title: Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system.
abstract: Haploid embryonic stem cells (ESCs) are useful for studying mammalian genes because disruption of only one allele can cause loss-of-function phenotypes. Here, we report the use of haploid ESCs and the CRISPR RNA-guided Cas9 nuclease gene-targeting system to manipulate mammalian genes. Co-transfection of haploid ESCs with vectors expressing Cas9 nuclease and single-guide RNAs (sgRNAs) targeting Tet1, Tet2, and Tet3 resulted in the complete disruption of all three genes and caused a loss-of-function phenotype with high efficiency (50%). Co-transfection of cells with vectors expressing Cas9 and sgRNAs targeting two loci on the same chromosome resulted in the creation of a large chromosomal deletion and a large inversion. Thus, the use of the CRISPR system in combination with haploid ESCs provides a powerful platform to manipulate the mammalian genome.
authors.affiliation: Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University , Maebashi, Gunma , Japan.
authors.forename: Takuro
authors.initials: T
authors.lastname: Horii
authors.affiliation: Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University , Maebashi, Gunma , Japan.
authors.forename: Sumiyo
authors.initials: S
authors.lastname: Morita
authors.affiliation: Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University , Maebashi, Gunma , Japan.
authors.forename: Mika
authors.initials: M
authors.lastname: Kimura
authors.affiliation: Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University , Maebashi, Gunma , Japan ; Department of Laboratory Sciences, Graduate School of Health Sciences, Gunma University , Maebashi, Gunma , Japan.
authors.forename: Ryouhei
authors.initials: R
authors.lastname: Kobayashi
authors.affiliation: Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University , Maebashi, Gunma , Japan.
authors.forename: Daiki
authors.initials: D
authors.lastname: Tamura
authors.affiliation: Division of Molecular and Cellular Medicine, National Cancer Center Research Institute , Chuo-ku, Tokyo , Japan.
authors.forename: Ryou-U
authors.initials: RU
authors.lastname: Takahashi
authors.affiliation: Laboratory of Epigenetics, Institute for Protein Research, Osaka University , Suita, Osaka , Japan.
authors.forename: Hironobu
authors.initials: H
authors.lastname: Kimura
authors.affiliation: Laboratory of Epigenetics, Institute for Protein Research, Osaka University , Suita, Osaka , Japan.
authors.forename: Isao
authors.initials: I
authors.lastname: Suetake
authors.affiliation: Division of Cancer Development System, National Cancer Center Research Institute , Chuo-ku, Tokyo , Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Ohata
authors.affiliation: Division of Cancer Development System, National Cancer Center Research Institute , Chuo-ku, Tokyo , Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Okamoto
authors.affiliation: Laboratory of Epigenetics, Institute for Protein Research, Osaka University , Suita, Osaka , Japan.
authors.forename: Shoji
authors.initials: S
authors.lastname: Tajima
authors.affiliation: Division of Molecular and Cellular Medicine, National Cancer Center Research Institute , Chuo-ku, Tokyo , Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ochiya
authors.affiliation: Department of Laboratory Sciences, Graduate School of Health Sciences, Gunma University , Maebashi, Gunma , Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Abe
authors.affiliation: Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University , Maebashi, Gunma , Japan.
authors.forename: Izuho
authors.initials: I
authors.lastname: Hatada
keywords: CRISPR/Cas; Embryonic stem cells; Genome engineering; Haploid; Tet1; Tet2; Tet3