An optimized prime editing system for efficient modification of the pig genome

文献类型： 外文期刊

第一作者： Qi, Yanan

作者： Qi, Yanan;Zhang, Ying;Tian, Shuangjie;Zong, Ruojun;Yan, Xinghui;Wang, Yu;Zhao, Jianguo;Qi, Yanan;Tian, Shuangjie;Zong, Ruojun;Yan, Xinghui;Wang, Yu;Zhao, Jianguo;Qi, Yanan;Zhang, Ying;Tian, Shuangjie;Zong, Ruojun;Yan, Xinghui;Wang, Yu;Zhao, Jianguo;Zhang, Ying;Zhao, Jianguo;Wang, Yanfang;Wang, Yanfang

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关键词： prime editing; pig; porcine embryonic fibroblasts; editing efficiency

期刊名称：SCIENCE CHINA-LIFE SCIENCES （ 影响因子：9.1； 五年影响因子：7.1 ）

ISSN： 1674-7305

年卷期： 2023 年

页码：

收录情况： SCI

摘要： Prime editing (PE) is a recent gene editing technology that can mediate insertions or deletions and all twelve types of base-to-base conversions. However, its low efficiency hampers the application in creating novel breeds and biomedical models, especially in pigs and other important farm animals. Here, we demonstrate that the pig genome is editable using the PE system, but the editing efficiency was quite low as expected. Therefore, we aimed to enhance PE efficiency by modulating both exogenous PE tools and endogenous pathways in porcine embryonic fibroblasts (PEFs). First, we modified the pegRNA by extending the duplex length and mutating the fourth thymine in a continuous sequence of thymine bases to cytosine, which significantly enhanced PE efficiency by improving the expression of pegRNA and targeted cleavage. Then, we targeted SAMHD1, a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that impedes the reverse transcription process in retroviruses, and found that treatment with its inhibitor, cephalosporin C zinc salt (CPC), increased PE efficiency up to 29-fold (4-fold on average), presumably by improving the reverse transcription process of Moloney murine leukemia virus reverse transcriptase (M-MLV RT) in the PE system. Moreover, PE efficiency was obviously improved by treatment with a panel of histone deacetylase inhibitors (HDACis). Among the four HDACis tested, panobinostat was the most efficient, with an efficiency up to 122-fold (7-fold on average), partly due to the considerable HDACi-mediated increase in transgene expression. In addition, the synergistic use of the three strategies further enhanced PE efficiency in PEFs. Our study provides novel approaches for optimization of the PE system and broadens the application scope of PE in agriculture and biomedicine.

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