title: CRISPR-Cas9-Mediated Genetic Screening in Mice with Haploid Embryonic Stem Cells Carrying a Guide RNA Library.
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.
authors.affiliation: 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.
authors.forename: Cuiqing
authors.initials: C
authors.lastname: Zhong
authors.affiliation: 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.
authors.forename: Qi
authors.initials: Q
authors.lastname: Yin
authors.affiliation: 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.
authors.forename: Zhenfei
authors.initials: Z
authors.lastname: Xie
authors.affiliation: 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.
authors.forename: Meizhu
authors.initials: M
authors.lastname: Bai
authors.affiliation: 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.
authors.forename: Rui
authors.initials: R
authors.lastname: Dong
authors.affiliation: Animal Core Facility, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Tang
authors.affiliation: 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.
authors.forename: Yu-Hang
authors.initials: YH
authors.lastname: Xing
authors.affiliation: 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.
authors.forename: Hongling
authors.initials: H
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Suming
authors.initials: S
authors.lastname: Yang
authors.affiliation: 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.
authors.forename: Ling-Ling
authors.initials: LL
authors.lastname: Chen
authors.affiliation: Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Marisa S
authors.initials: MS
authors.lastname: Bartolomei
authors.affiliation: Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK.
authors.forename: Anne
authors.initials: A
authors.lastname: Ferguson-Smith
authors.affiliation: 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.
authors.forename: Dangsheng
authors.initials: D
authors.lastname: Li
authors.affiliation: 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.
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.affiliation: 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.
authors.forename: Yuxuan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: 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.
authors.forename: Jinsong
authors.initials: J
authors.lastname: Li
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D019976:Cloning, Organism; D019175:DNA Methylation / Q000235:genetics; D015723:Gene Library; D005820:Genetic Testing; D006238:Haploidy; D006579:Heterozygote; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data; D000066450:Mouse Embryonic Stem Cells / Q000378:metabolism*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems