CRISPR/Cas9 nuclease-mediated gene knock-in in bovine-induced pluripotent cells

• Published in: Stem cells and development Vol. 24; no. 3; p. 393
• Main Authors: Heo, Young Tae, Quan, Xiaoyuan, Xu, Yong Nan, Baek, Soonbong, Choi, Hwan, Kim, Nam-Hyung, Kim, Jongpil
• Format: Journal Article
• Language: English
• Published: United States 01.02.2015
• Subjects: Animals; Animals, Genetically Modified; Benzamides - pharmacology; Blastocyst - cytology; Blastocyst - metabolism; Cattle - embryology; Cattle - genetics; Cells, Cultured; CRISPR-Cas Systems; Diphenylamine - analogs & derivatives; Diphenylamine - pharmacology; Fertilization in Vitro; Fibroblasts - cytology; Gene Knock-In Techniques; Genetic Engineering - methods; Genetic Enhancement; Genetic Loci - genetics; Genetic Vectors; Glycogen Synthase Kinase 3 - pharmacology; Glycogen Synthase Kinase 3 beta; Homologous Recombination; Induced Pluripotent Stem Cells - metabolism; Induced Pluripotent Stem Cells - transplantation; Mice; Mice, SCID; Polymorphism, Restriction Fragment Length; Pyridines - pharmacology; Pyrimidines - pharmacology; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Teratoma - etiology; Transcription Factors - genetics; Transcription Factors - metabolism; Valproic Acid - pharmacology
• ISSN: 1557-8534
• DOI: 10.1089/scd.2014.0278

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.