International Journal of Industrial Entomology and Biomaterials

Korean Society of Sericultural Science (한국잠사학회)
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Enhancement of antimicrobial peptide genes expression in Cactus mutated Bombyx mori cells by CRISPR/Cas9

  • Park, Jong Woo (Department of Agricultural Biology, National Institute of Agricultural Science, RDA) ;
  • Yu, Jeong Hee (Department of Agricultural Biology, National Institute of Agricultural Science, RDA) ;
  • Kim, Seong-Wan (Department of Agricultural Biology, National Institute of Agricultural Science, RDA) ;
  • Kweon, Hae Yong (Department of Agricultural Biology, National Institute of Agricultural Science, RDA) ;
  • Choi, Kwang-Ho (Department of Agricultural Biology, National Institute of Agricultural Science, RDA) ;
  • Kim, Seong-Ryul (Department of Agricultural Biology, National Institute of Agricultural Science, RDA)
  • Received : 2018.08.31
  • Accepted : 2018.09.11
  • Published : 2018.09.30
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Abstract

CRISPR/Cas9 gene editing system is an efficient method to mutation in a sequence specific manner. Here we report the direct transfection of the Cas9 nuclease and gene specific guide RNA can be used in BM-N cell line derived from Bombyx mori ovarian tissue to enfeeble function of endogenous gene in vitro. We have used gene editing system to negative regulation components of major signaling cascade, the Toll pathway, which controls B. mori resistance to microbe infections, such as fungi and gram positive bacteria. We demonstrate that the $I{\kappa}B-like$ protein Cactus may controls the activation of transcription factors such as Rel A and Rel B. The direct transfection of Cas9 nuclease and Cactus-specific guide-RNA complex may be used in BM-N cells to disrupt the function of endogenous genes in vitro. A mutation frequency of 30-40% was observed in the transfected cells, and various mutations caused the target region. Moreover, RT-PCR analysis revealed that Cactus gene was down regulated after these mutations. More importantly, mutation of BmCactus stimulated expression of lysozyme, moricin, and lebocin genes. These results suggest that the CRISPR/Cas9 systems are expected to efficiently induce site-specific mutations and it was possible to produce antimicrobial peptide through the gene editing.

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References

  1. Basua S, Aryana A, Overcasha JM, Samuela GH, Andersona MAE, Dahlemb TJ, et al. (2015) Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti. Proc Natl Acad Sci USA 112(13), 4038-4043. https://doi.org/10.1073/pnas.1502370112
  2. Doudna JA, Charpentier E (2014) The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213), 1078-1086.
  3. Harrison MH, Jenkins BV, O’Connor-Giles KM, Wildonger J (2014) A CRISPR view of development. Genes Dev 28, 1859-1872. https://doi.org/10.1101/gad.248252.114
  4. Jang IH, Chosa N, Kim SH, Nam HJ, Lemaitre B, Ochiai M, et al. (2006) A Spatzle-Processing Enzyme Required for Toll Signaling Activation in Drosophila Innate Immunity. Dev Cell 10, 45-55. https://doi.org/10.1016/j.devcel.2005.11.013
  5. Kistler KH, Vosshall LB, Matthews BJ (2015) Genome Engineering with CRISPR-Cas9 in the Mosquito Aedes aegypti. Cell Rep 11, 51-60. https://doi.org/10.1016/j.celrep.2015.03.009
  6. Ma J, Li C, Pan G, Li Z, Han B, Xu J, et al. (2013) Genome-wide transcriptional response of silkworm (Bombyx mori) to infection by the microsporidian Nosema bombycis. PLoS ONE 8(12), e84137. https://doi.org/10.1371/journal.pone.0084137
  7. 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. https://doi.org/10.1093/bioinformatics/btu743
  8. Park SW, Goo TW, Choi GH, Kang SW, Kim SW, Kim SR (2013) Functional analysis of Bombyx mori Decapentaplegic gene for bone differentiation in a mammalian cell. Int J Indust Entomol 27(1), 159-165. https://doi.org/10.7852/ijie.2013.27.1.159
  9. Qiu P, Pan PC, Govind S (1998) A role for the Drosophila Toll/Cactus pathway in larval hematopoiesis. Development 125, 1909-1920.
  10. Stokes BA, Yadav S, Shokal U, Smith LC, Eleftherianos I (2015) Bacterial and fungal pattern recognition receptors in homologous innate signaling pathways of insects and mammals. Front Microbiol 28(6), 19.
  11. Sun Y, Jiang Y, Wang Y, Li X, Yang R, Yu Z, et al. (2016) The Toll signaling pathway in the chinese oak silkworm, Antheraea pernyi: Innate immune responses to different microorganisms. PLoS ONE 11(8), e0160200. https://doi.org/10.1371/journal.pone.0160200
  12. Taniai K, Lee JH, Lee IH (2006) Bombyx mori cell line as a model of immune-system organs. Insect Mol Bio 15(3), 269-279. https://doi.org/10.1111/j.1365-2583.2006.00639.x
  13. Wua S, Zhanga X, Hea Y, Shuaia J, Chena X, Ling E (2010) Expression of antimicrobial peptide genes in Bombyx mori gut modulated by oral bacterial infection and development. Dev Comp Immunol 34 (2010), 1191-1198. https://doi.org/10.1016/j.dci.2010.06.013
  14. Zeng B, Zhan S, Wang Y, Huang Y, Xu J, Liu Q, et al. (2016) Expansion of CRISPR targeting sites in Bombyx mori. Insect Biochem Mol Biol. 72, 31-40. https://doi.org/10.1016/j.ibmb.2016.03.006
  15. Zhong X, Rao XJ, Yi HY, Lin XY, Huang XH, Yu1 XQ (2016) Coexpression of Dorsal and Rel2 negatively Regulates Antimicrobial Peptide Expression in the Tobacco Hornworm Manduca sexta. Sci Rep 6, 20654. https://doi.org/10.1038/srep20654
  16. Zhu L, Mon H, Xu J, Lee JM, Kusakabe T (2015) CRISPR/Cas9-mediated knockout of factors in non-homologous end joining pathway enhances gene targeting in silkworm cells. Sci Rep 5, 18103.