A synthetic homing endonuclease-based gene drive system in the human malaria mosquito

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 specificity. 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 vector. The translation of these achievements into control measures requires an effective technology to spread a genetic modification from laboratory mosquitoes to field populations. We have suggested previously that homing endonuclease genes (HEGs), a class of simple selfish genetic elements, could be exploited for this purpose. Here we demonstrate that a synthetic genetic element, consisting of mosquito regulatory regions and the homing endonuclease gene I-SceI, 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.

Figure 1. Analysis of HEG activity in transgenic mosquitoes

a, d Anticipated molecular events unfolding in Donor/Reporter (a) or Donor/Target (d) trans-heterozygous (TH) males. The Donor locus expresses I-SceI under the control of the male germline promoter β2-tubulin. The Reporter and the Target loci contain an I-SceI recognition site within the GFP gene. a, In Donor/Reporter TH males I-SceI activity is detected by scoring events that restore the GFP reading frame upon cleavage of the I-SceI recognition site. d, In Donor/Target TH males cleavage of the Target locus is followed by end resection and homing of the I-SceI gene from the homologous chromosome. This leads to the inactivation of the GFP reporter gene and can also lead to co-conversion of the NotI molecular marker (Pax; 3xP3 promoter, Act; Actin5C promoter). c, f Phenotypic analysis of progeny from crosses of Donor/Reporter (b, c) or Donor/Target (e, f) trans-heterozygote with wild-type (WT) mosquitoes. The column graphs show the percentage of GFP− and GFP+ individuals. The bar graphs on the right show, as a percentage of the total progeny, the GFP−RFP+CFP+, GFP−RFP−CFP+ and GFP−RFP−CFP− individuals observed. The inset (f, right panel) shows the molecular genotype of GFP−RFP+CFP+ individuals analyzed for the presence of the HEG and the NotI molecular markers.

Figure 2. HEG invasion in mosquito cage populations

a, b Temporal dynamics of GFP− mosquitoes in populations in which the HEG Donor allele was seeded at a frequency of 10% (a) or 50% (b) into a background of GFP+ mosquitoes carrying the HEG Target allele. The experimental points (Black) are overlaid onto predicted dynamics derived from a deterministic population genetic model (Dashed Red Line) and from 20 iterations of a stochastic model (Grey Lines). The dynamics of a cage population in which the HEG Donor and Control alleles were combined at a frequency of 50% is also shown (Dashed Blue Line). c, Molecular genotyping performed on individuals randomly collected from cage 1 at generations 3,6,9 and 12 using a set of PCR primers that specifically amplifies the HEG cassette (Primerset 1b, Supplementary Fig. 2). Presence of the NotI marker was determined by in vitro digestion of PCR products using NotI. The graph shows the fraction of mosquitoes carrying the HEG (Black) and the fraction carrying the HEG and the NotI marker on the same chromosome (Red) overlaid onto predictions from 20 stochastic simulations (Grey lines and Dashed Red Lines, respectively).