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Construction of a One-Vector Multiplex CRISPR/Cas9 Editing System to Inhibit Nucleopolyhedrovirus Replication in Silkworms

Virologica Sinica volume 34, pages 444–453 (2019)Cite this article

Abstract

Recently the developed single guide (sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology has opened a new avenue for antiviral therapy. The CRISPR/Cas9 system uniquely allows targeting of multiple genome sites simultaneously. However, there are relatively few applications of CRISPR/Cas9 multigene editing to target insect viruses. To address the need for sustained delivery of a multiplex CRISPR/Cas9-based genome-editing vehicle against insect viruses, we developed a one-vector (pSL1180-Cas9-U6-sgRNA) system that expresses multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus (BmNPV) in insect cells. We screened the immediate-early-1 gene (ie-1), the major envelope glycoprotein gene (gp64), and the late expression factor gene (lef-11), and identified multiple sgRNA editing sites through flow cytometry and viral DNA replication analysis. In addition, we constructed a multiplex editing vector (PSL1180-Cas9-sgIE1-sgLEF11-sgGP64, sgMultiple) to efficiently regulate multiplex gene-editing and inhibit BmNPV replication after viral infection. This is the first report of the application of a multiplex CRISPR/Cas9 system to inhibit insect virus replication. This multiplex system can significantly enhance the potential of CRISPR/Cas9-based multiplex genome engineering in insect virus.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (Nos. 31872427 and 31572466), China Agriculture Research System (CARS-18), Chongqing Special Postdoctoral Science Foundation (XmT2018020), and China Postdoctoral Science Foundation (2018M633309).

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Author notes

  1. Zhanqi Dong and Qi Qin have contributed equally to this work.

Affiliations

  1. State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716, China

    Zhanqi Dong, Qi Qin, Zhigang Hu, Peng Chen, Liang Huang, Xinling Zhang, Ting Tian, Cheng Lu & Minhui Pan

  2. Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture, Southwest University, Chongqing, 400716, China

    Cheng Lu & Minhui Pan

Authors
  1. Zhanqi Dong
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  2. Qi Qin
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  3. Zhigang Hu
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  4. Peng Chen
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  5. Liang Huang
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  6. Xinling Zhang
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  7. Ting Tian
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  8. Cheng Lu
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  9. Minhui Pan
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Contributions

ZD and ZH conceived and designed the experiments. LH, QQ, TT and XZ curated the Data. ZD, PC and QQ analyzed the date; ZD, CL and MP contributed to the writing of manuscript. All authors reviewed and approved the final manuscript for submission.

Corresponding authors

Correspondence to Cheng Lu or Minhui Pan.

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The authors declare that they have no conflict of interest.

Animal and Human Rights Statement

This article does not contain any studies with human or animal subjects performed by any of the authors.

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Dong, Z., Qin, Q., Hu, Z. et al. Construction of a One-Vector Multiplex CRISPR/Cas9 Editing System to Inhibit Nucleopolyhedrovirus Replication in Silkworms. Virol. Sin. 34, 444–453 (2019). https://doi.org/10.1007/s12250-019-00121-4

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  • DOI: https://doi.org/10.1007/s12250-019-00121-4

Keywords

  • Bombyx mori
  • Bombyx mori nucleopolyhedrovirus (BmNPV)
  • Antiviral therapeutic
  • CRISPR/Cas9
  • Multigene editing