Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system

Takuro Horii; Sumiyo Morita; Mika Kimura; Ryouhei Kobayashi; Daiki Tamura; Ryou-U Takahashi; Hironobu Kimura; Isao Suetake; Hirokazu Ohata; Koji Okamoto; Shoji Tajima; Takahiro Ochiya; Yumiko Abe; Izuho Hatada

doi:10.7717/peerj.230

Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system

PeerJ. 2013 Dec 23:1:e230. doi: 10.7717/peerj.230.

Authors

Affiliations

¹ Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University , Maebashi, Gunma , Japan.
² 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.
³ Division of Molecular and Cellular Medicine, National Cancer Center Research Institute , Chuo-ku, Tokyo , Japan.
⁴ Laboratory of Epigenetics, Institute for Protein Research, Osaka University , Suita, Osaka , Japan.
⁵ Division of Cancer Development System, National Cancer Center Research Institute , Chuo-ku, Tokyo , Japan.
⁶ Department of Laboratory Sciences, Graduate School of Health Sciences, Gunma University , Maebashi, Gunma , Japan.

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.

Keywords: CRISPR/Cas; Embryonic stem cells; Genome engineering; Haploid; Tet1; Tet2; Tet3.

Grants and funding

This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Ministry of Health, Labor and Welfare of Japan; the National Institute of Biomedical Innovation; the Asahi Glass Foundation; the Ichiro Kanehara Foundation; and the Takeda Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.