Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system
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-transfectio...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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PeerJ Inc.
2013-12-01
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| Series: | PeerJ |
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| Online Access: | https://peerj.com/articles/230.pdf |
| _version_ | 1827607966354767872 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Takuro Horii |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T07:04:27Z |
| format | Article |
| id | doaj.art-afb4ac3fd52f4d40abac7a2467ea58f0 |
| institution | Directory Open Access Journal |
| issn | 2167-8359 |
| language | English |
| last_indexed | 2024-03-09T07:04:27Z |
| publishDate | 2013-12-01 |
| publisher | PeerJ Inc. |
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| series | PeerJ |
| spelling | doaj.art-afb4ac3fd52f4d40abac7a2467ea58f02023-12-03T09:46:03ZengPeerJ Inc.PeerJ2167-83592013-12-011e23010.7717/peerj.230230Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA systemTakuro Horii0Sumiyo Morita1Mika Kimura2Ryouhei Kobayashi3Daiki Tamura4Ryou-u Takahashi5Hironobu Kimura6Isao Suetake7Hirokazu Ohata8Koji Okamoto9Shoji Tajima10Takahiro Ochiya11Yumiko Abe12Izuho Hatada13Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, JapanLaboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, JapanLaboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, JapanLaboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, JapanLaboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, JapanDivision of Molecular and Cellular Medicine, National Cancer Center Research Institute, Chuo-ku, Tokyo, JapanLaboratory of Epigenetics, Institute for Protein Research, Osaka University, Suita, Osaka, JapanLaboratory of Epigenetics, Institute for Protein Research, Osaka University, Suita, Osaka, JapanDivision of Cancer Development System, National Cancer Center Research Institute, Chuo-ku, Tokyo, JapanDivision of Cancer Development System, National Cancer Center Research Institute, Chuo-ku, Tokyo, JapanLaboratory of Epigenetics, Institute for Protein Research, Osaka University, Suita, Osaka, JapanDivision of Molecular and Cellular Medicine, National Cancer Center Research Institute, Chuo-ku, Tokyo, JapanDepartment of Laboratory Sciences, Graduate School of Health Sciences, Gunma University, Maebashi, Gunma, JapanLaboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, JapanHaploid 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.https://peerj.com/articles/230.pdfCRISPR/CasHaploidEmbryonic stem cellsGenome engineering Tet1 Tet2 |
| spellingShingle | 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 Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system PeerJ CRISPR/Cas Haploid Embryonic stem cells Genome engineering Tet1 Tet2 |
| title | Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system |
| title_full | Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system |
| title_fullStr | Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system |
| title_full_unstemmed | Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system |
| title_short | Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system |
| title_sort | genome engineering of mammalian haploid embryonic stem cells using the cas9 rna system |
| topic | CRISPR/Cas Haploid Embryonic stem cells Genome engineering Tet1 Tet2 |
| url | https://peerj.com/articles/230.pdf |
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