Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) system is continually optimized to achieve the most efficient gene editing effect. The Cas12iMax, a Cas12i variant, exhibits powerful DNA editing activity and enriches the gene editing toolbox. However, the application of Cas12iMax in large domestic animals has not yet been reported. To verify the efficiency and feasibility of multiple gene editing in large animals, we generated porcine fibroblasts with simultaneous knockouts of IGF2, ANPEP, CD163, and MSTN via Cas12iMax in one step. Phenotypically stable pigs were created through somatic cell nuclear transfer technology. They exhibited improved growth performance and muscle quality. Furthermore, we simultaneously edited three genes in bovine fibroblasts. A knockout of MSTN and PRNP was created and the amino acid Q-G in CD18 was precisely substituted. Meanwhile, no off-target phenomenon was observed by sum-type analysis or off-target detection. These results verified the effectiveness of Cas12iMax for gene editing in livestock animals and demonstrated the potential application of Cas12iMax in the field of animal trait improvement for agricultural production.
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Acknowledgement
This work was supported by the National Key Research and Development Program of China (2018YFE0201100, 2021YFA0805905, 2021YFA0805701, 2022YFA1103101), the National Natural Science Foundation of China (32102549), the National Key R&D Program of Ningxia (2021BEF02023), the China Agriculture Research System of MOF and MARA (CARS-36), the Agricultural Science and Technology Innovation Program (ASTIP-IAS06), the project from The Xinjiang Production and Construction Corps and Foundation of State Key Laboratory for Sheep Genetic Improvement and Healthy Production (2021ZD04). We express our gratitude to Prof. Aihua Zheng for his valuable assistance during the virus resistance experiments conducted on the model pigs. Additionally, we extend our appreciation to the staff of the Beijing Farm Animal Research Center for their diligent husbandry of the model pigs.
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Ren, J., Hai, T., Chen, Y. et al. Improve meat production and virus resistance by simultaneously editing multiple genes in livestock using Cas12iMax. Sci. China Life Sci. 67, 555–564 (2024). https://doi.org/10.1007/s11427-023-2407-0
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DOI: https://doi.org/10.1007/s11427-023-2407-0