Highly Efficient Temperature Inducible CRISPR-Cas9 Gene Targeting in Drosophila suzukii

文献类型： 外文期刊

第一作者： Yan, Ying

作者： Yan, Ying;Kobayashi, Yukino;Schetelig, Marc F.;Huang, Cong;Liu, Bo;Qian, Wanqiang;Wan, Fanghao;Huang, Cong;Liu, Bo;Qian, Wanqiang;Wan, Fanghao

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关键词： spotted-wing drosophila; insect transgenesis; targeted mutagenesis; conditional system; pigmentation; genetic control; gene drive

期刊名称：INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES （ 影响因子：5.924； 五年影响因子：6.132 ）

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年卷期： 2021 年 22 卷 13 期

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收录情况： SCI

摘要： The spotted-wing Drosophila (Drosophila suzukii 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 D. suzukii. Five independent transgenic lines were generated using a piggyBac construct containing the EGFP fluorescent marker gene and the Cas9 gene under the control of the D. melanogaster heat shock protein 70 promoter and 3'UTR. Heat-shock (HS) treated embryos were analyzed by reverse transcriptase PCR, revealing strong heat inducibility of the transgenic Cas9 expression. By injecting gRNA targeting EGFP into one selected line, 50.0% of G(0) flies showed mosaic loss-of-fluorescence phenotype, and 45.5% of G(0) flies produced G(1) 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 yellow 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 D. suzukii.

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