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Genetics: Early Online, published on September 3, 2013 as 10.1534/genetics.113.156737 Highly improved gene targeting by germline-specific Cas9 expression in Drosophila Shu Kondo1,2, Ryu Ueda1,2 1Invertebrate Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka, Japan. 2Department of Genetics, the Graduate University for Advanced Studies, Mishima, Shizuoka, Japan. Correspondence should be addressed to S.K. (skondo@nig.ac.jp). Copyright 2013. 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 mutate the target site. We demonstrate rapid generation of mutants in seven neuropeptide and two miRNA 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. Phenotypes of mutant animals have provided deeper insight into gene function than any other means. In Drosophila melanogaster , hundreds