Optimising homing endonuclease gene drive performance in a semi-refractory species: the Drosophila melanogaster experience

PLoS One. 2013;8(1):e54130. doi: 10.1371/journal.pone.0054130. Epub 2013 Jan 18.

Abstract

Homing endonuclease gene (HEG) drive is a promising insect population control technique that employs meganucleases to impair the fitness of pest populations. Our previous studies showed that HEG drive was more difficult to achieve in Drosophila melanogaster than Anopheles gambiae and we therefore investigated ways of improving homing performance in Drosophila. We show that homing in Drosophila responds to increased expression of HEGs specifically during the spermatogonia stage and this could be achieved through improved construct design. We found that 3'-UTR choice was important to maximise expression levels, with HEG activity increasing as we employed Hsp70, SV40, vasa and βTub56D derived UTRs. We also searched for spermatogonium-specific promoters and found that the Rcd-1r promoter was able to drive specific expression at this stage. Since Rcd-1 is a regulator of differentiation in other species, it suggests that Rcd-1r may serve a similar role during spermatogonial differentiation in Drosophila. Contrary to expectations, a fragment containing the entire region between the TBPH gene and the bgcn translational start drove strong HEG expression only during late spermatogenesis rather than in the germline stem cells and spermatogonia as expected. We also observed that the fraction of targets undergoing homing was temperature-sensitive, falling nearly four-fold when the temperature was lowered to 18°C. Taken together, this study demonstrates how a few simple measures can lead to substantial improvements in the HEG-based gene drive strategy and reinforce the idea that the HEG approach may be widely applicable to a variety of insect control programs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions / genetics
  • Animals
  • Animals, Genetically Modified
  • Drosophila Proteins / genetics*
  • Drosophila Proteins / metabolism
  • Drosophila melanogaster / enzymology
  • Drosophila melanogaster / genetics*
  • Endonucleases / genetics*
  • Endonucleases / metabolism
  • Female
  • Gene Expression Regulation, Enzymologic*
  • In Situ Hybridization
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Male
  • Microscopy, Fluorescence
  • Promoter Regions, Genetic / genetics
  • Spermatogenesis / genetics
  • Temperature
  • Testis / metabolism

Substances

  • 3' Untranslated Regions
  • Drosophila Proteins
  • Luminescent Proteins
  • Endonucleases

Grant support

This work was funded via a FNIH grant to Austin Burt (Imperial College). The FNIH funding originated from the generous support of the Bill & Melinda Gates Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.