BackgroundRewriting the genomes of living organisms has been a long-standing aim in the biological sciences. The revelation of the CRISPR/Cas9 technology has revolutionized the entire biological field. Since its emergence, this technology has been widely applied to induce gene knockouts, insertions, deletions, and base substitutions. However, the classical version of this system was imperfect for inducing or correcting desired mutations. A subsequent development generated more advanced classes, including cytosine and adenine base editors, which can be used to achieve single nucleotide substitutions. Nevertheless, these advanced systems still suffer from several limitations, such as the inability to edit loci without a suitable PAM sequence and to induce base transversions. On the other hand, the recently emerged prime editors (PEs) can achieve all possible single nucleotide substitutions as well as targeted insertions and deletions, which show promising potential to alter and correct the genomes of various organisms. Of note, the application of PE to edit livestock genomes has not been reported yet.ResultsIn this study, using PE, we successfully generated sheep with two agriculturally significant mutations, including the fecundity-relatedFecBBp.Q249R and the tail length-relatedTBXTp.G112W. Additionally, we applied PE to generate porcine blastocysts with a biomedically relevant point mutation (KCNJ5p.G151R) as a porcine model of human primary aldosteronism.ConclusionsOur study demonstrates the potential of the PE system to edit the genomes of large animals for the induction of economically desired mutations and for modeling human diseases. Although prime-edited sheep and porcine blastocysts could be generated, the editing frequencies are still unsatisfactory, highlighting the need for optimizations in the PE system for efficient generation of large animals with customized traits.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-023-09409-y.