Ir Adv 4245 Driver

[Salvator Grimard]

Thankyou for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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).

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.

Our results may have implications beyond malaria vector control. The role of doublesex in sex determination in all insect species so far analyzed, and the high degree of doublesex sequence conservation among members of the same species (in gene regions involved in sex-specific splicing), suggests that these sequences might be an Achilles heel present in many insect pests that could be targeted with gene editing approaches.

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.

Microdissection and phenotypic characterization were carried out using Olympus SZX7 optical microscopes. Mosquitoes were collected in Falcon tubes and anesthetized on ice 5 min before dissection. For phenotypic comparison, the legs of the mosquitoes were removed to achieve the profile orientation. Pictures were taken using a HiChrome-SMII GXCAM digital mounted camera (GT Vision). Pictures of gonads were taken using the EVOS imaging system (Thermo-Fisher).

Two cage trials were initiated using 300 wild-type females, 150 wild-type males and 150 dsxFCRISPRh/+ males. The wild-type and dsxFCRISPRh lines were reared in parallel and kept under the same conditions. For the starting generation only, age-matched male and female pupae were allowed to emerge in separate cages and were mixed only when all the pupae had emerged. Both dsxFCRISPRh and wild-type male pupae were screened for the presence of the RFP marker. Mosquitoes were left to mate for 5 days before they were blood fed on anesthetized mice. Two days after, the mosquitoes were set to lay in a 300-ml egg bowl filled with water and lined with filter paper. The eggs produced from the cage were photographed and counted using JMicroVision V1.27. Prior to counting, eggs were dispersed using gentle water spraying in the egg bowl to homogenize the population, and 650 eggs were randomly selected to seed the next generation. Larvae emerging from the 650 eggs were counted and screened for the presence of the RFP marker to score the transgenic rate of the progeny. The number of pupae used to seed the next generation was also recorded.

Libraries were prepared in accordance with the Illumina 16S Metagenomic Sequencing Library Preparation protocol and the Nextera XT Index Kit. AMPure XP beads were used to purify the amplicons. Dual indices and Illumina sequencing adapters were attached in a second PCR step using the Nextera XT Indexing Kit and purified with the AMPure XP beads. The resulting libraries were validated using an Agilent 2100 Bioanalyzer (DNA High Sensitivity kit, sample dilution 1:5) to determine size distribution and a Qubit 3.0 fluorometer to determine concentration of libraries. Indexed DNA libraries were normalized to 4 nM, pooled and loaded at a concentration of 9 pM onto an Illumina Flowcell v2 with 19% of ϕX control and sequenced using the Illumina MiSeq, 2 250 bp v2 paired end run.

We ran CRISPResso35 software v1.0.8 on raw sequencing data to detect mutations at the target site using parameter -q 30, setting the minimum average read quality score (phred33) to 30. Raw sequencing data was deposited in the NCBI BioProject database (accession code PRJNA476358). Resulting allele frequency tables were processed using ad hoc Python and R scripts to group, filter and visualize indels and substitutions in the amplicon. To visualize the frequency of the most abundant indels around the cut site in both cages over the four generations, we calculated the mean frequency of indels occurring within the target region, including 20 bp upstream and downstream of the target site. The top ten alleles with the highest mean frequency were then selected to show the change of frequency of each allele throughout four generations. To plot and show the distribution of indels and substitutions in the whole amplicon, we filtered out alleles with less than three reads.

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.

3a8082e126