Kwon Dong-Hyeok, Gim Gyeong-Min, Yum Soo-Young, Eom Kyeong-Hyeon, Lee Song-Jeon, Han Sang-Eun, Kim Hee-Soo, Kim Hyeong-Jong, Lee Woo-Sung, Choi Woo-Jae, Lee Ji-Hyun, Kim Do-Yoon, Jung Dae-Jin, Kim Dae-Hyun, Yi Jun-Koo, Moon Byeong-Ho, Lee Won-You, Jang Goo
Department of Theriogenology, College of Veterinary Medicine and the Research Institute for Veterinary Science, Seoul National University, Seoul, 08826, Republic of Korea.
Bk21 FOUR Future Veterinary Medicine Leading Education & Research Center, Seoul National University, Seoul, 08826, Republic of Korea.
BMC Genomics. 2025 Mar 5;26(1):217. doi: 10.1186/s12864-025-11381-8.
Livestock, particularly cattle, are crucial for biotechnology fields, such as genetic breeding, infectious diseases, bioreactors, and specific disease models. However, genetic engineering in cattle has lagged due to long gestation periods, single embryo pregnancies, and high rearing costs. Additionally, the slow validation of germline transmission and the absence of germline-competent embryonic stem cells hinder progress. With the development of genome editing technologies like ZFN, TALEN, and CRISPR-Cas9, recent advancements have shown that Cas9-expressing pigs and chickens have been successfully produced. We hypothesize that generating CRISPR/Cas9-expressing cattle and their resources will provide a powerful resource for bovine genome editing, advancing our understanding of bovine genetics and disease resistance.
In this study, two types of Cas9-expressing cattle were successfully produced: Cas9-RFP-fatty acid dehydrogenase I (FatI), Cas9-GFP-sgRNA for the prion protein (sgPRNP). Somatic cells from these cattle were induced to mutate multiple target genes when single-guide RNAs (sgRNAs) were transfected into the somatic cells. Additionally, semen from Cas9 expressing male cattle was frozen and used to fertilize wild-type oocytes, successfully transmitting the transgene (Cas9, reporter genes, FatI), and sgPRNP) to the next generation. Furthermore, the gene editing capabilities of Cas9, including knockout and high-efficiency knock-in, were confirmed in embryos derived from F1 semen through in vitro production.
These data demonstrate, for the first time, that Cas9-expressing cattle were successfully born, and this transgene was transmitted to the next-generation calves (F1) and F2 embryos. In addition, somatic and germ cells derived from F0 and F1generations were used to evaluate the potential for gene editing (knockout and knock-in) in multiple genes. PRNP-mutated F1 cattle are currently being raised as a resistance model for bovine spongiform encephalopathy. These transgenic bovine models and their derivatives will serve as a valuable resource for both in vitro and in vivo genome editing, advancing our genetic understanding of bovine genomics and diseases.
家畜，尤其是牛，在生物技术领域至关重要，如基因育种、传染病研究、生物反应器以及特定疾病模型。然而，由于妊娠期长、单胚胎妊娠以及饲养成本高，牛的基因工程发展滞后。此外，生殖系传递的验证缓慢以及缺乏具有生殖系能力的胚胎干细胞也阻碍了进展。随着锌指核酸酶（ZFN）、转录激活因子样效应物核酸酶（TALEN）和规律成簇间隔短回文重复序列/Cas9（CRISPR-Cas9）等基因组编辑技术的发展，最近的进展表明，已成功培育出表达Cas9的猪和鸡。我们推测，培育表达CRISPR/Cas9的牛及其资源将为牛基因组编辑提供强大资源，增进我们对牛遗传学和抗病性的理解。
在本研究中，成功培育出两种表达Cas9的牛：Cas9-红色荧光蛋白-脂肪酸脱氢酶I（FatI）、用于朊病毒蛋白的Cas9-绿色荧光蛋白-单向导RNA（sgPRNP）。当将单向导RNA（sgRNA）转染到这些牛的体细胞中时，体细胞被诱导使多个靶基因突变。此外，将表达Cas9的雄性牛的精液冷冻，用于使野生型卵母细胞受精，成功地将转基因（Cas9、报告基因、FatI和sgPRNP）传递给下一代。此外，通过体外生产，在源自F1精液的胚胎中证实了Cas9的基因编辑能力，包括基因敲除和高效基因敲入。
这些数据首次证明，表达Cas9的牛成功诞生，并且该转基因传递给了下一代犊牛（F1）和F2胚胎。此外，利用来自F0和F1代的体细胞和生殖细胞评估了多个基因进行基因编辑（敲除和敲入）的潜力。PRNP突变的F1牛目前作为牛海绵状脑病的抗性模型正在饲养中。这些转基因牛模型及其衍生物将成为体外和体内基因组编辑的宝贵资源，增进我们对牛基因组学和疾病的遗传学理解。
