title: Spermatogenic Cell-Specific Gene Mutation in Mice via CRISPR-Cas9.
abstract: Tissue-specific knockout technology enables the analysis of the gene function in specific tissues in adult mammals. However, conventional strategy for producing tissue-specific knockout mice is a time- and labor-consuming process, restricting rapid study of the gene function in vivo. CRISPR-Cas9 system from bacteria is a simple and efficient gene-editing technique, which has enabled rapid generation of gene knockout lines in mouse by direct injection of CRISPR-Cas9 into zygotes. Here, we demonstrate CRISPR-Cas9-mediated spermatogenic cell-specific disruption of Scp3 gene in testes in one step. We first generated transgenic mice by pronuclear injection of a plasmid containing Hspa2 promoter driving Cas9 expression and showed Cas9 specific expression in spermatogenic cells. We then produced transgenic mice carrying Hspa2 promoter driven Cas9 and constitutive expressed sgRNA targeting Scp3 gene. Male founders were infertile due to developmental arrest of spermatogenic cells while female founders could produce progeny normally. Consistently, male progeny from female founders were infertile and females could transmit the transgenes to the next generation. Our study establishes a CRISPR-Cas9-based one-step strategy to analyze the gene function in adult tissues by a temporal-spatial pattern.
authors.affiliation: School of Life Science and Technology, Shanghai Tech University, Shanghai 200031, China; Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, 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.
authors.forename: Meizhu
authors.initials: M
authors.lastname: Bai
authors.affiliation: Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, 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.
authors.forename: Dan
authors.initials: D
authors.lastname: Liang
authors.affiliation: Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, 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.
authors.forename: Yinghua
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, 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.
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.affiliation: Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, 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: School of Life Science and Technology, Shanghai Tech University, Shanghai 200031, China; Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, 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: jsli@sibcb.ac.cn.
authors.forename: Jinsong
authors.initials: J
authors.lastname: Li
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D005260:Female; D018703:Founder Effect; D055786:Gene Knockout Techniques; D007246:Infertility / Q000235:genetics; D008297:Male; D051379:Mice; D009154:Mutation; D009687:Nuclear Proteins / Q000172:deficiency / Q000235:genetics; D013091:Spermatogenesis / Q000235:genetics*
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C490062:Sycp3 protein, mouse
keywords: CRISPR-Cas9; Mouse; Spermatogenesis; Tissue-specific knockout