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One-step in vivo gene knock-out in porcine embryos using recombinant adeno-associated viruses

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机构： [1]Department of Pathology, Institute of Clinical Pathology, Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China. [2]Experimental Animal Center, West China Hospital, Sichuan University, Chengdu, China. [3]Security Department, West China Hospital, Sichuan University, Chengdu, China. [4]Institute of Respiratory Health, West China Hospital, Sichuan University, Chengdu, China. [5]Department of Toxicological Inspection, Sichuan Center for Disease Prevention and Control, Chengdu, China. [6]Laboratory of Liver Transplantation, Frontiers Science Center for Disease-related Molecular, Network, West China Hospital, Sichuan University, Chengdu, China. [7]State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, China.

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关键词： CRISPR/Cas9 recombinant adeno-associated viruses embryo gene delivery gene-edited pig animal model

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Introduction: Gene-edited pigs have become prominent models for studying human disease mechanisms, gene therapy, and xenotransplantation. CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated 9 (CRISPR/Cas9) technology is a widely employed tool for generating gene-edited pigs. Nevertheless, delivering CRISPR/Cas9 to pre-implantation embryos has traditionally posed challenges due to its reliance on intricate micromanipulation equipment and specialized techniques, resulting in high costs and time-consuming procedures. This study aims to introduce a novel one-step approach for generating genetically modified pigs by transducing CRISPR/Cas9 components into pre-implantation porcine embryos through oviductal injection of recombinant adeno-associated viruses (rAAV). Methods: We first used rAAV-1, rAAV-6, rAAV-8, rAAV-9 expressing EGFP to screen for rAAV serotypes that efficiently target porcine embryos, and then, to achieve efficient expression of CRISPR/Cas9 in vivo for a short period, we packaged sgRNAs targeting the GHR genes to self-complementary adeno-associated virus (scAAV), and Cas9 proteins to single-stranded adeno-associated virus (ssAAV). The efficiency of porcine embryos -based editing was then validated in vitro. The feasibility of this one-step method to produce gene-edited pigs using rAAV-CRISPR/Cas9 oviductal injection into sows within 24 h of conception was then validated. Results: Our research firstly establishes the efficient delivery of CRISPR/Cas9 to pig zygotes, both in vivo and in vitro, using rAAV6. Successful gene editing in pigs was achieved through oviductal injection of rAAV-CRISPR/Cas9. Conclusion: This method circumvents the intricate procedures involved in in vitro embryo manipulation and embryo transfers, providing a straightforward and cost-effective approach for the production of gene-edited pigs.Copyright © 2024 Gao, He, Zhu, Peng, Zhou, Deng, Liao, Ni, Li, Gao, Bu, Yang, Yang, Yang and Bao.

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出版当年[2023]版：

大类 | 2 区生物学

小类 | 2 区发育生物学 3 区细胞生物学

最新[2023]版：

大类 | 2 区生物学

小类 | 2 区发育生物学 3 区细胞生物学

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第一作者机构： [1]Department of Pathology, Institute of Clinical Pathology, Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China.

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