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Cell Stem Cell. 2015 Aug 6;17(2):221-32. doi: 10.1016/j.stem.2015.06.005. Epub 2015 Jul 9.

CRISPR-Cas9-Mediated Genetic Screening in Mice with Haploid Embryonic Stem Cells Carrying a Guide RNA Library.

Author information

1
Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China.
2
Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; School of Life Science and Technology, Shanghai Tech University, Shanghai 200031, China.
3
College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China; Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology; CAS Center for Excellence in Brain Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
4
Animal Core Facility, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
5
Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China; State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
6
Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
7
Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK.
8
Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China; Shanghai Information Center for Life Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
9
Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology; CAS Center for Excellence in Brain Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. Electronic address: liyang@picb.ac.cn.
10
Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. Electronic address: yuxuan.wu@sibcb.ac.cn.
11
Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; School of Life Science and Technology, Shanghai Tech University, Shanghai 200031, China. Electronic address: jsli@sibcb.ac.cn.

Erratum in

  • Cell Stem Cell. 2015 Aug 6;17(2):247.

Abstract

Mouse androgenetic haploid embryonic stem cells (AG-haESCs) can support full-term development of semi-cloned (SC) embryos upon injection into MII oocytes and thus have potential applications in genetic modifications. However, the very low birth rate of SC pups limits practical use of this approach. Here, we show that AG-haESCs carrying deletions in the DMRs (differentially DNA methylated regions) controlling two paternally repressed imprinted genes, H19 and Gtl2, can efficiently support the generation of SC pups. Genetic manipulation of these DKO-AG-haESCs in vitro using CRISPR-Cas9 can produce SC mice carrying multiple modifications with high efficiency. Moreover, transfection of DKO-AG-haESCs with a constitutively expressed sgRNA library and Cas9 allows functional mutagenic screening. DKO-AG-haESCs are therefore an effective tool for the introduction of organism-wide mutations in mice in a single generation.

PMID:
26165924
DOI:
10.1016/j.stem.2015.06.005
[Indexed for MEDLINE]
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