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Journal of Integrative Agriculture
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Highly Efficient Multiplex Precise Genome Editing in Porcine Cells Using epiCBE4max System

Xianghua Xu1*, Bo Li1*, Lu Jing5, Xuewen Xu1, Shengsong Xie1, 3, 4, Changchun Li2, Xinyun Li1, 3, 4, Xiaosong Han1, 2#, Jinxue Ruan1#

1 Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China

2 Yazhouwan National Laboratory, Sanya 572024, China

3 The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China

4 Hubei Hongshan Laboratory, Frontiers Science Center for Animal Breeding and Sustainable Production, Wuhan 430070, China

5 New Hope Liuhe Co., Ltd./Key Laboratory of Digital Intelligent Breeding Technological Innovation for Swine and Poultry, Ministry of Agriculture and Rural Affairs, Chengdu, Sichuan, 610023, China

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摘要  

碱基编辑器(BEs)在生物医学研究、疾病治疗和动植物分子育种等领域展现出重大应用价值。然而,如何在猪细胞中实现高效精准的多重基因编辑仍是一个亟待解决的重要问题。为解决这一问题,本研究成功开发了基于EBNA1/oriP附加体元件的CBE4max基因编辑系统(命名为epiCBE4max),该系统能够在猪细胞中实现高效精准的一步式多重碱基编辑。通过将具有自主复制功能的附加体元件整合至CBE4max系统骨架,epiCBE4max能够持久维持碱基编辑元件的高水平表达,从而显著提升编辑效率。研究团队针对与家畜生长性状和抗病能力相关的五个关键基因(ANTXR1ANPEPCD163CALRMSTN),设计并筛选出具有高活性的sgRNAs,随后在U6启动子调控下,将这些sgRNAs串联克隆至epiCBE4max载体,成功构建了五基因同时编辑载体:epiCBE4max-5×(U6+sg)。实验结果显示,将该系统通过电穿孔技术导入猪胎儿成纤维细胞(PFF)后,在获得的18个单克隆细胞系中,有高达94.44%17/18)的细胞系在所有五个靶基因位点都表现出有效的编辑效果其中CALR基因位点的编辑效率尤为显著(达89.67%;其中44.44%8/18)的细胞系在五个靶位点均实现纯合编辑,且未检测到明显的脱靶效应。本研究建立的epiCBE4max编辑技术为猪细胞提供了高效、可靠的多重基因编辑新工具,不仅为家畜遗传改良、疾病模型构建提供了技术支撑,也为异种器官移植和功能基因组学研究开辟了新途径,具有重要的科学价值和应用前景。后续研究将把该方法与体细胞核移植技术相结合,培育高品质基因编辑猪,从而推动畜牧育种等领域的可持续发展。



Online: 08 December 2025  
Fund: 

This work was supported by the Biological Breeding-National Science and Technology Major Project, China (2023ZD0404703), the National Natural Science Foundation of China (32302705), the Science and Technology Development Project of Xinjiang, China (2022LQ01003), the Natural Science Foundation of Hubei Province of China (2023AFB1034).

About author:  Xianghua Xu, E-mail: xuxianghua@webmail.hzau.edu.cn; Bo Li, E-mail: 103.bo.hzau.edu.com@webmail.hzau.edu.cn; #Correspondence Jinxue Ruan, E-mail: ruanjinxue@mail.hzau.edu.cn; Xiaosong Han, E-mail: xshan@mail.hzau.edu.cn * These authors contributed equally to this study.

Cite this article: 

Xianghua Xu, Bo Li, Lu Jing, Xuewen Xu, Shengsong Xie, Changchun Li, Xinyun Li, Xiaosong Han, Jinxue Ruan. 2025. Highly Efficient Multiplex Precise Genome Editing in Porcine Cells Using epiCBE4max System. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.12.011

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