Establishment of a baculovirus-inducible CRISPR/Cas9 system for antiviral research in transgenic silkworms
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.
KeywordsInducible CRISPR/Cas9 Transgenic Antiviral therapy BmNPV ATAD3A-KO
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.
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.
- Blissard GW, Rohrmann GF (1990) Baculovirus diversity and molecular biology. Annu Rev Entomol 35:127–155. https://doi.org/10.1146/annurev.en.35.010190.001015 CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1128/JVI.02465-16 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1083/jcb.200609158 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1016/j.celrep.2015.06.049 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1534/genetics.112.144402 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1038/71978 CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1093/neuonc/not077 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1016/j.antiviral.2014.01.017 CrossRefPubMedGoogle Scholar