Efficient and Safe Editing of Porcine Endogenous Retrovirus Genomes by Multiple-Site Base-Editing Editor
Shuwen Zheng,
Haiwen Zhong,
Xiaoqing Zhou,
Min Chen,
Wansheng Li,
Yin Zi,
Yue Chi,
Jinling Wang,
Wei Zheng,
Qingjian Zou,
Liangxue Lai,
Chengcheng Tang
Affiliations
Shuwen Zheng
Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen 529020, China
Haiwen Zhong
College of Animal Science, South China Agricultural University, Guangzhou 510642, China
Xiaoqing Zhou
Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen 529020, China
Min Chen
Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen 529020, China
Wansheng Li
Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen 529020, China
Yin Zi
Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen 529020, China
Yue Chi
Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen 529020, China
Jinling Wang
Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen 529020, China
Wei Zheng
Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen 529020, China
Qingjian Zou
Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen 529020, China
Liangxue Lai
Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen 529020, China
Chengcheng Tang
Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen 529020, China
Gene-modified miniature pigs serve as alternative tissue and organ donors for xenotransplantation to alleviate the shortage of human allogenic organs. However, the high copy number of porcine endogenous retrovirus (PERV) genomes integrates with the porcine genome, which has a potential risk of cross-species transmission and hinders the clinical practice of xenotransplantation. Recently, CRISPR/Cas9 has been used to inactivate PERVs. However, Cas9 also triggers severe DNA damage at multiple integrated PERV sites in the porcine genome, which induces senescence and apoptosis of porcine cells. In this study, the cytosine base editor (CBE), an efficient and safe editor that does not cause DNA double strand breaks (DSBs), was used for PERV editing to reduce cytotoxic effects. Seven sgRNAs were set to target gag and pol loci of PERVs to induce premature stop codons. We found that approximately 10% of cell clones were completely inactivated for PERVs in pig ST cells, and the plasmid that was used for editing the PERVs did not integrate into host genome and influence the karyotype of the modified cells. Our studies offer a powerful and safe strategy for further generating PERV-knockout pigs using base editors.
