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Drivers traveling on Nutley Street North or South over I-66 should stay alert for yield signs and merging traffic when entering the circles. No traffic pattern changes will occur with this work for drivers traveling from I-66 East to Nutley Street South; from Nutley Street North to I-66 East; from I-66 West to Nutley Street North; and from Nutley Street South to I-66 West.
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However, several technical and scientific issues remain before these proof-of-principle demonstrations are advanced to effect vector population suppression. The development of a Y-drive has so far proven difficult because of the complete transcriptional shut down of the sex chromosomes during meiosis, which prevents the expression of a Y-linked sex distorter during gamete formation6,7. A gene drive designed to disrupt the A. gambiae fertility gene AGAP007280 initially increased in frequency, but the selection of nuclease-resistant, functional variants that could be detected as early as generation 2 completely blocked the spread of the drive2. Resistant variants comprised small insertions or deletions (indels) of differing length generated by nonhomologous end joining repair following nuclease activity at the target site. The development of resistance to any nuclease-based gene drive was predicted3 and is regarded as the main technical obstacle for the use of gene drives for vector control8,9,10,11,12 (Supplementary Table 1). Gene drive targets with functional or structural constraints that might prevent the development of resistant variants could offer a route to successful population control. With this in mind, we evaluated the potential for disruption of the sex determination pathway in A. gambiae mosquitoes to selectively block the formation of the female splice transcript of the gene doublesex (dsx).
Two cages were set up with a starting population of 300 wild-type females, 150 wild-type males and 150 dsxFCRISPRh/+ males, seeding each cage with a dsxFCRISPRh allele frequency of 12.5%. (a) The frequency of dsxFCRISPRh mosquitoes was scored for each generation. The drive allele reached 100% prevalence in both cage 2 (blue) and cage 1 (red) at generation 7 and 11, respectively, in agreement with a deterministic model (black line) that takes into account the parameter values retrieved from the fecundity assays. Twenty stochastic simulations were run (gray lines) assuming a maximum population size of 650 individuals. (b) Total egg output deriving from each generation of the cage was measured and normalized relative to the output from the starting generation. Suppression of the reproductive output of each cage led the population to collapse completely (black arrows) by generation 8 (cage 2) or generation 12 (cage 1). Parameter estimates included in the model are provided in Supplementary Table 5.
The development of a gene drive capable of collapsing a human malaria vector population to levels that cannot support malaria transmission is a long-sought scientific and technical goal22. The gene drive dsxFCRISPRh targeting exon 5 of dsx has several features that make it suitable for future field testing. Specifically, this drive has high inheritance bias, heterozygous individuals are fully fertile, homozygous females are sterile and unable to bite, and we found no evidence for nuclease-resistant functional variants at the drive target site. We note that these proof-of-principle experiments cannot conclude that this drive is resistance proof. This is in contrast to a recent study in Drosophila that targeted the transformer gene, upstream of doublesex. Invasion of the drive in transformer was rapidly compromised by the accumulation of large numbers of functional and nonfunctional resistant alleles23.
Our doublesex gene drive now needs to be rigorously evaluated in large confined spaces that more closely mimic native ecological conditions, in accordance with the recommendations of the US National Academy of Sciences24. Under such conditions, competition for resources or mating success may disproportionately affect individuals harboring the gene drive, resulting in invasion dynamics substantially different from those observed in insectary cage experiments. Indeed, previous work with other genetically manipulated insects would suggest that in the less ideal conditions present in field cages and natural landscapes (competition for food, presence of predators and environmental stressors), heterozygous female mosquitoes carrying the drive allele might have a further reduction in fitness as result of the combined effect of the genetic background of the laboratory strain and the presence of the drive construct itself (Supplementary Table 1)25,26,27. To mimic less ideal conditions, we modeled varying levels of additional reduction in fitness (over the experimentally observed value of reproduction rate) associated with the heterozygous gene drive and evaluated the effects on penetrance of the doublesex gene drive (Supplementary Fig. 10). An additional reduction in fitness (over the experimentally observed value) of up to 40% would still allow the drive to reach 100% frequency and cause population suppression, albeit more slowly. Further reductions in fitness would result in different equilibrium frequencies that might still cause a large reproductive load on the population.
All mosquitoes were housed at Imperial College London in an insectary that is compliant with Arthropod Containment Guidelines Level 2 (ref. 33). All GM work was performed under institutionally approved biosafety and GM protocols. In particular, GM mosquitoes containing constructs with the potential to show gene drive were housed in dedicated cubicles, separated by at least six doors from the external environment and requiring two levels of security card access. Moreover, because of its location in a city with a northern temperate climate, A. gambiae mosquitoes housed in the insectary are also ecologically contained. The physical and ecological containment of the insectary are compliant with guidelines set out in a recent commentary calling for safeguards in the study of synthetic gene drive technologies34.
RCME of the dsxFCRISPRh construct into the dsx locus was confirmed using primers binding the drive cassette (hCas9-F and RFP-R) and the neighboring genomic integration site (dsxin4-F and dsxex5-R1). Primer sequences can be found in Supplementary Table 4.
To model the results of the cage experiments, we use discrete-generation recursion equations for the genotype frequencies, treating males and females separately. Fij(t) and Mij(t) denote the frequency of females (or males) of genotype i/j in the total female (or male) population. We consider three alleles, W (wild-type), D (driver) and R (nonfunctional resistant), and therefore six genotypes.
Here df and dm are the rates of transmission of the driver allele in the two sexes and uf and um are the fractions of nondrive gametes that are nonfunctional resistant (R alleles) from meiotic end-joining. In all other genotypes, inheritance is Mendelian.
Parental effects. We consider that further cleavage of the W allele and repair can occur in the embryo if nuclease is present, due to one or both contributing gametes derived from a parent with one or two driver alleles. The presence of parental nuclease is assumed to affect somatic cells and therefore female fitness but has no effect in germline cells that would alter gene transmission. Previously, embryonic EJ effects (maternal only) were modeled as acting immediately in the zygote. Here, we consider that experimental measurements of female individuals of different genotypes and origins show a range of fitnesses, suggesting that individuals may be mosaics with intermediate phenotypes. We therefore model genotypes W/X (X = W, D, R) with parental nuclease as individuals with an intermediate reduced fitness , or depending on whether nuclease was derived from a transgenic mother, father or both. We assume that parental effects are the same whether the parent(s) had one or two drive alleles. For simplicity, a baseline reduced fitness of w10, w01, w11 is assigned to all genotypes W/X (X = W, D, R) with maternal, paternal and maternal/paternal effects, with fitness estimated as the product of mean egg production values and hatching rates relative to wild type in Supplementary Table 5 in the deterministic model. In the stochastic version of the model, egg production from female individuals with different parentage is sampled with replacement from experimental values.
Recursion equations. We first consider the gamete contributions from each genotype, including parental effects on fitness. In addition to W and R gametes that are derived from parents that have no drive allele and therefore have no deposited nuclease, gametes from W/D females and W/D, D/R and D/D males carry nuclease that is transmitted to the zygote, and these are denoted W*, D* and R*. The proportion ei of type i alleles in eggs produced by females participating in reproduction are given in terms of male and female genotype frequencies below. Frequencies of mosaic individuals with parental effects (i.e., reduced fitness) due to nuclease from mothers, fathers or both are denoted by superscripts 10, 01 or 11. 59ce067264