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.