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

Mengyu Gao; YuTing He; XingLong Zhu; WanLiu Peng; YanYan Zhou; Yang Deng; Guangneng Liao; Wei Ni; Yi Li; Jun Gao; Hong Bu; Jiayin Yang; Guang Yang; Yang Yang; Ji Bao

doi:10.3389/fcell.2024.1376936

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

Front Cell Dev Biol. 2024 Mar 15:12:1376936. doi: 10.3389/fcell.2024.1376936. eCollection 2024.

Authors

Mengyu Gao¹, YuTing He¹, XingLong Zhu¹, WanLiu Peng¹, YanYan Zhou¹, Yang Deng¹, Guangneng Liao², Wei Ni³, Yi Li⁴, Jun Gao⁵, Hong Bu¹, Jiayin Yang⁶, Guang Yang², Yang Yang⁷, Ji Bao¹

Affiliations

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.

Abstract

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.

Keywords: CRISPR/Cas9; animal model; embryo gene delivery; gene-edited pig; recombinant adeno-associated viruses.

Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This project was supported by National Natural Scientific Foundations of China (81770618), National Natural Scientific Foundations of China (82070640), the Postdoctoral Research and Development Fund of West China Hospital, Sichuan University (2023HXBH039), 1.3.5 project for disciplines of excellence, West China Hospital Sichuan University (ZYJC21014).