Construction and application of an HSP70 promoter-inducible genome editing system in transgenic silkworm to induce resistance to Nosema bombycis
The microsporidian Nosema bombycis is an obligate intracellular parasitic fungus that causes devastating disease in sericulture. To date, no efficient biotechnological method to inhibit the proliferation of microspores has been established. Here, we developed a powerful genetic engineering technique involving microsporidia-inducible genome editing in transgenic silkworm that confers resistance to N. bombycis. This system includes an HSP70 promoter-induced expression of the Cas9 protein line and a target BmATAD3A gene line. The double-positive HSP70-Cas9(+)×sgATAD3A(+) lines were obtained by hybridization and activation of the CRISPR/Cas9 system under the condition of microsporidia infection, although it is silenced in uninfected individuals. Genome editing analysis showed that the system could efficiently edit the BmATAD3A gene and induce large deletions. It is notable that the HSP70-induced system could effectively improve the survival rate of transgenic silkworm after microsporidia infection and inhibit the expression of key microsporidia genes. Moreover, no significant developmental differences between the transgenic silkworms infected with microsporidia and normal individuals were observed. In this study, we effectively inhibited microsporidia proliferation in transgenic individuals through disruptive techniques, thereby providing a method for microsporidia treatment and prevention, paving the way for economically advantageous insect breeding.
KeywordsMicrosporidia Silkworm CRISPR/Cas9 Transgenic Inducible genome editing
Z.D., J.L., and L.H. performed vector cloning, sequencing, cell culturing, and PCR. Z.D., J.L., and Z.H. conducted transgenic injections. N.Z., X.H, Z.H., 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. 31902214, 31872427, and 31572466), the China Agriculture Research System (CARS-18), Chongqing Special Postdoctoral Science Foundation (No. XmT2018020), Natural Science Foundation of Chongqing (cstc2019jcyj-msxm2371), and the China Postdoctoral Science Foundation (No. 2018M633309).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any experiments with human participants or animals performed by any of the authors.
- Bence M, Jankovics F, Lukacsovich T, Erdelyi M (2017) Combining the auxin-inducible degradation system with CRISPR/Cas9-based genome editing for the conditional depletion of endogenous Drosophila melanogaster proteins. FEBS J 284(7):1056–1069. https://doi.org/10.1111/febs.14042 CrossRefPubMedGoogle Scholar
- Dong Z, Huang L, Dong F, Hu Z, Qin Q, Long J, Cao M, Chen P, Lu C, Pan MH (2018) Establishment of a baculovirus-inducible CRISPR/Cas9 system for antiviral research in transgenic silkworms. Appl Microbiol Biotechnol 102(21):9255–9265. https://doi.org/10.1007/s00253-018-9295-8 CrossRefPubMedGoogle Scholar
- Han B, Weiss LM (2017) Microsporidia: obligate intracellular pathogens within the fungal kingdom. Microbiol Spectr 5(2). https://doi.org/10.1128/microbiolspec.FUNK-0018-2016
- Huang Y, Chen J, Sun B, Zheng R, Li B, Li Z, Tan Y, Wei J, Pan G, Li C, Zhou Z (2018b) Engineered resistance to Nosema bombycis by in vitro expression of a single-chain antibody in Sf9-III cells. PLoS One 13(2):e0193065. https://doi.org/10.1371/journal.pone.0193065 CrossRefPubMedPubMedCentralGoogle Scholar
- Li W, Evans JD, Huang Q, Rodriguez-Garcia C, Liu J, Hamilton M, Grozinger CM, Webster TC, Su S, Chen YP (2016) Silencing the honey bee (Apis mellifera) naked cuticle gene (nkd) improves host immune function and reduces Nosema ceranae infections. Appl Environ Microbiol 82(22):6779–6787. https://doi.org/10.1128/AEM.02105-16 CrossRefPubMedPubMedCentralGoogle Scholar
- Li Z, Pan G, Ma Z, Han B, Sun B, Ni Q, Chen J, Li T, Liu T, Long M, Li C, Zhou Z (2017) Comparative proteomic analysis of differentially expressed proteins in the Bombyx mori fat body during the microsporidia Nosema bombycis infection. J Invertebr Pathol 149:36–43. https://doi.org/10.1016/j.jip.2017.06.009 CrossRefPubMedGoogle Scholar
- Luo X, Li M, Su B (2016) Application of the genome editing tool CRISPR/Cas9 in non-human primates. Dongwuxue Yanjiu 37(4):214–219. https://doi.org/10.13918/j.issn.2095-8137.2016.4.214 CrossRefPubMedGoogle Scholar
- Ma Z, Li C, Pan G, Li Z, Han B, Xu J, Lan X, Chen J, Yang D, Chen Q, Sang Q, Ji X, Li T, Long M, Zhou Z (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 CrossRefPubMedPubMedCentralGoogle Scholar
- Martin-Hernandez R, Higes M, Sagastume S, Juarranz A, Dias-Almeida J, Budge GE, Meana A, Boonham N (2017) Microsporidia infection impacts the host cell’s cycle and reduces host cell apoptosis. PLoS One 12(2):e0170183. https://doi.org/10.1371/journal.pone.0170183 CrossRefPubMedPubMedCentralGoogle Scholar
- Paldi N, Glick E, Oliva M, Zilberberg Y, Aubin L, Pettis J, Chen Y, Evans JD (2010) Effective gene silencing in a microsporidian parasite associated with honeybee (Apis mellifera) colony declines. Appl Environ Microbiol 76(17):5960–5964. https://doi.org/10.1128/AEM.01067-10 CrossRefPubMedPubMedCentralGoogle Scholar
- Sarfati C, Bourgeois A, Menotti J, Liegeois F, Moyou-Somo R, Delaporte E, Derouin F, Ngole EM, Molina JM (2006) Prevalence of intestinal parasites including microsporidia in human immunodeficiency virus-infected adults in Cameroon: a cross-sectional study. Am J Trop Med Hyg 74(1):162–164CrossRefGoogle Scholar
- Vyas VK, Bushkin GG, Bernstein DA, Getz MA, Sewastianik M, Barrasa MI, Bartel DP, Fink GR (2018) New CRISPR mutagenesis strategies reveal variation in repair mechanisms among fungi. mSphere 3(2). https://doi.org/10.1128/mSphere.00154-18
- Wang Q, Song Y, Chai Y, Pan G, Li T, Yuan Y, Yuan R (2014) Electrochemical immunosensor for detecting the spore wall protein of Nosema bombycis based on the amplification of hemin/G-quadruplex DNAzyme concatamers functionalized Pt@Pd nanowires. Biosens Bioelectron 60:118–123. https://doi.org/10.1016/j.bios.2014.03.075 CrossRefPubMedGoogle Scholar