Search

The contemporary effective population size Ne is important in many disciplines including population genetics, conservation science and pest management. One of the most popular methods of estimating this quantity uses temporal changes in allele frequency due to genetic drift. A significant assumption of the existing methods is the independence among loci while constructing confidence intervals (CI), which restricts the types of species or genetic data applicable to the methods. Although genetic linkage does not bias point Ne estimates, applying these methods to linked loci can yield unreliable CI that are far too narrow. We extend the current methods to enable the use of many linked loci to produce precise contemporary Ne estimates, while preserving the targeted CI width and coverage. This is achieved by deriving the covariance of changes in allele frequency at linked loci in the face of recombination and sampling errors, such that the extra sampling variance due to between-locus correlation is properly handled. Extensive simulations are used to verify the new method. We apply the method to two temporally spaced genomic data sets of Anopheles mosquitoes collected from a cluster of villages in Burkina Faso between 2012 and 2014. With over 33,000 linked loci considered, the Ne estimate for Anopheles coluzzii is 9,242 (95% CI 5,702-24,282), and for Anopheles gambiae it is 4,826 (95% CI 3,602-7,353).

Only female insects transmit diseases such as malaria, dengue and Zika; therefore, control methods that bias the sex ratio of insect offspring have long been sought. Genetic elements such as sex-chromosome drives can distort sex ratios to produce unisex populations that eventually collapse, but the underlying molecular mechanisms are unknown. We report a male-biased sex-distorter gene drive (SDGD) in the human malaria vector Anopheles gambiae. We induced super-Mendelian inheritance of the X-chromosome-shredding I-PpoI nuclease by coupling this to a CRISPR-based gene drive inserted into a conserved sequence of the doublesex (dsx) gene. In modeling of invasion dynamics, SDGD was predicted to have a quicker impact on female mosquito populations than previously developed gene drives targeting female fertility. The SDGD at the dsx locus led to a male-only population from a 2.5% starting allelic frequency in 10–14 generations, with population collapse and no selection for resistance. Our results support the use of SDGD for malaria vector control., A sex-distorter gene drive causes population collapse in the malaria mosquito.

Synthetic gene drive constructs are being developed to control disease vectors, invasive species, and other pest species. In a well-mixed random mating population a sufficiently strong gene drive is expected to eliminate a target population, but it is not clear whether the same is true when spatial processes play a role. In species with an appropriate biology it is possible that drive-induced reductions in density might lead to increased inbreeding, reducing the efficacy of drive, eventually leading to suppression rather than elimination, regardless of how strong the drive is. To investigate this question we analyse a series of explicitly solvable stochastic models considering a range of scenarios for the relative timing of mating, reproduction, and dispersal and analyse the impact of two different types of gene drive, a Driving Y chromosome and a homing construct targeting an essential gene. We find in all cases a sufficiently strong Driving Y will go to fixation and the population will be eliminated, except in the one life history scenario (reproduction and mating in patches followed by dispersal) where low density leads to increased inbreeding, in which case the population persists indefinitely, tending to either a stable equilibrium or a limit cycle. These dynamics arise because Driving Y males have reduced mating success, particularly at low densities, due to having fewer sisters to mate with. Increased inbreeding at low densities can also prevent a homing construct from eliminating a population. For both types of drive, if there is strong inbreeding depression, then the population cannot be rescued by inbreeding and it is eliminated. These results highlight the potentially critical role that low-density-induced inbreeding and inbreeding depression (and, by extension, other sources of Allee effects) can have on the eventual impact of a gene drive on a target population.

Evolution of resistance is a major barrier to successful deployment of gene drive systems to suppress natural populations, which could greatly reduce the burden of many vector borne diseases. Multiplexed guide RNAs that require resistance mutations in all target cut sites is a promising anti-resistance strategy, since in principle resistance would only arise in unrealistically large populations. Using novel stochastic simulations that accurately model evolution at very large population sizes, we explore the probability of resistance due to three important mechanisms: 1) non-homologous end-joining mutations, 2) single nucleotide mutants arising de novo or, 3) single nucleotide polymorphisms pre-existing as standing variation. Our results explore the relative importance of these mechanisms and highlight a complexity of the mutation-selection-drift balance between haplotypes with complete resistance and those with an incomplete number of resistant alleles. We find this leads to a qualitatively new phenomenon where weakly deleterious naturally occurring variants greatly amplify the probability of multi-site resistance. This challenges the intuition that many target sites would guarantee prevention of resistance, where in the face of standing genetic variation, it can be probable even in not very large populations. This result has broad application to resistance arising in many multi-site evolutionary scenarios including multi-drug resistance to antibiotics, antivirals and cancer treatments, as well as the evolution of vaccine escape mutations in large populations.

CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying (‘homing’) therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel adult lethal gene drive (ALGD) in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations., Author summary Gene drives have the potential to be applied as a novel control strategy of disease-transmitting mosquitoes, by spreading genetic traits that suppress or modify the target population. Many gene drive elements work by recognising and cutting a specific target sequence in the mosquito genome and copying themselves into that target sequence allowing the gene drive to increase in frequency in the population. Like other mosquito control interventions, efficacy will greatly depend on minimising the development of resistance to the gene drive mechanism—most likely via a change in the target sequence that prevents further cutting. One strategy to reduce resistance is to target sequences that are highly conserved, which implies that changes cannot easily be tolerated. We developed a strategy that simulates high selection pressure, under which resistance is most likely to emerge, and therefore provides a stringent test of its propensity to arise. Unlike previous results with another gene drive, we recovered a resistant allele within a few generations of gene drive exposure and at high frequency. Our results show that conserved sequences can vary hugely in ability to tolerate mutations and highlights the need to functionally validate future candidate gene drive target sites for their robustness to resistance.

DNA sequences that are exactly conserved over long evolutionary time scales have been observed in a variety of taxa. Such sequences are likely under strong functional constraint and they have been useful in the field of comparative genomics for identifying genome regions with regulatory function. A potential new application for these ultra-conserved elements has emerged in the development of gene drives to control mosquito populations. Many gene drives work by recognising and inserting at a specific target sequence in the genome, often imposing a reproductive load as a consequence. They can therefore select for target sequence variants that provide resistance to the drive. Focusing on highly conserved, highly constrained sequences lowers the probability that variant, gene drive-resistant alleles can be tolerated. Here we search for conserved sequences of 18bp and over in an alignment of 21 Anopheles genomes, spanning an evolutionary timescale of 100 million years, and characterise the resulting sequences according to their location and function. Over 8000 ultra-conserved elements were found across the alignment, with a maximum length of 164 bp. Length-corrected gene ontology analysis revealed that genes containing Anopheles ultra-conserved elements were over-represented in categories with structural or nucleotide binding functions. Known insect transcription factor binding sites were found in 48% of intergenic Anopheles ultra-conserved elements. When we looked at the genome sequences of 1142 wild-caught mosquitoes we found that 15% of the Anopheles ultra-conserved elements contained no polymorphisms. Our list of Anopheles ultra-conserved elements should provide a valuable starting point for the selection and testing of new targets for gene-drive modification in the mosquitoes that transmit malaria.

The contemporary effective population size Ne is important in many disciplines including population genetics, conservation science and pest management. One of the most popular methods of estimating this quantity uses temporal changes in allele frequency due to genetic drift. A significant assumption of the existing methods is the independence among loci while constructing confidence intervals (CI), which restricts the types of species or genetic data applicable to the methods. Although genetic linkage does not bias point Ne estimates, applying these methods to linked loci can yield unreliable CI that are far too narrow. We extend the current methods to enable the use of many linked loci to produce precise contemporary Ne estimates, while preserving the targeted CI width and coverage. This is achieved by deriving the covariance of changes in allele frequency at linked loci in the face of recombination and sampling errors, such that the extra sampling variance due to between‐locus correlation is properly handled. Extensive simulations are used to verify the new method. We apply the method to two temporally spaced genomic data sets of Anopheles mosquitoes collected from a cluster of villages in Burkina Faso between 2012 and 2014. With over 33,000 linked loci considered, the Ne estimate for Anopheles coluzzii is 9,242 (95% CI 5,702–24,282), and for Anopheles gambiae it is 4,826 (95% CI 3,602–7,353).

Synthetic gene drive constructs could, in principle, provide the basis for highly efficient interventions to control disease vectors and other pest species. This efficiency derives in part from leveraging natural processes of dispersal and gene flow to spread the construct and its impacts from one population to another. However, sometimes (for example, with invasive species) only specific populations are in need of control, and impacts on non-target populations would be undesirable. Many gene drive designs use nucleases that recognise and cleave specific genomic sequences, and one way to restrict their spread would be to exploit sequence differences between target and non-target populations. In this paper we propose and model a series of low threshold double drive designs for population suppression, each consisting of two constructs, one imposing a reproductive load on the population and the other inserted into a differentiated locus and controlling the drive of the first. Simple deterministic, discrete-generation computer simulations are used to assess the alternative designs. We find that the simplest double drive designs are significantly more robust to pre-existing cleavage resistance at the differentiated locus than single drive designs, and that more complex designs incorporating sex ratio distortion can be more efficient still, even allowing for successful control when the differentiated locus is neutral and there is up to 50% pre-existing resistance in the target population. Similar designs can also be used for population replacement, with similar benefits. A population genomic analysis of CRISPR PAM sites in island and mainland populations of the malaria mosquito Anopheles gambiae indicates that the differentiation needed for our methods to work can exist in nature. Double drives should be considered when efficient but localised population genetic control is needed and there is some genetic differentiation between target and non-target populations., Author summary Some disease vectors, invasive species, and other pests cannot be satisfactorily controlled with existing interventions, and new methods are required. Synthetic gene drive systems that are able to spread though populations because they are inherited at a greater-than-Mendelian rate have the potential to form the basis for new, highly efficient pest control measures. The most efficient such strategies use natural gene flow to spread a construct throughout a species’ range, but if control is only desired in a particular location then these approaches may not be appropriate. As some of the most promising gene drive designs use nucleases to target specific DNA sequences, it ought to be possible to exploit sequence differences between target and non-target populations to restrict the spread and impact of a gene drive. In this paper we propose using two-construct “double drive” designs that exploit pre-existing sequence differences between target and non-target populations. Our approaches maintain the efficiencies associated with only small release rates being needed and can work if the differentiated locus is selectively neutral and if the differentiation is far from complete, and therefore expand the range of options to be considered in developing genetic approaches to control pest species.

Homing-based gene drives use a germline source of nuclease to copy themselves at specific target sites in a genome and bias their inheritance. Such gene drives can be designed to spread and deliberately suppress populations of malaria mosquitoes by impairing female fertility. However, strong unintended fitness costs of the drive and a propensity to generate resistant mutations can limit a gene drive’s potential to spread. Alternative germline regulatory sequences in the drive element confer improved fecundity of carrier individuals and reduced propensity for target site resistance. This is explained by reduced rates of end-joining repair of DNA breaks from parentally deposited nuclease in the embryo, which can produce heritable mutations that reduce gene drive penetrance. We tracked the generation and selection of resistant mutations over the course of a gene drive invasion of a population. Improved gene drives show faster invasion dynamics, increased suppressive effect and later onset of target site resistance. Our results show that regulation of nuclease expression is as important as the choice of target site when developing a robust homing-based gene drive for population suppression., Author summary Gene drives are selfish genetic elements that are able to drastically bias their own inheritance. They can rapidly invade populations, even starting from a very low frequency. Recent advances have allowed the engineering of gene drives deliberately designed to spread genetic traits of choice into populations of malaria-transmitting mosquito species–for example traits that impair a mosquito’s ability to reproduce or its ability to transmit parasites. The class of gene drive in question uses a very precise cutting and copying mechanism, termed ‘homing’, that allows it to increase its numbers in the cells that go on to form sperm or eggs, thereby increasing the chances that a copy of the gene drive is transmitted to offspring. However, while this type of gene drive can rapidly invade a mosquito population, mosquitoes can also eventually become resistant to the gene drive in some cases. Here we show that restricting the cutting activity of the gene drive to the germline tissue is crucial to maintaining its potency and we illustrate how failure to restrict this activity can lead to the generation of mutations that can make mosquitoes resistant to the gene drive.

Vector population control using insecticides is a key element of current strategies to prevent malaria transmission in Africa. The introduction of effective insecticides, such as the organophosphate pirimiphos-methyl, is essential to overcome the recurrent emergence of resistance driven by the highly diverse Anopheles genomes. Here, we use a population genomic approach to investigate the basis of pirimiphos-methyl resistance in the major malaria vectors Anopheles gambiae and A. coluzzii. A combination of copy number variation and a single non-synonymous substitution in the acetylcholinesterase gene, Ace1, provides the key resistance diagnostic in an A. coluzzii population from Côte d’Ivoire that we used for sequence-based association mapping, with replication in other West African populations. The Ace1 substitution and duplications occur on a unique resistance haplotype that evolved in A. gambiae and introgressed into A. coluzzii, and is now common in West Africa primarily due to selection imposed by other organophosphate or carbamate insecticides. Our findings highlight the predictive value of this complex resistance haplotype for phenotypic resistance and clarify its evolutionary history, providing tools to for molecular surveillance of the current and future effectiveness of pirimiphos-methyl based interventions., Author summary Control of mosquito populations via insecticidal tools or interventions is a mainstay of campaigns to reduce malaria transmission. However, especially in sub-Saharan Africa, continued insecticidal selection pressure on the most important species of Anopheles malaria mosquitoes has favoured the evolutionary selection of increasingly effective resistance mechanisms. We investigate the genetic basis of resistance to the organophosphate pirimiphos-methyl, the dominant insecticide now used for indoor residual spraying campaigns in Africa. Genome-wide association analysis of a population from Cote d’Ivoire showed that resistant specimens share a unique combination of mutations in one gene, the acetylcholinesterase enzyme, which constitute the prime cause of pirimiphos-methyl resistance. Further testing of these mutations in diagnostic assays involving two major malaria vectors, A. coluzzii and A. gambiae, validate their use as informative predictors of pirimiphos-methyl resistance. Using data from a large collection of whole genome sequenced specimens from a broader range of locations (Burkina-Faso, Côte d’Ivoire, Ghana, and Guinea), our evolutionary analyses demonstrate that these mutations emerged in A. gambiae and transferred into A. coluzzii by inter-specific hybridisation. Our results show how resistance mechanisms in key malaria vectors have developed and spread, and provide validated tools for molecular surveillance to inform public health campaigns.

DNA sequences that are exactly conserved over long evolutionary time scales have been observed in a variety of taxa. Such sequences are likely under strong functional constraint and they have been useful in the field of comparative genomics for identifying genome regions with regulatory function. A potential new application for these ultra-conserved elements has emerged in the development of gene drives to control mosquito populations. Many gene drives work by recognising and inserting at a specific target sequence in the genome, often imposing a reproductive load as a consequence. They can therefore select for target sequence variants that provide resistance to the drive. Focusing on highly conserved, highly constrained sequences lowers the probability that variant, gene drive-resistant alleles can be tolerated.Here we search for conserved sequences of 18bp and over in an alignment of 21Anophelesgenomes, spanning an evolutionary timescale of 100 million years, and characterise the resulting sequences according to their location and function. Over 8000 ultra-conserved elements were found across the alignment, with a maximum length of 164 bp. Length-corrected gene ontology analysis revealed that genes containingAnophelesultra-conserved elements were over-represented in categories with structural or nucleotide binding functions. Known insect transcription factor binding sites were found in 48% of intergenicAnophelesultra-conserved elements. When we looked at the genome sequences of 1142 wild-caught mosquitoes we found that 15% of theAnophelesultra-conserved elements contained no polymorphisms. Our list ofAnophelesultra-conserved elements should provide a valuable starting point for the selection and testing of new targets for gene-drive modification in the mosquitoes that transmit malaria.

Background Population suppression gene drive has been proposed as a strategy for malaria vector control. A CRISPR-Cas9-based transgene homing at the doublesex locus (dsxFCRISPRh) has recently been shown to increase rapidly in frequency in, and suppress, caged laboratory populations of the malaria mosquito vector Anopheles gambiae. Here, problem formulation, an initial step in environmental risk assessment (ERA), was performed for simulated field releases of the dsxFCRISPRh transgene in West Africa. Methods Building on consultative workshops in Africa that previously identified relevant environmental and health protection goals for ERA of gene drive in malaria vector control, 8 potentially harmful effects from these simulated releases were identified. These were stratified into 46 plausible pathways describing the causal chain of events that would be required for potential harms to occur. Risk hypotheses to interrogate critical steps in each pathway, and an analysis plan involving experiments, modelling and literature review to test each of those risk hypotheses, were developed. Results Most potential harms involved increased human (n = 13) or animal (n = 13) disease transmission, emphasizing the importance to subsequent stages of ERA of data on vectorial capacity comparing transgenics to non-transgenics. Although some of the pathways (n = 14) were based on known anatomical alterations in dsxFCRISPRh homozygotes, many could also be applicable to field releases of a range of other transgenic strains of mosquito (n = 18). In addition to population suppression of target organisms being an accepted outcome for existing vector control programmes, these investigations also revealed that the efficacy of population suppression caused by the dsxFCRISPRh transgene should itself directly affect most pathways (n = 35). Conclusions Modelling will play an essential role in subsequent stages of ERA by clarifying the dynamics of this relationship between population suppression and reduction in exposure to specific potential harms. This analysis represents a comprehensive identification of plausible pathways to potential harm using problem formulation for a specific gene drive transgene and organism, and a transparent communication tool that could inform future regulatory studies, guide subsequent stages of ERA, and stimulate further, broader engagement on the use of population suppression gene drive to control malaria vectors in West Africa.

Only female insects transmit diseases such as malaria, dengue and Zika; therefore, control methods that bias the sex ratio of insect offspring have long been sought. Genetic elements such as sex-chromosome drives can distort sex ratios to produce unisex populations that eventually collapse, but the underlying molecular mechanisms are unknown. We report a male-biased sex-distorter gene drive (SDGD) in the human malaria vector Anopheles gambiae. We induced super-Mendelian inheritance of the X-chromosome-shredding I-PpoI nuclease by coupling this to a CRISPR-based gene drive inserted into a conserved sequence of the doublesex (dsx) gene. In modeling of invasion dynamics, SDGD was predicted to have a quicker impact on female mosquito populations than previously developed gene drives targeting female fertility. The SDGD at the dsx locus led to a male-only population from a 2.5% starting allelic frequency in 10-14 generations, with population collapse and no selection for resistance. Our results support the use of SDGD for malaria vector control.

This study uses population genomic data to estimate demographic and selection parameters in two sister lineages of the wild yeast Saccharomyces paradoxus and compare their evolution. We first estimate nucleotide and recombinational diversities in each of the two lineages to infer their population size and frequency of sex and then analyze the rate of mutation accumulation since divergence from their inferred common ancestor to estimate the generation time and efficacy of selection. We find that one of the lineages has significantly higher silent nucleotide diversity and lower linkage disequilibrium, indicating a larger population with more frequent sexual generations. The same lineage also shows shorter generation time and higher efficacy of purifying selection, the latter consistent with the finding of larger population size and more frequent sex. Similar analyses are also performed on the ancestries of individual strains within lineages and we find significant differences between strains implying variation in rates of mitotic cell divisions. Our sample includes some strains originating in the Chernobyl nuclear-accident exclusion zone, which has been subjected to high levels of radiation for nearly 30 years now. We find no evidence, however, for increased rates of mutation. Finally, there is a positive correlation between rates of mutation accumulation and length of growing period, as measured by latitude of the place of origin of strains. Our study illustrates the power of genomic analyses in estimating population and life history parameters and testing predictions based on population genetic theory.

BackgroundGene drives based on CRISPR-Cas9 technology are increasingly being considered as tools for reducing the capacity of mosquito populations to transmit malaria, and one of the most promising options is driving endonuclease genes that reduce the fertility of female mosquitoes. In particular, there is much interest in constructs that target the conserved mosquito doublesex (dsx) gene such that the emergence of functional drive-resistant alleles is unlikely. Proof of principle that these constructs can lead to substantial population suppression has been obtained in population cages, and they are being evaluated for use in sub-Saharan Africa. Here, we use simulation modelling to understand the factors affecting the spread of this type of gene drive over a one million-square kilometre area of West Africa containing substantial environmental and social heterogeneity.ResultsWe found that a driving endonuclease gene targeting female fertility could lead to substantial reductions in malaria vector populations on a regional scale. The exact level of suppression is influenced by additional fitness costs of the transgene such as the somatic expression of Cas9, and its deposition in sperm or eggs leading to damage to the zygote. In the absence of these costs, or of emergent drive-resistant alleles that restore female fertility, population suppression across the study area is predicted to stabilise at ~ 95% 4 years after releases commence. Small additional fitness costs do not greatly affect levels of suppression, though if the fertility of females whose offspring transmit the construct drops by more than ~ 40%, then population suppression is much less efficient. We show the suppression potential of a drive allele with high fitness costs can be enhanced by engineering it also to express male bias in the progeny of transgenic males. Irrespective of the strength of the drive allele, the spatial model predicts somewhat less suppression than equivalent non-spatial models, in particular in highly seasonal regions where dry season stochasticity reduces drive efficiency. We explored the robustness of these results to uncertainties in mosquito ecology, in particular their method of surviving the dry season and their dispersal rates.ConclusionsThe modelling presented here indicates that considerable suppression of vector populations can be achieved within a few years of using a female sterility gene drive, though the impact is likely to be heterogeneous in space and time.

Despite impressive progress, malaria continues to impose a substantial burden of mortality and morbidity, particularly in sub-Saharan Africa, and new tools will be needed to achieve elimination. Gene drive is a natural process by which some genes are inherited at a greater-than-Mendelian rate and can spread through a population even if they cause harm to the organisms carrying them. Many different synthetic gene drive systems have been proposed to suppress the number of mosquitoes and/or reduce vector competence. As with any control measure, due attention should be paid to the possible evolution of resistance. No gene drive construct has yet been reported that is ‘field-ready’ for release, and when such constructs are developed, they should be assessed on a case-by-case basis. Gene drive approaches to vector control promise to have a number of key features that motivate their continued development, and scrutiny, by all concerned.

The sustainability of malaria control in Africa is threatened by the rise of insecticide resistance in Anopheles mosquitoes, which transmit the disease(1). To gain a deeper understanding of how mosquito populations are evolving, here we sequenced the genomes of 765 specimens of Anopheles gambiae and Anopheles coluzzii sampled from 15 locations across Africa, and identified over 50 million single nucleotide polymorphisms within the accessible genome. These data revealed complex population structure and patterns of gene flow, with evidence of ancient expansions, recent bottlenecks, and local variation in effective population size. Strong signals of recent selection were observed in insecticide-resistance genes, with several sweeps spreading over large geographical distances and between species. The design of new tools for mosquito control using gene-drive systems will need to take account of high levels of genetic diversity in natural mosquito populations.

Background The introduction of new malaria control interventions has often led to the evolution of resistance, both of the parasite to new drugs and of the mosquito vector to new insecticides, compromising the efficacy of the interventions. Recent progress in molecular and population biology raises the possibility of new genetic-based interventions, and the potential for resistance to evolve against these should be considered. Here, population modelling is used to determine the main factors affecting the likelihood that resistance will evolve against a synthetic, nuclease-based driving Y chromosome that produces a male-biased sex ratio. Methods A combination of deterministic differential equation models and stochastic analyses involving branching processes and Gillespie simulations is utilized to assess the probability that resistance evolves against a driving Y that otherwise is strong enough to eliminate the target population. The model considers resistance due to changes at the target site such that they are no longer cleaved by the nuclease, and due to trans-acting autosomal suppressor alleles. Results The probability that resistance evolves increases with the mutation rate and the intrinsic rate of increase of the population, and decreases with the strength of drive and any pleiotropic fitness costs of the resistant allele. In seasonally varying environments, the time of release can also affect the probability of resistance evolving. Trans-acting suppressor alleles are more likely to suffer stochastic loss at low frequencies than target site resistant alleles. Conclusions As with any other intervention, there is a risk that resistance will evolve to new genetic approaches to vector control, and steps should be taken to minimize this probability. Two design features that should help in this regard are to reduce the rate at which resistant mutations arise, and to target sequences such that if they do arise, they impose a significant fitness cost on the mosquito. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-1932-7) contains supplementary material, which is available to authorized users.

There is a need for new interventions against the ongoing burden of vector-borne diseases such as malaria and dengue. One suggestion has been to develop genes encoding effector molecules that block parasite development within the vector, and then use the nuclease-based homing reaction as a form of gene drive to spread those genes through target populations. If the effector gene reduces the fitness of the mosquito and does not contribute to the drive, then loss-of-function mutations in the effector will eventually replace functional copies, but protection may nonetheless persist sufficiently long to provide a public health benefit. Here, we present a quantitative model allowing one to predict the duration of protection as a function of the probabilities of different molecular processes during the homing reaction, various fitness effects, and the efficacy of the effector in blocking transmission. Factors that increase the duration of protection include reducing the frequency of pre-existing resistant alleles, the probability of nonrecombinational DNA repair, the probability of homing-associated loss of the effector, the fitness costs of the nuclease and effector, and the completeness of parasite blocking. For target species that extend over an area much larger than the typical dispersal distance, the duration of protection is expected to be highest at the release site, and decrease away from there, eventually falling to zero, as effector-less drive constructs replace effector-containing ones. We also model an alternative strategy of using the nuclease to target an essential gene, and then linking the effector to a sequence that restores the essential function and is resistant to the nuclease. Depending upon parameter values, this approach can prolong the duration of protection. Our models highlight the key design criteria needed to achieve a desired level of public health benefit.

Gene drive is a natural process of biased inheritance that, in principle, could be used to control pest and vector populations. As with any form of pest control, attention should be paid to the possibility of resistance evolving. For nuclease-based gene drive aimed at suppressing a population, resistance could arise by changes in the target sequence that maintain function, and various strategies have been proposed to reduce the likelihood that such alleles arise. Even if these strategies are successful, it is almost inevitable that alleles will arise at the target site that are resistant to the drive but do not restore function, and the impact of such sequences on the dynamics of control has been little studied. We use population genetic modelling of a strategy targeting a female fertility gene to demonstrate that such alleles may be expected to accumulate, and thereby reduce the reproductive load on the population, if nuclease expression per se causes substantial heterozygote fitness effects or if parental (especially paternal) deposition of nuclease either reduces offspring fitness or affects the genotype of their germline. All these phenomena have been observed in synthetic drive constructs. It will, therefore, be important to allow for non-functional resistance alleles in predicting the dynamics of constructs in cage populations and the impacts of any field release.

BackgroundMeasures of linkage disequilibrium (LD) play a key role in a wide range of applications from disease association to demographic history estimation. The true population LD cannot be measured directly and instead can only be inferred from genetic samples, which are unavoidably subject to measurement error. Previous studies ofr2(a measure of LD), such as the bias due to finite sample size and its variance, were based on the special case that the true population-wise LD is zero. These results generally do not hold for non-zero$$ {r}_{true}^2 $$rtrue2values, which are more common in real genetic data.ResultsThis work generalises the estimation ofr2to all levels of LD, and for both phased and unphased data. First, we provide new formulae for the effect of finite sample size on the observedr2values. Second, we find a new empirical formula for the variance of the observedr2, equals to 2E[r2](1 − E[r2])/n, wherenis the diploid sample size. Third, we propose a new routine, Constrained ML, a likelihood-based method to directly estimate haplotype frequencies andr2from diploid genotypes under Hardy-Weinberg Equilibrium. While serving the same purpose as the pre-existing Expectation-Maximisation algorithm, the new routine can have better convergence and is simpler to use. A new likelihood-ratio test is also introduced to test for the absence of a particular haplotype. Extensive simulations are run to support these findings.ConclusionMost inferences on LD will benefit from our new findings, from point and interval estimation to hypothesis testing. Genetic analyses utilisingr2information will become more accurate as a result.

Background The persistence of malaria in large parts of sub-Saharan Africa has motivated the development of novel tools to complement existing control programmes, including gene-drive technologies to modify mosquito vector populations. Here, we use a stochastic simulation model to explore the potential of using a driving-Y chromosome to suppress vector populations in a 106 km2 area of West Africa including all of Burkina Faso. Results The consequence of driving-Y introductions is predicted to vary across the landscape, causing elimination of the target species in some regions and suppression in others. We explore how this variation is determined by environmental conditions, mosquito behaviour, and the properties of the gene-drive. Seasonality is particularly important, and we find population elimination is more likely in regions with mild dry seasons whereas suppression is more likely in regions with strong seasonality. Conclusions Despite the spatial heterogeneity, we suggest that repeated introductions of modified mosquitoes over a few years into a small fraction of human settlements may be sufficient to substantially reduce the overall number of mosquitoes across the entire geographic area. Electronic supplementary material The online version of this article (10.1186/s12915-019-0645-5) contains supplementary material, which is available to authorized users.

Some genes or gene complexes are transmitted from parents to offspring at a greater-than-Mendelian rate, and can spread and persist in populations even if they cause some harm to the individuals carrying them. Such genes may be useful for controlling populations or species that are harmful. Driving-Y chromosomes may be particularly potent in this regard, as they produce a male-biased sex ratio that, if sufficiently extreme, can lead to population elimination. To better understand the potential of such genes to spread over a landscape, we have developed a series of reaction–diffusion models of a driving-Y chromosome in 1-D and radially-symmetric 2-D unbounded domains. The wild-type system at carrying capacity is found to be unstable to the introduction of driving-Y males for all models investigated. Numerical solutions exhibit travelling wave pulses and fronts, and analytical and semi-analytical solutions for the asymptotic wave speed under bounded initial conditions are derived. The driving-Y male invades the wild-type equilibrium state at the front of the wave and completely replaces the wild-type males, leaving behind, at the tail of the wave, a reduced- or zero-population state of females and driving-Y males only. In our simplest model of a population with one life stage and density-dependent mortality, wave speed depends on the strength of drive and the diffusion rate of Y-drive males, and is independent of the population dynamic consequences (suppression or elimination). Incorporating an immobile juvenile stage of fixed duration into the model reduces wave speed approximately in proportion to the relative time spent as a juvenile. If females mate just once in their life, storing sperm for subsequent reproduction, then wave speed depends on the movement of mated females as well as Y-drive males, and may be faster or slower than in the multiple-mating model, depending on the relative duration of juvenile and adult life stages. Numerical solutions are shown for parameter values that may in part be representative for Anopheles gambiae, the primary vector of malaria in sub-Saharan Africa.

Estimating recent effective population size is of great importance in characterising and predicting the evolution of natural populations. Methods based on nucleotide diversity may underestimate current day effective population sizes due to historical bottlenecks, whilst methods that reconstruct demographic history typically only detect long-term variations. However, soft selective sweeps, which leave a fingerprint of mutational history by recurrent mutations on independent haplotype backgrounds, holds promise of an estimate more representative of recent population history. Here we present a simple and robust method of estimation based only on knowledge of the number of independent recurrent origins and the current frequency of the beneficial allele in a population sample, independent of the strength of selection and age of the mutation. Using a forward time theoretical framework, we show the mean number of origins is a function ofθ= 2Nμand current allele frequency, through a simple equation, and the distribution is approximately Poisson. This estimate is robust to whether mutants pre-existed before selection arose, and is equally accurate for diploid populations with incomplete dominance. For fast (e.g., seasonal) demographic changes compared to time scale for fixation of the mutant allele, and for moderate peak-to-trough ratios, we show our constant population size estimate can be used to bound the maximum and minimum population size. Applied to the Vgsc gene ofAnopheles gambiae, we estimate an effective population size of roughly 6 × 107, and including seasonal demographic oscillations, a minimum effective population size greater than 6 × 106and a maximum less than 3 × 109.

Complete population collapse of malaria vector Anopheles gambiae in cages is achieved using a gene drive that targets doublesex. Supplementary information The online version of this article (doi:10.1038/nbt.4245) contains supplementary material, which is available to authorized users., In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. We found that CRISPR–Cas9-targeted disruption of the intron 4–exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR–Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7–11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive. Supplementary information The online version of this article (doi:10.1038/nbt.4245) contains supplementary material, which is available to authorized users.

CRISPR-Cas9 nuclease-based gene drives rely on inducing chromosomal breaks in the germline that are repaired in ways that lead to a biased inheritance of the drive. Gene drives designed to impair female fertility can suppress populations of the mosquito vector of malaria. However, strong unintended fitness costs, due to ectopic nuclease expression, and high levels of resistant mutations, limited the potential of the first generation of gene drives to spread.Here we show that changes to regulatory sequences in the drive element, designed to contain nuclease expression to the germline, confer improved fecundity over previous versions and generate drastically lower rates of target site resistance. We employed a genetic screen to show that this effect is explained by reduced rates of end-joining repair of DNA breaks at the target site caused by deposited nuclease in the embryo.Highlighting the impact of deposited Cas9, many of the mutations arising from this source of nuclease activity in the embryo are heritable, thereby having the potential to generate resistant target sites that reduce the penetrance of the gene drive.Finally, in cage invasion experiments these gene drives show improved invasion dynamics compared to first generation drives, resulting in greater than 90% suppression of the reproductive output and a delay in the emergence of target site resistance, even at a resistance-prone target sequence. We shed light on the dynamics of generation and selection of resistant alleles in a population by tracking, longitudinally, the frequency of resistant alleles in the face of an invading gene drive. Our results illustrate important considerations for future gene drive design and should expedite the development of gene drives robust to resistance.

The nature of selection acting on a population is in large measure determined by the distribution of fitness effects of new mutations. In this study, we use DNA sequences from four closely related clades of Saccharomyces paradoxus and Saccharomyces cerevisiae to identify and polarize new mutations and estimate their fitness effects. By progressively restricting the analyses to narrower categories of sites, we further seek to characterize sites with predictable mutational effects, that is, unconditionally deleterious, neutral or beneficial. Consistent with previous studies on S. paradoxus, we have failed to find evidence for mutations with beneficial effects, even in regions that were divergent in two outgroup clades, perhaps a consequence of the relatively unchallenged, predominantly asexual and highly inbred lifestyle of this species. On the other hand, there is abundant evidence of deleterious mutations, varying in severity of effect from strongly deleterious to very mild, particularly in regions conserved in the outgroup taxa, indicating a history of persistent purifying selection. Narrowing the analysis down to individual amino acids reduces further the range of effects: for example, mutations changing cysteine are predicted to be nearly always strongly deleterious, whereas those changing arginine, serine, and tyrosine are expected to be nearly neutral. The proportion of mutations with deleterious effects for a particular amino acid is correlated with long-term stasis of that amino acid among highly divergent sequences from a variety of organisms, showing that functionality of sites tends to persist through the diversification of clades and that our findings are also relevant to longer evolutionary times and other taxa.

In male heterogametic species the Y chromosome is transmitted solely from fathers to sons, and is selected for based only on its impacts on male fitness. This fact can be exploited to develop efficient pest control strategies that use Y-linked editors to disrupt the fitness of female descendants. In simple “strategic” population models we show that Y-linked editors can be substantially more efficient than other self-limiting strategies and, while not as efficient as gene drive approaches, are expected to have less impact on non-target populations with which there is some gene flow. Efficiency can be further augmented by simultaneously releasing an autosomal X-shredder construct, in either the same or different males. Y-linked editors may be attractive option to consider when efficient control of a species is desired in some locales but not others.

Drive is a process of accelerated inheritance from one generation to the next that allows some genes to spread rapidly through populations even if they do not contribute to—or indeed even if they detract from—organismal survival and reproduction. Genetic elements that can spread by drive include gametic and zygotic killers, meiotic drivers, homing endonuclease genes, B chromosomes, and transposable elements. The fact that gene drive can lead to the spread of fitness-reducing traits (including lethality and sterility) makes it an attractive process to consider exploiting to control disease vectors and other pests. There are a number of efforts to develop synthetic gene drive systems, particularly focused on the mosquito-borne diseases that continue to plague us.

Versatile molecular tools for creating driving transgenes and other invasive genetic factors present regulatory, ethical, and environmental challenges that should be addressed to ensure their safe use. In this article, we discuss driving transgenes and invasive genetic factors that can potentially spread after their introduction into a small proportion of individuals in a population. The potential of invasive genetic factors to increase their number in natural populations presents challenges that require additional safety measures not provided by previous recommendations regarding accidental release of arthropods. In addition to providing physical containment, invasive genetic factors require greater attention to strain management, including their distribution and identity confirmation. In this study, we focus on insects containing such factors with recommendations for investigators who are creating them, institutional biosafety committees charged with ensuring safety, funding agencies providing support, those managing insectaries handling these materials who are responsible for containment, and other persons who will be receiving insects—transgenic or not—from these facilities. We give specific examples of efforts to modify mosquitoes for mosquito-borne disease control, but similar considerations are relevant to other arthropods that are important to human health, the environment, and agriculture.

Driving endonuclease genes (DEGs) spread through a population by a non-Mendelian mechanism. In a heterozygote, the protein encoded by a DEG causes a double-strand break in the homologous chromosome opposite to where its gene is inserted and when the break is repaired using the homologue as a template the DEG heterozygote is converted to a homozygote. Some DEGs occur naturally while several classes of endonucleases can be engineered to spread in this way, with CRISPR-Cas9 based systems being particularly flexible. There is great interest in using driving endonuclease genes to impose a genetic load on insects that vector diseases or are economic pests to reduce their population density, or to introduce a beneficial gene such as one that might interrupt disease transmission. This paper reviews both the population genetics and population dynamics of DEGs. It summarises the theory that guides the design of DEG constructs intended to perform different functions. It also reviews the studies that have explored the likelihood of resistance to DEG phenotypes arising, and how this risk may be reduced. The review is intended for a general audience and mathematical details are kept to a minimum. Electronic supplementary material The online version of this article (doi:10.1186/s12915-017-0420-4) contains supplementary material, which is available to authorized users.