Laboratory of Marine Life Science and Genetics, Graduate School of Agricultural Science, Tohoku University, Sendai, 980-0845, Japan.
Fish Physiol Biochem. 2021 Aug;47(4):849-855. doi: 10.1007/s10695-021-00945-6. Epub 2021 Mar 20.
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，现在可应用于许多生物体，包括非常规模型。然而，引入基因组编辑组件既费时又费力，有时还需要微注射的特殊技能。在这项研究中，我们开发了一种通过注射到母体卵巢（IMO）将外源蛋白递送到卵子中的技术，这导致 CRISPR/Cas9 递送到卵生动物的卵子中，包括鱼类。为了测试该技术，我们研究了是否可以通过 IMO 将标记有 GFP 或荧光素酶（Luc）的外源蛋白和荧光标记的 RNP（Cas9 和 sgRNA 复合物）递送到卵子中。当 GFP-Luc 或 Cas9-Luc 通过 IMO 递送到时，通过 GFP 荧光或荧光素酶活性证实其掺入受精卵中；蛋白质在卵黄中积累。Cas9-RNP（靶向酪氨酸酶）也被递送到卵子中。然而，还没有观察到靶基因酪氨酸酶的基因组编辑。这大概是因为 IMO 递送的 RNP 被包裹在卵黄颗粒中，没有进入胚胎核。因此，本报告表明，包括 Cas9-RNP 在内的外源分子可通过 IMO 成功递送到受精卵中。将递送到的 RNP 转移到核中将是通过 IMO 递送系统成功进行基因组编辑的关键。
