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Construction and application of an HSP70 promoter-inducible genome editing system in transgenic silkworm to induce resistance to Nosema bombycis

  • Zhanqi Dong
  • Jiangqiong Long
  • Liang Huang
  • Zhigang Hu
  • Peng Chen
  • Nan Hu
  • Ning Zheng
  • Xuhua Huang
  • Cheng LuEmail author
  • Minhui PanEmail author
Applied genetics and molecular biotechnology

Abstract

The microsporidian Nosema bombycis is an obligate intracellular parasitic fungus that causes devastating disease in sericulture. To date, no efficient biotechnological method to inhibit the proliferation of microspores has been established. Here, we developed a powerful genetic engineering technique involving microsporidia-inducible genome editing in transgenic silkworm that confers resistance to N. bombycis. This system includes an HSP70 promoter-induced expression of the Cas9 protein line and a target BmATAD3A gene line. The double-positive HSP70-Cas9(+)×sgATAD3A(+) lines were obtained by hybridization and activation of the CRISPR/Cas9 system under the condition of microsporidia infection, although it is silenced in uninfected individuals. Genome editing analysis showed that the system could efficiently edit the BmATAD3A gene and induce large deletions. It is notable that the HSP70-induced system could effectively improve the survival rate of transgenic silkworm after microsporidia infection and inhibit the expression of key microsporidia genes. Moreover, no significant developmental differences between the transgenic silkworms infected with microsporidia and normal individuals were observed. In this study, we effectively inhibited microsporidia proliferation in transgenic individuals through disruptive techniques, thereby providing a method for microsporidia treatment and prevention, paving the way for economically advantageous insect breeding.

Keywords

Microsporidia Silkworm CRISPR/Cas9 Transgenic Inducible genome editing 

Notes

Author contributions

Z.D., J.L., and L.H. performed vector cloning, sequencing, cell culturing, and PCR. Z.D., J.L., and Z.H. conducted transgenic injections. N.Z., X.H, Z.H., and J.L. participated in mortality analyses and DNA replication assays. Z.D., M.P., and C.L. conceived the experimental design and participated in data analysis. Z.D., M.P., P.C., and C.L. were involved in the preparation of the manuscript. The final manuscript was reviewed and approved by all authors.

Funding Information

This study was funded by The National Natural Science Foundation of China (Grant Nos. 31902214, 31872427, and 31572466), the China Agriculture Research System (CARS-18), Chongqing Special Postdoctoral Science Foundation (No. XmT2018020), Natural Science Foundation of Chongqing (cstc2019jcyj-msxm2371), and the China Postdoctoral Science Foundation (No. 2018M633309).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any experiments with human participants or animals performed by any of the authors.

Supplementary material

253_2019_10135_MOESM1_ESM.pdf (113 kb)
ESM 1 (PDF 113 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Silkworm Genome BiologySouthwest UniversityChongqingChina
  2. 2.Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of AgricultureSouthwest UniversityChongqingChina
  3. 3.The General Extension Station of Sericulture Technology of Guangxi Zhuang Autonomous RegionNanningChina

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