title: Efficient gene knock-out and knock-in with transgenic Cas9 in Drosophila.
abstract: Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor.
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Zhaoyu
authors.initials: Z
authors.lastname: Xue
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Mengda
authors.initials: M
authors.lastname: Ren
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Menghua
authors.initials: M
authors.lastname: Wu
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Junbiao
authors.initials: J
authors.lastname: Dai
authors.affiliation: Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Yikang S
authors.initials: YS
authors.lastname: Rong
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China gaogu@mail.tsinghua.edu.cn.
authors.forename: Guanjun
authors.initials: G
authors.lastname: Gao
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001426:Bacterial Proteins / Q000235:genetics*; D003851:Deoxyribonucleases / Q000235:genetics*; D004330:Drosophila / Q000235:genetics*; D015870:Gene Expression; D055879:Gene Knock-In Techniques; D055786:Gene Knockout Techniques; D023061:Gene Order; D018390:Gene Targeting; D018095:Germ-Line Mutation; D059765:Homologous Recombination; D008845:Microinjections; D019076:Transgenes; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D001426:Bacterial Proteins; D003851:Deoxyribonucleases; D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: Cas9; Drosophila; gRNA; knock-in; knock-out