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Applied Microbiology and Biotechnology

, Volume 102, Issue 21, pp 9255–9265 | Cite as

Establishment of a baculovirus-inducible CRISPR/Cas9 system for antiviral research in transgenic silkworms

  • Zhanqi Dong
  • Liang Huang
  • Feifan Dong
  • Zhigang Hu
  • Qi Qin
  • Jiangqiong Long
  • Mingya Cao
  • Peng Chen
  • Cheng Lu
  • Min-Hui Pan
Applied genetics and molecular biotechnology

Abstract

The CRISPR/Cas9 system is a powerful genetic engineering technique that has been widely used in gene therapy, as well as in the development of novel antimicrobials and transgenic insects. However, several challenges, including the lack of effective host target genes and the off-target effects, limit the application of CRISPR/Cas9 in insects. To mitigate these difficulties, we established a highly efficient virus-inducible CRISPR/Cas9 system in transgenic silkworms. This system includes the baculovirus-inducible promoter 39K, which directs transcription of the gene encoding, the Cas9 protein, and the U6 promoter which targets the sgATAD3A site of the ATPase family AAA domain-containing protein 3 (ATAD3A) gene. The double-positive transgenic line sgATAD3A×39K-Cas9 (ATAD3A-KO) was obtained by hybridization; antiviral activity in this hybrid transgenic line is induced only after Bombyx mori nucleopolyhedrovirus (BmNPV) infection. The BmNPV-inducible system significantly reduced off-target effects and did not affect the economically important characteristics of the transgenic silkworms. Most importantly, this novel system efficiently and consistently edited target genes, inhibiting BmNPV replication after the transgenic silkworms were inoculated with occlusion bodies (OBs). The suppression of BmNPV by the virus-inducible system was comparable to that of the stably expressed CRISPR/Cas9 system. Therefore, we successfully established a highly efficient BmNPV-inducible ATAD3A-KO transgenic silkworm line, with improved gene targeting specificity and antiviral efficiency. Our study thereby provides insights into the treatment of infectious diseases and into the control of insect pests.

Keywords

Inducible CRISPR/Cas9 Transgenic Antiviral therapy BmNPV ATAD3A-KO 

Notes

Author contributions

Z.D., F.D., and L.H. performed vector cloning, sequencing, cell culturing, and PCR. Z.D., F.D., and Z.H. conducted transgenic injections. M.C., Z.H., Q.Q., 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

This study was funded by The National Natural Science Foundation of China (Grant Nos. 31472153 and 31572466) and the China Agriculture Research System (CARS-18).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

This article does not contain any experiments with human participants or animals (except invertebrates, which are exempt from ethical concerns) performed by any of the authors.

Supplementary material

253_2018_9295_MOESM1_ESM.pdf (395 kb)
ESM 1 (PDF 395 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Silkworm Genome BiologySouthwest UniversityChongqingChina
  2. 2.Joint National Laboratory for Antibody Drug Engineering, Institute of ImmunologyHenan University School of MedicineKaifengChina
  3. 3.Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of AgricultureSouthwest UniversityChongqingChina

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