也被掺入鸡蛋中。然而，尚未观察到靶基因酪氨酸酶的基因组编辑。这可能是因为 IMO 提供的 RNP 被包裹在蛋黄颗粒中，没有进入胚胎细胞核。因此，本报告表明，包括 Cas9-RNP 在内的外源分子已被 IMO 成功递送到受精卵中。将传递的 RNP 转移到细胞核中对于通过 IMO 传递系统成功进行基因组编辑至关重要。