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.
Highlights
Some arthropods pose serious threats to human and animal health as well as to agriculture
We observed that while this vector yielded high levels of Homing endonuclease gene (HEG) activity with promoters expressing later in spermatogenesis, there was little or no activity when used with promoters targeted to the early germline stem cell and spermatogonial stages
Previous studies establishing the feasibility of HEG-based gene drive in Diptera indicated that the process was more efficient in Anopheles gambiae than in Drosophila melanogaster
Summary
Some arthropods pose serious threats to human and animal health as well as to agriculture. Techniques for engineering HEG target specificity have recently been developed for gene therapy [3,4]. Burt proposed that such methods could be applied to engineer HEGs that recognise and cleave sequences within coding sequences of genes in insect genomes, with the subsequent invasion of these HEGs into a population leading to the inactivation of target genes and the subsequent decline in fitness of the targeted population [2]. HEG gene drive could be effective if activity was restricted to the male germline to target genes required for female fertility/viability or engineered to destroy the X-chromosome by cutting at multiple Xspecific sites [5,6]
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