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Homozygous editing of multiple genes for accelerated generation of xenotransplantation pigs

文献类型： 外文期刊

作者： Duan, Xiaoyue 1 ; Chen, Chaolei 2 ; Du, Chang 2 ; Guo, Liang 1 ; Liu, Jun 2 ; Hou, Naipeng 1 ; Li, Pan 1 ; Qi, Xiaolan 3 ; Gao, Fei 1 ; Du, Xuguang 1 ; Song, Jiangping 1 ; Wu, Sen 1 ;

作者机构： 1.China Agr Univ, Sanya Inst, Sanya 572024, Peoples R China

2.China Agr Univ, Coll Biol Sci, State Key Lab Anim Biotech Breeding, Beijing 100193, Peoples R China

3.Lanzhou Univ, Chinese Acad Agr Sci, Lanzhou Vet Res Inst, State Key Lab Anim Dis Control & Prevent,Coll Vet, Lanzhou 730000, Peoples R China

4.Chinese Acad Med Sci & Peking Union Med Coll, Fuwai Hosp, Anim Expt Ctr, Beijing Key Lab Preclin Res & Evaluat Cardiovasc I, Beijing 100037, Peoples R China

5.China Agr Univ, Coll Biol Sci, State Key Lab Anim Biotech Breeding, Natl Engn Lab Anim Breeding,Frontiers Sci Ctr Mol, Beijing 100193, Peoples R China

期刊名称：GENOME RESEARCH （ 影响因子：5.5； 五年影响因子：7.3 ）

ISSN： 1088-9051

年卷期： 2025 年 35 卷 5 期

页码：

收录情况： SCI

摘要： Although CRISPR-Cas-based genome editing has made significant strides over the past decade, achieving simultaneous homozygous gene editing of multiple targets in primary cells remains a significant challenge. In this study, we optimized a coselection strategy to enhance homozygous gene editing rates in the genomes of primary porcine fetal fibroblasts (PFFs). The strategy utilizes the expression of a surrogate reporter (eGFP) to select for cells with the highest reporter expression, thereby improving editing efficiency. For simultaneous multigene editing, we targeted the most challenging site for selection, whereas other target sites did not require selection. Using this approach, we successfully obtained single-cell PFF clones (three of 10) with seven or more homozygously edited genes, including GGTA1, CMAH, B4GALNT2, CD46, CD47, THBD, and GHR. Importantly, cells edited using this strategy can be efficiently used for somatic cell nuclear transfer (SCNT) to generate healthy xenotransplantation pigs in <5 months, a process that previously required years of breeding or multiple rounds of SCNT.

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