title: CRISPR/Cas9 nuclease-mediated gene knock-in in bovine-induced pluripotent cells.
abstract: Efficient and precise genetic engineering in livestock such as cattle holds great promise in agriculture and biomedicine. However, techniques that generate pluripotent stem cells, as well as reliable tools for gene targeting in livestock, are still inefficient, and thus not routinely used. Here, we report highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, we generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3β and MEK inhibitor (2i) treatment. We observed that these bovine iPSCs are highly similar to naïve pluripotent stem cells with regard to gene expression and developmental potential in teratomas. Moreover, CRISPR/Cas9 nuclease, which was specific for the bovine NANOG locus, showed highly efficient editing of the bovine genome in bovine iPSCs and embryos. To conclude, CRISPR/Cas9 nuclease-mediated homologous recombination targeting in bovine pluripotent cells is an efficient gene editing method that can be used to generate transgenic livestock in the future.
authors.affiliation: 1 Department of Animal Sciences, Center for the Animal Bioreactor and Xenotransplantation, Chungbuk National University , Cheongju, South Korea .
authors.forename: Young Tae
authors.initials: YT
authors.lastname: Heo
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Quan
authors.forename: Yong Nan
authors.initials: YN
authors.lastname: Xu
authors.forename: Soonbong
authors.initials: S
authors.lastname: Baek
authors.forename: Hwan
authors.initials: H
authors.lastname: Choi
authors.forename: Nam-Hyung
authors.initials: NH
authors.lastname: Kim
authors.forename: Jongpil
authors.initials: J
authors.lastname: Kim
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001549:Benzamides / Q000494:pharmacology; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D064113:CRISPR-Cas Systems; D002417:Cattle / Q000196:embryology / Q000235:genetics*; D002478:Cells, Cultured; D004159:Diphenylamine / Q000031:analogs & derivatives / Q000494:pharmacology; D005307:Fertilization in Vitro; D005347:Fibroblasts / Q000166:cytology; D055879:Gene Knock-In Techniques; D005818:Genetic Engineering / Q000379:methods*; D024861:Genetic Enhancement; D056426:Genetic Loci / Q000235:genetics; D005822:Genetic Vectors; D038362:Glycogen Synthase Kinase 3 / Q000494:pharmacology; D000071679:Glycogen Synthase Kinase 3 beta; D059765:Homologous Recombination; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism* / Q000637:transplantation; D051379:Mice; D016513:Mice, SCID; D012150:Polymorphism, Restriction Fragment Length; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013724:Teratoma / Q000209:etiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014635:Valproic Acid / Q000494:pharmacology
chemical_list: D001549:Benzamides; C473711:Chir 99021; D011725:Pyridines; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014635:Valproic Acid; C506614:mirdametinib; D004159:Diphenylamine; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3