title: Highly improved gene targeting by germline-specific Cas9 expression in Drosophila.
abstract: We report a simple yet extremely efficient platform for systematic gene targeting by the RNA-guided endonuclease Cas9 in Drosophila. The system comprises two transgenic strains: one expressing Cas9 protein from the germline-specific nanos promoter and the other ubiquitously expressing a custom guide RNA (gRNA) that targets a unique site in the genome. The two strains are crossed to form an active Cas9-gRNA complex specifically in germ cells, which cleaves and mutates the target site. We demonstrate rapid generation of mutants in seven neuropeptide and two microRNA genes in which no mutants have been described. Founder animals stably expressing Cas9-gRNA transmitted germline mutations to an average of 60% of their progeny, a dramatic improvement in efficiency over the previous methods based on transient Cas9 expression. Simultaneous cleavage of two sites by co-expression of two gRNAs efficiently induced internal deletion with frequencies of 4.3-23%. Our method is readily scalable to high-throughput gene targeting, thereby accelerating comprehensive functional annotation of the Drosophila genome.
authors.affiliation: Invertebrate Genetics Laboratory, National Institute of Genetics and Department of Genetics, the Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan.
authors.forename: Shu
authors.initials: S
authors.lastname: Kondo
authors.forename: Ryu
authors.initials: R
authors.lastname: Ueda
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D029721:Drosophila Proteins / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics*; D005136:Eye Proteins / Q000235:genetics; D005260:Female; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D017344:Genes, Insect; D018095:Germ-Line Mutation; D008297:Male; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016601:RNA-Binding Proteins / Q000235:genetics; D058727:RNA, Small Untranslated
chemical_list: D018528:ATP-Binding Cassette Transporters; D064130:CRISPR-Associated Proteins; D029721:Drosophila Proteins; D005136:Eye Proteins; D016601:RNA-Binding Proteins; C059254:w protein, Drosophila; C070574:nos protein, Drosophila; D058727:RNA, Small Untranslated
keywords: CRISPR; Cas9; Drosophila