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

PeerJ

Yumiko Abe¹  Koji Okamoto²  Hirokazu Ohata²  Takahiro Ochiya³  Ryou-u Takahashi³  Hironobu Kimura⁴  Shoji Tajima⁴  Isao Suetake⁴  Izuho Hatada⁵  Daiki Tamura⁵  Mika Kimura⁵  Ryouhei Kobayashi⁵  Takuro Horii⁵  Sumiyo Morita⁵ 

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

关键词: CRISPR/Cas;    Haploid;    Embryonic stem cells;    Genome engineering;   

DOI  :  10.7717/peerj.230

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown