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Silkworm genetic sexing through W chromosome-linked, targeted gene integration

文献类型： 外文期刊

作者： Zhang, Zhongjie 1 ; Niu, Baolong 3 ; Ji, Dongfeng 3 ; Li, Muwang 4 ; Li, Kai 2 ; James, Anthony A. 5 ; Tan, Anjiang 1 ; H 1 ;

作者机构： 1.Chinese Acad Sci, Shanghai Inst Plant Physiol & Ecol, Ctr Excellence Mol Plant Sci, Key Lab Insect Dev & Evolutionary Biol, Shanghai 200032, Peoples R China

2.East China Normal Univ, Sch Life Sci, Shanghai 200062, Peoples R China

3.Zhejiang Acad Agr Sci, Sericultural Res Inst, Hangzhou 310021, Zhejiang, Peoples R China

4.Jiangsu Univ Sci & Technol, Sericultural Res Inst, Zhenjiang 212018, Peoples R China

5.Univ Calif Irvine, Dept Microbiol & Mol Genet, Irvine, CA 92697 USA

6.Univ Calif Irvine, Dept Mol Biol & Biochem, Irvine, CA 92697 USA

关键词： Lepidoptera; transgenesis; W chromosome; female lethality

期刊名称：PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA （ 影响因子：11.205； 五年影响因子：12.291 ）

ISSN： 0027-8424

年卷期： 2018 年 115 卷 35 期

页码：

收录情况： SCI

摘要： Sex separation methods are critical for genetic sexing systems in commercial insect production and sterile insect techniques. Integration of selectable marker genes into a sex chromosome is particularly useful in insects with a heterogametic sex determination system. Here, we describe targeted gene integration of fluorescent marker expression cassettes into a randomly amplified polymorphic DNA (RAPD) marker region in the W chromosome of the lepidopteran model insect Bombyx mori using transcriptional activator-like effector nuclease (TALEN)-mediated genome editing. This silkworm strain shows ubiquitous female-specific red or green fluorescence from the embryonic to adult stages. Furthermore, we developed a binary, female-specific, embryonic lethality system combining the TALEN and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. This system includes one strain with TALEN-mediated, W-specific Cas9 expression driven by the silkworm germ cell-specific nanos (nos) promoter and another strain with U6-derived single-guide RNA (sgRNA) expression targeting transformer 2 (tra2), an essential gene for silkworm embryonic development. Filial 1 (F1) hybrids exhibit complete female-specific lethality during embryonic stages. Our study provides a promising approach for B. mori genetic sexing and sheds light on developing sterile insect techniques in other insect species, especially in lepidopteran pests with WZ/ZZ sex chromosome systems.

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