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 LuEmail author
  • Min-Hui PanEmail author
Applied genetics and molecular biotechnology


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.


Inducible CRISPR/Cas9 Transgenic Antiviral therapy BmNPV ATAD3A-KO 


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.


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)


  1. Blissard GW, Rohrmann GF (1990) Baculovirus diversity and molecular biology. Annu Rev Entomol 35:127–155. CrossRefPubMedGoogle Scholar
  2. Cao MY, Kuang XX, Li HQ, Lei XJ, Xiao WF, Dong ZQ, Zhang J, Hu N, Chen TT, Lu C, Pan MH (2016) Screening and optimization of an efficient B. mori nucleopolyhedrovirus inducible promoter. J Biotechnol 231:72–80. CrossRefPubMedGoogle Scholar
  3. Carson DD, Guarino LA, Summers MD (1988) Functional mapping of an AcNPV immediately early gene which augments expression of the IE-1 trans-activated 39K gene. Virology 162(2):444–451CrossRefGoogle Scholar
  4. Chen S, Hou C, Bi H, Wang Y, Xu J, Li M, James AA, Huang Y, Tan A (2017) Transgenic clustered regularly interspaced short palindromic repeat/Cas9-mediated viral gene targeting for antiviral therapy of B. mori nucleopolyhedrovirus. J Virol 91(8):e02465–e02416. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Cheng Y, Wang XY, Du C, Gao J, Xu JP (2014) Expression analysis of several antiviral related genes to BmNPV in different resistant strains of silkworm, B mori. J Insect Sci 14:76. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Dong ZQ, Zhang J, Chen XM, He Q, Cao MY, Wang L, Li HQ, Xiao WF, Pan CX, Lu C, Pan MH (2014) B. mori nucleopolyhedrovirus ORF79 is a per os infectivity factor associated with the PIF complex. Virus Res 184:62–70. CrossRefPubMedGoogle Scholar
  7. Dong C, Qu L, Wang H, Wei L, Dong Y, Xiong S (2015a) Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication. Antivir Res 118:110–117. CrossRefPubMedGoogle Scholar
  8. Dong XL, Liu TH, Wang W, Pan CX, Wu YF, Du GY, Chen P, Lu C, Pan MH (2015b) BmREEPa is a novel gene that facilitates BmNPV entry into silkworm cells. PLoS One 10(12):e0144575. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Dong ZQ, Hu N, Zhang J, Chen TT, Cao MY, Li HQ, Lei XJ, Chen P, Lu C, Pan MH (2015c) Oligomerization of baculovirus LEF-11 is involved in viral DNA replication. PLoS One 10(12):e0144930. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Dong ZQ, Chen TT, Zhang J, Hu N, Cao MY, Dong FF, Jiang YM, Chen P, Lu C, Pan MH (2016) Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells. Antivir Res 130:50–57. CrossRefPubMedGoogle Scholar
  11. Dong XL, Wu YF, Liu TH, Wang W, Pan CX, Adur M, Zhang MJ, Pan MH, Lu C (2017a) B. mori protein BmREEPa and BmPtchd could form a complex with BmNPV envelope protein GP64. Biochem Biophys Res Commun 490(4):1254–1259. CrossRefPubMedGoogle Scholar
  12. Dong ZQ, Hu N, Dong FF, Chen TT, Jiang YM, Chen P, Lu C, Pan MH (2017b) Baculovirus LEF-11 hijack host ATPase ATAD3A to promote virus multiplication in B. mori cells. Sci Rep 7:46187. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Dong Z, Dong F, Yu X, Huang L, Jiang Y, Hu Z, Chen P, Lu C, Pan M (2018) Excision of nucleopolyhedrovirus form transgenic silkworm using the CRISPR/Cas9 system. Front Microbiol 9:209. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Ebina H, Misawa N, Kanemura Y, Koyanagi Y (2013) Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep 3:2510. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Guarino LA, Dong W, Xu B, Broussard DR, Davis RW, Jarvis DL (1992) Baculovirus phosphoprotein pp31 is associated with virogenic stroma. J Virol 66(12):7113–7120PubMedPubMedCentralGoogle Scholar
  16. He J, Mao CC, Reyes A, Sembongi H, Di Re M, Granycome C, Clippingdale AB, Fearnley IM, Harbour M, Robinson AJ, Reichelt S, Spelbrink JN, Walker JE, Holt IJ (2007) The AAA+ protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization. J Cell Biol 176(2):141–146. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Horn C, Wimmer EA (2000) A versatile vector set for animal transgenesis. Dev Genes Evol 210(12):630–637CrossRefGoogle Scholar
  18. Jiang L, Xia Q (2014) The progress and future of enhancing antiviral capacity by transgenic technology in the silkworm B. mori. Insect Biochem Mol Biol 48:1–7. CrossRefPubMedGoogle Scholar
  19. Jiang L, Wang G, Cheng T, Yang Q, Jin S, Lu G, Wu F, Xiao Y, Xu H, Xia Q (2012) Resistance to B. mori nucleopolyhedrovirus via overexpression of an endogenous antiviral gene in transgenic silkworms. Arch Virol 157(7):1323–1328. CrossRefPubMedGoogle Scholar
  20. Jiang L, Zhao P, Cheng T, Sun Q, Peng Z, Dang Y, Wu X, Wang G, Jin S, Lin P, Xia Q (2013) A transgenic animal with antiviral properties that might inhibit multiple stages of infection. Antivir Res 98(2):171–173. CrossRefPubMedGoogle Scholar
  21. Kelly BJ, King LA, Possee RD (2007) Introduction to baculovirus molecular biology. Methods Mol Biol 388:25–54. CrossRefPubMedGoogle Scholar
  22. Kennedy EM, Kornepati AV, Cullen BR (2015) Targeting hepatitis B virus cccDNA using CRISPR/Cas9. Antivir Res 123:188–192. CrossRefPubMedGoogle Scholar
  23. Kokoza V, Ahmed A, Wimmer EA, Raikhel AS (2001) Efficient transformation of the yellow fever mosquito Aedes aegypti using the piggyBac transposable element vector pBac[3xP3-EGFP afm]. Insect Biochem Mol Biol 31(12):1137–1143CrossRefGoogle Scholar
  24. Liu TH, Dong XL, Pan CX, Du GY, Wu YF, Yang JG, Chen P, Lu C, Pan MH (2016) A newly discovered member of the Atlastin family, BmAtlastin-n, has an antiviral effect against BmNPV in B. mori. Sci Rep 6:28946. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Ma H, Dang Y, Wu Y, Jia G, Anaya E, Zhang J, Abraham S, Choi JG, Shi G, Qi L, Manjunath N, Wu H (2015) A CRISPR-based screen identifies genes essential for West-Nile-virus-induced cell death. Cell Rep 12(4):673–683. CrossRefPubMedPubMedCentralGoogle Scholar
  26. McLean KJ, Jacobs-Lorena M (2016) Genetic control of malaria mosquitoes. Trends Parasitol 32(3):174–176. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Mistretta TA, Guarino LA (2005) Transcriptional activity of baculovirus very late factor 1. J Virol 79(3):1958–1960. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Naito Y, Hino K, Bono H, Ui-Tei K (2015) CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics 31(7):1120–1123. CrossRefPubMedGoogle Scholar
  29. Pelosse M, Crocker H, Gorda B, Lemaire P, Rauch J, Berger I (2017) MultiBac: from protein complex structures to synthetic viral nanosystems. BMC Biol 15(1):99. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Regev A, Rivkin H, Gurevitz M, Chejanovsky N (2006) New measures of insecticidal efficacy and safety obtained with the 39K promoter of a recombinant baculovirus. FEBS Lett 580(30):6777–6782. CrossRefPubMedGoogle Scholar
  31. Ricroch AE (2017) What will be the benefits of biotech wheat for European agriculture? Methods Mol Biol 1679:25–35. CrossRefPubMedGoogle Scholar
  32. Sarkar A, Atapattu A, Belikoff EJ, Heinrich JC, Li X, Horn C, Wimmer EA, Scott MJ (2006) Insulated piggyBac vectors for insect transgenesis. BMC Biotechnol 6:27. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Stahley MR, Stivers JT (2010) Mechanism and specificity of DNA strand exchange catalyzed by vaccinia DNA topoisomerase type I. Biochemistry 49(13):2786–2795. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Subbaiah EV, Royer C, Kanginakudru S, Satyavathi VV, Babu AS, Sivaprasad V, Chavancy G, Darocha M, Jalabert A, Mauchamp B, Basha I, Couble P, Nagaraju J (2013) Engineering silkworms for resistance to baculovirus through multigene RNA interference. Genetics 193(1):63–75. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme JC, Couble P (2000) Germline transformation of the silkworm B. mori L. using a piggyBac transposon-derived vector. Nat Biotechnol 18(1):81–84. CrossRefPubMedGoogle Scholar
  36. Taning CN, Van Eynde B, Yu N, Ma S, Smagghe G (2017) CRISPR/Cas9 in insects: applications, best practices and biosafety concerns. J Insect Physiol 98:245–257. CrossRefPubMedGoogle Scholar
  37. Thomas JL, Da Rocha M, Besse A, Mauchamp B, Chavancy G (2002) 3×P3-EGFP marker facilitates screening for transgenic silkworm B. mori L. from the embryonic stage onwards. Insect Biochem Mol Biol 32(3):247–253CrossRefGoogle Scholar
  38. Tsubota T, Sezutsu H (2017) Genome editing of silkworms. Methods Mol Biol 1630:205–218. CrossRefPubMedGoogle Scholar
  39. Yang H, Fan W, Wei H, Zhang J, Zhou Z, Li J, Lin J, Ding N, Zhong B (2008) Transgenic breeding of anti-B. mori L. nuclear polyhedrosis virus silkworm B. mori. Acta Biochim Biophys Sin Shanghai 40(10):873–876CrossRefGoogle Scholar
  40. Yang JG, Liu TH, Dong XL, Wu YF, Zhang Q, Zhou L, Chen P, Lu C, Pan MH (2017) In vivo RNA interference of BmNHR96 enhances the resistance of transgenic silkworm to BmNPV. Biochem Biophys Res Commun 493(1):332–339. CrossRefPubMedGoogle Scholar
  41. You WC, Chiou SH, Huang CY, Chiang SF, Yang CL, Sudhakar JN, Lin TY, Chiang IP, Shen CC, Cheng WY, Lin JC, Shieh SH, Chow KC (2013) Mitochondrial protein ATPase family, AAA domain containing 3A correlates with radioresistance in glioblastoma. Neuro-Oncology 15(10):1342–1352. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Zhang J, He Q, Zhang CD, Chen XY, Chen XM, Dong ZQ, Li N, Kuang XX, Cao MY, Lu C, Pan MH (2014a) Inhibition of BmNPV replication in silkworm cells using inducible and regulated artificial microRNA precursors targeting the essential viral gene lef-11. Antivir Res 104:143–152. CrossRefPubMedGoogle Scholar
  43. Zhang P, Wang J, Lu Y, Hu Y, Xue R, Cao G, Gong C (2014b) Resistance of transgenic silkworm to BmNPV could be improved by silencing ie-1 and lef-1 genes. Gene Ther 21(1):81–88. CrossRefPubMedGoogle Scholar
  44. Zhang B, Chen XF, Huang X, Yang X (2016) Research advances on animal genetics in China in 2015. Yi Chuan 38(6):467–507. CrossRefPubMedGoogle Scholar

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

Personalised recommendations