Cas9-expressing cattle using all-in-one CRISPR/Cas9 for bovine genome editing

Background: 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.Results: In this study, two types of CRISPR/Cas9-expressing cattle were successfully produced: Cas9-RFP-FatI, Cas9-GFP-sgPRNP. Somatic cells from these cattle were induced to mutate several target genes when single-guide RNAs (sgRNAs) were transfected into the somatic cells. Additionally, semen from CRISPR/Cas9 expressing male cattle was frozen and used to fertilize wild-type oocytes, successfully transmitting the transgene (Cas9, reporter genes, fatty acid dehydrogenase I [FatI]), and sgRNA for the prion protein [PRNP]) 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.Conclusion: 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. Less...

研究背景：家畜（尤其是牛）在遗传育种、传染病研究、生物反应器构建以及特定疾病模型开发等生物技术领域中具有至关重要的价值。然而，牛的基因工程研究进展相对迟缓，其原因在于漫长的妊娠期、单胚胎妊娠特性以及高昂的饲养成本。此外，生殖系传递的验证周期漫长，且缺乏具备生殖系整合能力的胚胎干细胞，进一步阻碍了相关研究的推进。随着锌指核酸酶（ZFN）、转录激活因子样效应物核酸酶（TALEN）以及CRISPR-Cas9等基因组编辑技术的发展，近期已有研究成功培育出表达Cas9的猪与鸡。本研究提出如下假说：培育表达CRISPR/Cas9的牛及其相关资源，将为牛基因组编辑研究提供强有力的工具，助力我们深入理解牛的遗传学特性与疾病抗性机制。

研究结果：本研究成功培育出两种表达CRISPR/Cas9的牛：Cas9-RFP-FatI与Cas9-GFP-sgPRNP。将单引导RNA（sgRNA）转染至这些牛的体细胞后，可诱导多个靶基因发生突变。此外，研究人员将表达CRISPR/Cas9的公牛精液冷冻保存，用于使野生型卵母细胞受精，成功将外源转基因（Cas9、报告基因、脂肪酸脱氢酶I（FatI）以及靶向朊蛋白（PRNP）的sgRNA）传递至子代。进一步通过体外胚胎生产实验证实，源自F1代公牛精液的胚胎中，Cas9的基因编辑能力（包括基因敲除与高效敲入）均得到验证。

研究结论：本研究数据首次证实，表达Cas9的牛成功诞生，且该外源转基因可传递至子代犊牛（F1代）以及F2代胚胎。此外，研究人员利用F0与F1代的体细胞及生殖细胞，评估了其在多基因编辑（敲除与敲入）中的应用潜力。目前，携带PRNP突变的F1代牛已作为牛海绵状脑病抗性模型进行饲养。这些转基因牛模型及其衍生资源，将为体外与体内基因组编辑研究提供宝贵的研究工具，推动我们对牛基因组学与相关疾病的遗传学认知进一步深化。