Highly Efficient Temperature Inducible CRISPR-Cas9 Gene Targeting in .

The spotted-wing Drosophila ( Matsumura) is native to eastern Asia, but has become a global threat to fruit production. In recent years, CRISPR/Cas9 targeting was established in this species allowing for functional genomic and genetic control studies. Here, we report the generation and characterization of Cas9-expressing strains of . Five independent transgenic lines were generated using a construct containing the fluorescent marker gene and the gene under the control of the promoter and 3'UTR. Heat-shock (HS) treated embryos were analyzed by reverse transcriptase PCR, revealing strong heat inducibility of the transgenic expression. By injecting gRNA targeting into one selected line, 50.0% of G flies showed mosaic loss-of-fluorescence phenotype, and 45.5% of G flies produced G mutants without HS. Such somatic and germline mutagenesis rates were increased to 95.4% and 85.7%, respectively, by applying a HS. Parental flies receiving HS resulted in high inheritance of the mutation (92%) in their progeny. Additionally, targeting the endogenous gene led to the lack of pigmentation and male lethality. We discuss the potential use of these efficient and temperature-dependent Cas9-expressing strains for the genetic studies in .