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Optimization of CRISPR/Cas9 Gene Editing System in Sheep (Ovis aries) Oocytes via Microinjection

文献类型： 外文期刊

作者： Wang, Haitao 1 ; Yang, Hengqian 1 ; Li, Tingting 1 ; Chen, Yan 1 ; Chen, Jieran 1 ; Zhang, Xiaosheng 3 ; Zhang, Jinlong 3 ; Zhang, Yuting 1 ; Zhang, Na 1 ; Ma, Runlin 1 ; Huang, Xun 1 ; Liu, Qiuyue 1 ;

作者机构： 1.Chinese Acad Sci, Inst Genet & Dev Biol, State Key Lab Mol Dev Biol, Beijing 100101, Peoples R China

2.Qingdao Agr Univ, Coll Anim Sci & Technol, Qingdao 266109, Peoples R China

3.Tianjin Acad Agr Sci, Inst Anim Sci & Vet, Tianjin 300381, Peoples R China

4.Tianjin Key Lab Anim Mol Breeding & Biotechnol, Tianjin 300381, Peoples R China

关键词： CRISPR/Cas9; microinjection; gene editing; sheep

期刊名称：INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES （ 影响因子：4.9； 五年影响因子：5.7 ）

ISSN： 1661-6596

年卷期： 2025 年 26 卷 3 期

页码：

收录情况： SCI

摘要： The CRISPR/Cas9 system has become a powerful tool for molecular design breeding in livestock such as sheep. However, the efficiency of the Cas9 system combined with zygote microinjection remains suboptimal. In this study, mature sheep oocytes were used for microinjection to assess the impact of various factors on Cas9 editing efficiency. We found that the in vitro maturation efficiency of oocytes is related to environmental factors such as air temperature, pressure, and humidity. Our results indicate that high-efficiency gene editing can be achieved when targeting the SOCS2, DYA, and TBXT, using a microinjection mixture with a concentration of 10 ng/mu L Cas9 and sgRNA. By optimizing the injection capillary, we significantly reduced the oocyte invalidation rate post-microinjection to 3.1-5.3%. Furthermore, we observed that using either Cas9 protein or mRNA in the microinjection process resulted in different genotypes in the edited oocytes. Importantly, parthenogenetic activation did not appear to affect the editing efficiency. Using this high-efficiency system, we successfully generated SOCS2 or DYA gene-edited sheep, with all lambs confirmed to be genetically modified. This study presents a highly efficient method for producing gene-edited sheep, potentially enabling more precise and effective strategies for livestock breeding.

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