Homozygous editing of multiple genes for accelerated generation of xenotransplantation pigs
- Xiaoyue Duan1,2,6,
- Chaolei Chen2,6,
- Chang Du2,6,
- Liang Guo1,2,6,
- Jun Liu2,
- Naipeng Hou1,
- Pan Li1,2,
- Xiaolan Qi3,
- Fei Gao1,2,
- Xuguang Du1,2,
- Jiangping Song1,4 and
- Sen Wu1,2,5
- 1Sanya Institute of China Agricultural University, Sanya, 572024, China;
- 2State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Beijing 100193, China;
- 3State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730000, China;
- 4Beijing Key Laboratory of Preclinical Research and Evaluation for Cardiovascular Implant Materials, Animal Experimental Center, Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, China;
- 5State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, National Engineering Laboratory for Animal Breeding, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing, 100193, China
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↵6 These authors contributed equally to this work.
Abstract
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.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.279709.124.
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Freely available online through the Genome Research Open Access option.
- Received June 19, 2024.
- Accepted February 18, 2025.
This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
