title: Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9.
abstract: The ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.
authors.affiliation: Gene Regulatory Laboratory, School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Xingjie
authors.initials: X
authors.lastname: Ren
authors.forename: Jin
authors.initials: J
authors.lastname: Sun
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Housden
authors.forename: Yanhui
authors.initials: Y
authors.lastname: Hu
authors.forename: Charles
authors.initials: C
authors.lastname: Roesel
authors.forename: Shuailiang
authors.initials: S
authors.lastname: Lin
authors.forename: Lu-Ping
authors.initials: LP
authors.lastname: Liu
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Yang
authors.forename: Decai
authors.initials: D
authors.lastname: Mao
authors.forename: Lingzhu
authors.initials: L
authors.lastname: Sun
authors.forename: Qujie
authors.initials: Q
authors.lastname: Wu
authors.forename: Jun-Yuan
authors.initials: JY
authors.lastname: Ji
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Xi
authors.forename: Stephanie E
authors.initials: SE
authors.lastname: Mohr
authors.forename: Jiang
authors.initials: J
authors.lastname: Xu
authors.forename: Norbert
authors.initials: N
authors.lastname: Perrimon
authors.forename: Jian-Quan
authors.initials: JQ
authors.lastname: Ni
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D064113:CRISPR-Cas Systems / Q000235:genetics*; D030541:Databases, Genetic; D029721:Drosophila Proteins / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D023281:Genomics / Q000379:methods*; D005854:Germ Cells; D016296:Mutagenesis / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics
chemical_list: D029721:Drosophila Proteins; D016601:RNA-Binding Proteins; C070574:nos protein, Drosophila
keywords: HRMA; nanos-Cas9