A synthetic homing endonuclease-based gene drive system in the human malaria mosquito (original) (raw)

Nature volume 473, pages 212–215 (2011)Cite this article

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Abstract

Genetic methods of manipulating or eradicating disease vector populations have long been discussed as an attractive alternative to existing control measures because of their potential advantages in terms of effectiveness and species specificity1,2,3. The development of genetically engineered malaria-resistant mosquitoes has shown, as a proof of principle, the possibility of targeting the mosquito’s ability to serve as a disease vector4,5,6,7. The translation of these achievements into control measures requires an effective technology to spread a genetic modification from laboratory mosquitoes to field populations8. We have suggested previously that homing endonuclease genes (HEGs), a class of simple selfish genetic elements, could be exploited for this purpose9. Here we demonstrate that a synthetic genetic element, consisting of mosquito regulatory regions10 and the homing endonuclease gene I-SceI11,12,13, can substantially increase its transmission to the progeny in transgenic mosquitoes of the human malaria vector Anopheles gambiae. We show that the I-SceI element is able to invade receptive mosquito cage populations rapidly, validating mathematical models for the transmission dynamics of HEGs. Molecular analyses confirm that expression of I-SceI in the male germline induces high rates of site-specific chromosomal cleavage and gene conversion, which results in the gain of the I-SceI gene, and underlies the observed genetic drive. These findings demonstrate a new mechanism by which genetic control measures can be implemented. Our results also show in principle how sequence-specific genetic drive elements like HEGs could be used to take the step from the genetic engineering of individuals to the genetic engineering of populations.

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Figure 1: Analysis of HEG activity in transgenic mosquitoes.

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Figure 2: HEG invasion in mosquito cage populations.

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Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Data deposits

The plasmids pHome-T and pHome-D have been deposited to GenBank under the accession numbers HQ159398 and HQ159399.

References

  1. Curtis, C. F. Possible use of translocations to fix desirable genes in insect pest populations. Nature 218, 368–369 (1968)
    Article ADS CAS Google Scholar
  2. Hamilton, W. D. Extraordinary sex ratios. A sex-ratio theory for sex linkage and inbreeding has new implications in cytogenetics and entomology. Science 156, 477–488 (1967)
    Article ADS CAS Google Scholar
  3. Alphey, L. et al. Malaria control with genetically manipulated insect vectors. Science 298, 119–121 (2002)
    Article ADS CAS Google Scholar
  4. Corby-Harris, V. et al. Activation of Akt signaling reduces the prevalence and intensity of malaria parasite infection and lifespan in Anopheles stephensi mosquitoes. PLoS Pathog. 6, e1001003 (2010)
    Article Google Scholar
  5. Ito, J., Ghosh, A., Moreira, L. A., Wimmer, E. A. & Jacobs-Lorena, M. Transgenic anopheline mosquitoes impaired in transmission of a malaria parasite. Nature 417, 452–455 (2002)
    Article ADS CAS Google Scholar
  6. Moreira, L. A. et al. Bee venom phospholipase inhibits malaria parasite development in transgenic mosquitoes. J. Biol. Chem. 277, 40839–40843 (2002)
    Article CAS Google Scholar
  7. Li, F., Patra, K. P. & Vinetz, J. M. An anti-chitinase malaria transmission-blocking single-chain antibody as an effector molecule for creating a _Plasmodium falciparum_-refractory mosquito. J. Infect. Dis. 192, 878–887 (2005)
    Article CAS Google Scholar
  8. Sinkins, S. P. & Gould, F. Gene drive systems for insect disease vectors. Nature Rev. Genet. 7, 427–435 (2006)
    Article CAS Google Scholar
  9. Burt, A. Site-specific selfish genes as tools for the control and genetic engineering of natural populations. Proc. R. Soc. Lond. B 270, 921–928 (2003)
    Article CAS Google Scholar
  10. Catteruccia, F., Benton, J. P. & Crisanti, A. An Anopheles transgenic sexing strain for vector control. Nature Biotechnol. 23, 1414–1417 (2005)
    Article CAS Google Scholar
  11. Jacquier, A. & Dujon, B. An intron-encoded protein is active in a gene conversion process that spreads an intron into a mitochondrial gene. Cell 41, 383–394 (1985)
    Article CAS Google Scholar
  12. Bellaiche, Y., Mogila, V. & Perrimon, N. I-SceI endonuclease, a new tool for studying DNA double-strand break repair mechanisms in Drosophila . Genetics 152, 1037–1044 (1999)
    CAS PubMed PubMed Central Google Scholar
  13. Windbichler, N. et al. Homing endonuclease mediated gene targeting in Anopheles gambiae cells and embryos. Nucleic Acids Res. 35, 5922–5933 (2007)
    Article CAS Google Scholar
  14. Stoddard, B. L. Homing endonuclease structure and function. Q. Rev. Biophys. 38, 49–95 (2005)
    Article CAS Google Scholar
  15. Goddard, M. R., Greig, D. & Burt, A. Outcrossed sex allows a selfish gene to invade yeast populations. Proc. R. Soc. Lond. B 268, 2537–2542 (2001)
    Article CAS Google Scholar
  16. Meredith, J. M. et al. Site-specific integration and expression of an anti-malarial gene in transgenic Anopheles gambiae significantly reduces Plasmodium infections. PLoS ONE 6, e14587 (2011)
    Article ADS CAS Google Scholar
  17. Burt, A. & Koufopanou, V. Homing endonuclease genes: the rise and fall and rise again of a selfish element. Curr. Opin. Genet. Dev. 14, 609–615 (2004)
    Article CAS Google Scholar
  18. Chen, C. H. et al. A synthetic maternal-effect selfish genetic element drives population replacement in Drosophila . Science 316, 597–600 (2007)
    Article ADS CAS Google Scholar
  19. McMeniman, C. J. et al. Stable introduction of a life-shortening Wolbachia infection into the mosquito Aedes aegypti . Science 323, 141–144 (2009)
    Article ADS CAS Google Scholar
  20. Ashworth, J. et al. Computational redesign of endonuclease DNA binding and cleavage specificity. Nature 441, 656–659 (2006)
    Article ADS CAS Google Scholar
  21. Jarjour, J. et al. High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display. Nucleic Acids Res. 37, 6871–6880 (2009)
    Article CAS Google Scholar
  22. Ashworth, J. et al. Computational reprogramming of homing endonuclease specificity at multiple adjacent base pairs. Nucleic Acids Res. 38, 5601–5608 (2010)
    Article CAS Google Scholar
  23. Thyme, S. B. et al. Exploitation of binding energy for catalysis and design. Nature 461, 1300–1304 (2009)
    Article ADS CAS Google Scholar
  24. Gao, H. et al. Heritable targeted mutagenesis in maize using a designed endonuclease. Plant J. 61, 176–187 (2010)
    Article CAS Google Scholar
  25. Grizot, S. et al. Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease. Nucleic Acids Res. 37, 5405–5419 (2009)
    Article CAS Google Scholar
  26. Munoz, I. G. et al. Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus. Nucleic Acids Res. 39, 729–743 (2010)
    Article Google Scholar
  27. Redondo, P. et al. Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases. Nature 456, 107–111 (2008)
    Article ADS CAS Google Scholar
  28. Arnould, S. et al. Engineered I-CreI derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells. J. Mol. Biol. 371, 49–65 (2007)
    Article CAS Google Scholar
  29. Rosen, L. E. et al. Homing endonuclease I-CreI derivatives with novel DNA target specificities. Nucleic Acids Res. 34, 4791–4800 (2006)
    Article ADS CAS Google Scholar
  30. Li, H., Pellenz, S., Ulge, U., Stoddard, B. L. & Monnat, R. J., Jr Generation of single-chain LAGLIDADG homing endonucleases from native homodimeric precursor proteins. Nucleic Acids Res. 37, 1650–1662 (2009)
    Article CAS Google Scholar
  31. Sheng, G., Thouvenot, E., Schmucker, D., Wilson, D. S. & Desplan, C. Direct regulation of rhodopsin 1 by Pax-6/eyeless in Drosophila: evidence for a conserved function in photoreceptors. Genes Dev. 11, 1122–1131 (1997)
    Article CAS Google Scholar
  32. Lobo, N. F., Clayton, J. R., Fraser, M. J., Kafatos, F. C. & Collins, F. H. High efficiency germ-line transformation of mosquitoes. Nature Protocols 1, 1312–1317 (2006)
    Article CAS Google Scholar
  33. Catteruccia, F., Godfray, H. C. & Crisanti, A. Impact of genetic manipulation on the fitness of Anopheles stephensi mosquitoes. Science 299, 1225–1227 (2003)
    Article ADS CAS Google Scholar
  34. Scalley-Kim, M., McConnell-Smith, A. & Stoddard, B. L. Coevolution of a homing endonuclease and its host target sequence. J. Mol. Biol. 372, 1305–1319 (2007)
    Article CAS Google Scholar
  35. Thyme, S. B. et al. Exploitation of binding energy for catalysis and design. Nature 461, 1300–1304 (2009)
    Article ADS CAS Google Scholar
  36. Doyon, J. B., Pattanayak, V., Meyer, C. B. & Liu, D. R. Directed evolution and substrate specificity profile of homing endonuclease I-SceI. J. Am. Chem. Soc. 128, 2477–2484 (2006)
    Article CAS Google Scholar
  37. Argast, G. M., Stephens, K. M., Emond, M. J. & Monnat, R. J., Jr I-_Ppo_I and I-_Cre_I homing site sequence degeneracy determined by random mutagenesis and sequential in vitro enrichment. J. Mol. Biol. 280, 345–353 (1998)
    Article CAS Google Scholar
  38. Ulge, U. Y., Baker, D. A. & Monnat, R. J. Comprehensive computational design of mCreI homing endonuclease cleavage specificity for genome engineering. Nucleic Acids Res. 10.1093/nar/gkr022 (1 February 2011)
  39. McConnell Smith, A. et al. Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease. Proc. Natl Acad. Sci. USA 106, 5099–5104 (2009)
    Article ADS CAS Google Scholar
  40. Das, R. & Baker, D. Macromolecular modeling with Rosetta. Annu. Rev. Biochem. 77, 363–382 (2008)
    Article CAS Google Scholar
  41. Studier, F. W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005)
    Article CAS Google Scholar

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Acknowledgements

We thank M. Ashburner, S. Russell, D. Huen and S. Chan for comments, assistance and for plasmids. We thank M. P. Calos for providing the pET11phiC31polyA plasmid. We thank M. J. Fraser Jr for providing the pBSII-IFP2-orf plasmid. We thank J. Meredith and P. Eggleston for providing the docking strain. We thank A. Hall, T. Nolan, K. Magnusson, D. Rogers and S. Fuchs for assistance. We thank S. Arshiya Quadri and M. Szeto for experimental support and the members of the laboratories of D. Baker, R. Monnat, A. Scharenberg and B. Stoddard for their collective support of HEG engineering. A. F. M. Hackmann provided graphics support. Funded by a grant from the Foundation for the National Institutes of Health through the Vector-Based Control of Transmission: Discovery Research (VCTR) program of the Grand Challenges in Global Health initiative and by NIH RL1 awards GM084433 to D.B. and CA133831 to R.J.M.

Author information

Author notes

  1. Austin Burt and Andrea Crisanti: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Life Sciences, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK
    Nikolai Windbichler, Miriam Menichelli, Philippos Aris Papathanos, Austin Burt & Andrea Crisanti
  2. Department of Biochemistry, University of Washington, Seattle, 98195, Washington, USA
    Summer B. Thyme & David Baker
  3. Graduate Program in Biomolecular Structure and Design, University of Washington, Seattle, 98195, Washington, USA
    Summer B. Thyme & David Baker
  4. Department of Pathology, University of Washington, Seattle, 98195, Washington, USA
    Hui Li, Umut Y. Ulge & Raymond J. Monnat
  5. Graduate Program in Molecular and Cellular Biology, University of Washington, Seattle, 98195, Washington, USA
    Umut Y. Ulge & Raymond J. Monnat
  6. Department of Genome Sciences, University of Washington, Seattle, 98195, Washington, USA
    Blake T. Hovde & Raymond J. Monnat
  7. Howard Hughes Medical Institute, University of Washington, Seattle, 98195, Washington, USA
    David Baker
  8. Department of Life Sciences, Imperial College London, Silwood Park Campus, Ascot, SL5 7PY, UK
    Austin Burt
  9. Department of Experimental Medicine, University of Perugia, Via Del Giochetto, 06122 Perugia, Italy
    Andrea Crisanti

Authors

  1. Nikolai Windbichler
  2. Miriam Menichelli
  3. Philippos Aris Papathanos
  4. Summer B. Thyme
  5. Hui Li
  6. Umut Y. Ulge
  7. Blake T. Hovde
  8. David Baker
  9. Raymond J. Monnat
  10. Austin Burt
  11. Andrea Crisanti

Contributions

N.W. designed the experiments. N.W., M.M. and P.A.P. performed the experiments. N.W. and P.A.P. generated the transgenic lines. M.M. maintained mosquito populations. N.W. analysed the data. A.B. and N.W. generated the population dynamic models. A.C. and A.B. inspired the work and wrote the paper together with N.W. HEG redesign and target site cleavage analyses were performed by S.B.T., H.L., U.Y.U. (contributed equally) and B.T.H. with guidance from D.B. and R.J.M. All authors read and approved the final manuscript.

Corresponding author

Correspondence toAndrea Crisanti.

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The authors declare no competing financial interests.

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Windbichler, N., Menichelli, M., Papathanos, P. et al. A synthetic homing endonuclease-based gene drive system in the human malaria mosquito.Nature 473, 212–215 (2011). https://doi.org/10.1038/nature09937

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  1. Nikolai Windbichler 8 July 2011, 06:06
    Similar observations have been made in Drosophila by Sang Chan, Daniel Naujoks, David Huen and Steven Russell published in Genetics:
    http://www.genetics.org/con...

Editorial Summary

Manipulating an insect vector

Genetic approaches to manipulating or eradicating disease vectors have been proposed as alternatives to malaria eradication. The success of this approach depends on efficient spread of a genetic modification in field populations. Windbichler et al. show that a synthetic genetic element consisting of mosquito regulatory elements and the homing endonuclease gene I-SceI can spread from a small number of individual Anopheles gambiae mosquitoes into large receptive populations in just a few generations. This is the first demonstration of a synthetic gene drive system in the main human malaria vector — and a similar approach should be applicable to many other pest species.