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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

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

by
Haitao Wang
1,†,
Hengqian Yang
1,2,†,
Tingting Li
1,
Yan Chen
1,
Jieran Chen
1,
Xiaosheng Zhang
3,4,
Jinlong Zhang
3,4,
Yuting Zhang
1,
Na Zhang
1,
Runlin Ma
1,
Xun Huang
1 and
Qiuyue Liu
1,*
1
State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
2
College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China
3
Institute of Animal Sciences and Veterinary, Tianjin Academy of Agriculture Sciences, Tianjin 300381, China
4
Tianjin Key Laboratory of Animal Molecular Breeding and Biotechnology, Tianjin 300381, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2025, 26(3), 1065; https://doi.org/10.3390/ijms26031065
Submission received: 6 December 2024 / Revised: 20 January 2025 / Accepted: 23 January 2025 / Published: 26 January 2025
(This article belongs to the Section Molecular Biology)
Review Reports Versions Notes

Abstract

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/μL 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.
Keywords: CRISPR/Cas9; microinjection; gene editing; sheep CRISPR/Cas9; microinjection; gene editing; sheep

Share and Cite

MDPI and ACS Style

Wang, H.; Yang, H.; Li, T.; Chen, Y.; Chen, J.; Zhang, X.; Zhang, J.; Zhang, Y.; Zhang, N.; Ma, R.; et al. Optimization of CRISPR/Cas9 Gene Editing System in Sheep (Ovis aries) Oocytes via Microinjection. Int. J. Mol. Sci. 2025, 26, 1065. https://doi.org/10.3390/ijms26031065

AMA Style

Wang H, Yang H, Li T, Chen Y, Chen J, Zhang X, Zhang J, Zhang Y, Zhang N, Ma R, et al. Optimization of CRISPR/Cas9 Gene Editing System in Sheep (Ovis aries) Oocytes via Microinjection. International Journal of Molecular Sciences. 2025; 26(3):1065. https://doi.org/10.3390/ijms26031065

Chicago/Turabian Style

Wang, Haitao, Hengqian Yang, Tingting Li, Yan Chen, Jieran Chen, Xiaosheng Zhang, Jinlong Zhang, Yuting Zhang, Na Zhang, Runlin Ma, and et al. 2025. "Optimization of CRISPR/Cas9 Gene Editing System in Sheep (Ovis aries) Oocytes via Microinjection" International Journal of Molecular Sciences 26, no. 3: 1065. https://doi.org/10.3390/ijms26031065

APA Style

Wang, H., Yang, H., Li, T., Chen, Y., Chen, J., Zhang, X., Zhang, J., Zhang, Y., Zhang, N., Ma, R., Huang, X., & Liu, Q. (2025). Optimization of CRISPR/Cas9 Gene Editing System in Sheep (Ovis aries) Oocytes via Microinjection. International Journal of Molecular Sciences, 26(3), 1065. https://doi.org/10.3390/ijms26031065

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