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One-step base editing in multiple genes by direct embryo injection for pig trait improvement

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Abstract

The precise and simultaneous acquisition of multiple beneficial alleles in the genome is in great demand for the development of elite pig breeders. Cytidine base editors (CBEs) that convert C:G to T:A have emerged as powerful tools for single-nucleotide replacement. Whether CBEs can effectively mediate C-to-T substitution at multiple sites/loci for trait improvement by direct zygote injection has not been verified in large animals. Here, we determined the editing efficiency of four CBE variants in porcine embryonic fibroblast cells and embryos. The findings showed that hA3A-BE3-Y130F and hA3A-eBE-Y130F consistently resulted in increased base-editing efficiency and low toxic effects in embryonic development. Further, we verified that using a one-step approach, direct zygote microinjection of the CBE system can generate pigs harboring multiple point mutations. Our process resulted in a stop codon in CD163 and myostatin (MSTN) and introduced a beneficial allele in insulin-like growth factor-2 (IGF2). The pigs showed disrupted expression of CD163 and MSTN and increased expression of IGF2, which significantly improved growth performance and infectious disease resistance. Our approach allows immediate introduction of multiple mutations in transgene-free animals to comprehensively improve economic traits through direct embryo microinjection, providing a potential new route to produce elite pig breeders.

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Acknowledgements

This work was supported by the National Natural Science Foundation for Distinguished Young Scholars (31925036), the National Key Research and Development Program of China (2020YFC1316602), the National Natural Science Foundation of China (81671274, 31272440, and 31801031), the National Transgenic Project of China (2016ZX08009003-006-007), and the Elite Youth Program of the Chinese Academy of Agricultural Sciences (ASTIP-IAS05).

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  1. Contributed equally to this work

Authors and Affiliations

  1. State Key Laboratory of Stem cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China

    Ruigao Song, Yu Wang, Qiantao Zheng, Jing Yao, Chunwei Cao & Jianguo Zhao

  2. University of Chinese Academy of Sciences, Beijing, 100049, China

    Yu Wang, Jing Yao & Jianguo Zhao

  3. State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Yu Wang & Yanfang Wang

  4. Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, 100101, China

    Jing Yao & Jianguo Zhao

  5. Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, 100101, China

    Jing Yao & Jianguo Zhao

  6. The Third Hospital of Shanxi Medical University, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Taiyuan, 030032, China

    Ruigao Song

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  1. Ruigao Song
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  2. Yu Wang
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  3. Qiantao Zheng
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  6. Yanfang Wang
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Correspondence to Yanfang Wang or Jianguo Zhao.

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Song, R., Wang, Y., Zheng, Q. et al. One-step base editing in multiple genes by direct embryo injection for pig trait improvement. Sci. China Life Sci. 65, 739–752 (2022). https://doi.org/10.1007/s11427-021-2013-8

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