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

• DOI: 10.3389/fcell.2024.1376936
• Publication Date: 2024-03-15T04:55:58Z
• Authors: 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
• Keywords: Adeno-associated virus; Xenotransplantation; Gene targeting; Gene knockin

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 generating gene-edited pigs. Nevertheless, delivering CRISPR/Cas9 to pre-implantation embryos has traditionally posed challenges due its reliance on intricate micromanipulation equipment specialized techniques, resulting in high costs time-consuming procedures. This study aims introduce novel one-step approach genetically modified by transducing components into porcine through oviductal injection of recombinant adeno-associated viruses (rAAV). Methods: We first used rAAV-1, rAAV-6, rAAV-8, rAAV-9 expressing EGFP screen rAAV serotypes that efficiently target embryos, then, achieve efficient expression vivo period, we packaged sgRNAs targeting the GHR genes self-complementary virus (scAAV), Cas9 proteins single-stranded (ssAAV). The efficiency -based editing was then validated vitro . feasibility this method produce using rAAV-CRISPR/Cas9 sows within 24 h conception validated. Results: Our research firstly establishes delivery pig zygotes, both , rAAV6. Successful achieved rAAV-CRISPR/Cas9. Conclusion: circumvents procedures involved embryo manipulation transfers, providing straightforward cost-effective production