Article

A Synthetic Gene Drive System for Local, Reversible Modification and Suppression of Insect Populations

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Abstract

Replacement of wild insect populations with genetically modified individuals unable to transmit disease provides a self-perpetuating method of disease prevention but requires a gene drive mechanism to spread these traits to high frequency [1-3]. Drive mechanisms requiring that transgenes exceed a threshold frequency in order to spread are attractive because they bring about local but not global replacement, and transgenes can be eliminated through dilution of the population with wild-type individuals [4-6]. These features are likely to be important in many social and regulatory contexts [7-10]. Here we describe the first creation of a synthetic threshold-dependent gene drive system, designated maternal-effect lethal underdominance (UD(MEL)), in which two maternally expressed toxins, located on separate chromosomes, are each linked with a zygotic antidote able to rescue maternal-effect lethality of the other toxin. We demonstrate threshold-dependent replacement in single- and two-locus configurations in Drosophila. Models suggest that transgene spread can often be limited to local environments. They also show that in a population in which single-locus UD(MEL) has been carried out, repeated release of wild-type males can result in population suppression, a novel method of genetic population manipulation.

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... Strategies to split a gene drive transgene into multiple pieces are predicted to limit the spatial distribution of the invading gene [25,26], but do not prevent the long-term establishment of one or more transgenes in nature, even during field-testing when potential hazards are unknown. Remediation in the form of additional large-scale mosquito releases are a potential reversal mechanism for current gene drive approaches [25,27]. However, this is far from ideal, as a field trial to evaluate a gene drive transgene may be forced to conclude abruptly owing to factors outside the control of the research team (natural disaster, armed conflict, political change, etc.). ...
... while the act of gene drive is predicted to be temporally restricted, the transgene components themselves are not. Thus, to remove these transgenic sequences from a field population would require sustained inundative releases of wild-type individuals at the conclusion of any trial [27]. While not explicitly tested here, it may be possible to accelerate the removal of split drive components using the same SEM mechanisms as described here. ...
... How to conduct such trials while preventing the long-term establishment of complete or partial gene drive transgenes remains unknown [13]. Given that any such field trial might end for political, social or financial reasons in addition to scientific ones, remedial releases of additional organisms as required by other approaches [9,27,45] may not be possible. Indeed, the requirement for remediation at the conclusion of any gene drive field trial poses clear challenges for the principles of fairness and justice in regard to the affected communities [13]. ...
Article
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Gene drive systems have long been sought to modify mosquito populations and thus combat malaria and dengue. Powerful gene drive systems have been developed in laboratory experiments, but may never be used in practice unless they can be shown to be acceptable through rigorous field-based testing. Such testing is complicated by the anticipated difficulty in removing gene drive transgenes from nature. Here, we consider the inclusion of self-elimination mechanisms into the design of homing-based gene drive transgenes. This approach not only caused the excision of the gene drive transgene, but also generates a transgene-free allele resistant to further action by the gene drive. Strikingly, our models suggest that this mechanism, acting at a modest rate (10%) as part of a single-component system, would be sufficient to cause the rapid reversion of even the most robust homing-based gene drive transgenes, without the need for further remediation. Modelling also suggests that unlike gene drive transgenes themselves, self-eliminating transgene approaches are expected to tolerate substantial rates of failure. Thus, self-elimination technology may permit rigorous field-based testing of gene drives by establishing strict time limits on the existence of gene drive transgenes in nature, rendering them essentially biodegradable. This article is part of the theme issue ‘Novel control strategies for mosquito-borne diseases'.
... Fitness valleys were first described for reciprocal chromosomal translocation (RCL) systems [35,36], with translocation heterozygotes having lower fitness than either homozygote (underdominance). Various toxin-antidote and related systems have since been proposed to generate a fitness valley, including killer-rescue systems [37], one-locus one-toxin-antidote (1L1T) systems [38], one-locus two-toxin-antidote (1L2T) systems [6,39], and two-locus two-toxin-antidote (2L2T) systems [6,39]. If introduced above their frequency threshold, these systems are The fitness-valley component consists of two loci C and D, with alleles C and D engineered to reduce fitness by expressing a toxin (toxin load of s t ) and an element modifying the population (the payload, which reduces fitness by s p ). ...
... Fitness valleys were first described for reciprocal chromosomal translocation (RCL) systems [35,36], with translocation heterozygotes having lower fitness than either homozygote (underdominance). Various toxin-antidote and related systems have since been proposed to generate a fitness valley, including killer-rescue systems [37], one-locus one-toxin-antidote (1L1T) systems [38], one-locus two-toxin-antidote (1L2T) systems [6,39], and two-locus two-toxin-antidote (2L2T) systems [6,39]. If introduced above their frequency threshold, these systems are The fitness-valley component consists of two loci C and D, with alleles C and D engineered to reduce fitness by expressing a toxin (toxin load of s t ) and an element modifying the population (the payload, which reduces fitness by s p ). ...
Article
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Engineered gene-drive techniques for population modification and/or suppression have the potential for tackling complex challenges, including reducing the spread of diseases and invasive species. Gene-drive systems with low threshold frequencies for invasion, such as homing-based gene drive, require initially few transgenic individuals to spread and are therefore easy to introduce. The self-propelled behavior of such drives presents a double-edged sword, however, as the low threshold can allow transgenic elements to expand beyond a target population. By contrast, systems where a high threshold frequency must be reached before alleles can spread—above a fitness valley—are less susceptible to spillover but require introduction at a high frequency. We model a proposed drive system, called “daisy quorum drive,” that transitions over time from a low-threshold daisy-chain system (involving homing-based gene drive such as CRISPR-Cas9) to a high-threshold fitness-valley system (requiring a high frequency—a “quorum”—to spread). The daisy-chain construct temporarily lowers the high thresholds required for spread of the fitness-valley construct, facilitating use in a wide variety of species that are challenging to breed and release in large numbers. Because elements in the daisy chain only drive subsequent elements in the chain and not themselves and also carry deleterious alleles (“drive load”), the daisy chain is expected to exhaust itself, removing all CRISPR elements and leaving only the high-threshold fitness-valley construct, whose spread is more spatially restricted. Developing and analyzing both discrete patch and continuous space models, we explore how various attributes of daisy quorum drive affect the chance of modifying local population characteristics and the risk that transgenic elements expand beyond a target area. We also briefly explore daisy quorum drive when population suppression is the goal. We find that daisy quorum drive can provide a promising bridge between gene-drive and fitness-valley constructs, allowing spread from a low frequency in the short term and better containment in the long term, without requiring repeated introductions or persistence of CRISPR elements.
... The project failed due to severe fitness deficits of the released strain, which was not a transgenic strain. If there is a fitness cost associated with a transgenic gene drive element, the release ratio required for fixation increases substantially (Gould et al., 2008;Akbari et al., 2013), though in these circumstances, release over multiple generations permits a lower release ratio to achieve fixation, though with a high number of released insects overall (Magori and Gould, 2006;Akbari et al., 2013). ...
... The project failed due to severe fitness deficits of the released strain, which was not a transgenic strain. If there is a fitness cost associated with a transgenic gene drive element, the release ratio required for fixation increases substantially (Gould et al., 2008;Akbari et al., 2013), though in these circumstances, release over multiple generations permits a lower release ratio to achieve fixation, though with a high number of released insects overall (Magori and Gould, 2006;Akbari et al., 2013). ...
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The last century has witnessed the introduction, establishment and expansion of mosquito-borne diseases into diverse new geographic ranges. Malaria is transmitted by female Anopheles mosquitoes. Despite making great strides over the past few decades in reducing the burden of malaria, transmission is now on the rise again, in part owing to the emergence of mosquito resistance to insecticides, antimalarial drug resistance and, more recently, the challenges of the COVID-19 pandemic, which resulted in the reduced implementation efficiency of various control programs. The utility of genetically engineered gene drive mosquitoes as tools to decrease the burden of malaria by controlling the disease-transmitting mosquitoes is being evaluated. To date, there has been remarkable progress in the development of CRISPR/Cas9-based homing endonuclease designs in malaria mosquitoes due to successful proof-of-principle and multigenerational experiments. In this review, we examine the lessons learnt from the development of current CRISPR/Cas9-based homing endonuclease gene drives, providing a framework for the development of gene drive systems for the targeted control of wild malaria-transmitting mosquito populations that overcome challenges such as with evolving drive-resistance. We also discuss the additional substantial works required to progress the development of gene drive systems from scientific discovery to further study and subsequent field application in endemic settings.
... Several synthetic RNAi-based TA gene drives have been developed theoretically or tested on mosquitos and flies to control diseases spread by those vectors. Some of these developed drives require a significant introduction threshold to ensure their spread, which makes them suitable to small secluded area populations with reduced gene flow [42][43][44][45][46][47]. The main RNAi-based TA drive systems developed to date are summarized in Table 1. ...
... Population replacement [42] UD MEL Theoretical design on Aedes aegypti UD MEL system comprises two unlinked constructs. The maternally expressed toxin is designed on the first construct, whereas its zygotically expressed antidote is present on a separate construct Population replacement [47] Combined MEDEAunderdominance system ...
Article
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Ongoing pest and disease outbreaks pose a serious threat to human, crop, and animal lives, emphasizing the need for constant genetic discoveries that could serve as mitigation strategies. Gene drives are genetic engineering approaches discovered decades ago that may allow quick, super-Mendelian dissemination of genetic modifications in wild populations, offering hopes for medicine, agriculture, and ecology in combating diseases. Following its first discovery, several naturally occurring selfish genetic elements were identified and several gene drive mechanisms that could attain relatively high threshold population replacement have been proposed. This review provides a comprehensive overview of the recent advances in gene drive research with a particular emphasis on CRISPR-Cas gene drives, the technology that has revolutionized the process of genome engineering. Herein, we discuss the benefits and caveats of this technology and place it within the context of natural gene drives discovered to date and various synthetic drives engineered. Later, we elaborate on the strategies for designing synthetic drive systems to address resistance issues and prevent them from altering the entire wild populations. Lastly, we highlight the major applications of synthetic CRISPR-based gene drives in different living organisms, including plants, animals, and microorganisms.
... Such systems would ideally be capable of enacting local population control by: (a) effectively spreading into populations to the extent required to achieve the desired epidemiological or ecological effect and (b) being recallable from the environment in the event of unwanted consequences, public disfavor, or the end of a trial period. Two varieties of these systems have been recently engineered: (1) threshold-dependent systems that tend to spread when introduced above a certain population frequency (Akbari et al. 2013;Buchman et al. 2018) and (2) temporally self-limiting systems that display transient drive activity before being eliminated by virtue of a fitness cost (Gould et al. 2008;Li et al. 2020). ...
... Finally, lessons from the field trials discussed here have implications for the spectrum of CRISPR-based gene drives, from those that are nonlocalized to those that are self-limiting. Recent attention has focused on CRISPR-based homing gene drives, for their ability to spread widely and their potential to control vector-borne diseases on a wide scale Kyrou et al. 2018); however, there are also threshold-dependent gene drive systems that can now be engineered using CRISPR, such as chromosomal translocations (Buchman et al. 2018) and various forms of underdominance (Akbari et al. 2013), as well as temporally self-limiting gene drive systems, such as split drive (Li et al. 2020), which display transient drive activity before being eliminated by virtue of a fitness cost. The CRISPR revolution has also enabled gene drive countermeasures to be engineered, such as homing-based drive remediation systems, ERACR (element for the reversal of the autocatalytic chain reaction) and e-CHACR (erasing construct hitchhiking on the autocatalytic chain reaction) (Gantz and Bier 2016;Xu et al. 2020). ...
Chapter
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The discovery of CRISPR-based gene editing and its application to homing-based gene drive has been greeted with excitement, for its potential to control mosquito-borne diseases on a wide scale, and concern, for the invasiveness and potential irreversibility of a release. At the same time, CRISPR-based gene editing has enabled a range of self-limiting gene drive systems to be engineered with much greater ease, including (1) threshold-dependent systems, which tend to spread only when introduced above a certain threshold population frequency, and (2) temporally self-limiting systems, which display transient drive activity before being eliminated by virtue of a fitness cost. As these CRISPR-based gene drive systems are yet to be field-tested, plenty of open questions remain to be addressed, and insights can be gained from precedents set by field trials of other novel genetics-based and biological control systems, such as trials of Wolbachia-transfected mosquitoes, intended for either population replacement or suppression, and trials of genetically sterile male mosquitoes, either using the RIDL system (release of insects carrying a dominant lethal gene) or irradiation. We discuss lessons learned from these field trials and implications for a phased exploration of gene drive technology, including homing-based gene drive, chromosomal translocations, and split gene drive as a system potentially suitable for an intermediate release.
... Therefore, in order to understand potential consequences of gene-drive spillovers, explicit incorporation of migration between populations into gene-drive models is required. Some studies have examined preferential-transmission migration models with genetic architectures that have not been CRISPR-based [8, [34][35][36][37][38][39]. Among these studies, some have found that polymorphic equilibria can exist for low migration rates, but not necessarily for high migration rates. ...
... Several approaches have been recently proposed for mitigating spillovers, involving complex gene drive architectures and deployment strategies [39,[62][63][64][65][66][67][68][69], as well as for implementing countermeasures to halt an ongoing gene drive [10, [70][71][72]. A few of these genedrive architectures have been demonstrated in laboratory settings [10, 38,53,60,73]. However, performance of mitigation strategies in local confinement of a gene drive has only been examined through proof-of-concept mathematical models, and their behaviors outside the specific population and environmental conditions examined are still not well understood. ...
Article
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The prospect of utilizing CRISPR-based gene-drive technology for controlling populations has generated much excitement. However, the potential for spillovers of gene-drive alleles from the target population to non-target populations has raised concerns. Here, using mathematical models, we investigate the possibility of limiting spillovers to non-target populations by designing differential-targeting gene drives, in which the expected equilibrium gene-drive allele frequencies are high in the target population but low in the non-target population. We find that achieving differential targeting is possible with certain configurations of gene-drive parameters, but, in most cases, only under relatively low migration rates between populations. Under high migration, differential targeting is possible only in a narrow region of the parameter space. Because fixation of the gene drive in the non-target population could severely disrupt ecosystems, we outline possible ways to avoid this outcome. We apply our model to two potential applications of gene drives—field trials for malaria-vector gene drives and control of invasive species on islands. We discuss theoretical predictions of key requirements for differential targeting and their practical implications.
... Gene drive occurs when specific alleles are transmitted to viable, fertile progeny at rates greater than those of competing allelic variants. When alleles of genes conferring traits of interest are linked with a synthetic genetic element that mediates self-sustaining drive, spread to high frequency in otherwise wildtype (WT) populations can be achieved for population modification [1][2][3][4][5][6][7][8] and population suppression [9][10][11], forms of genetic population management. These drive mechanisms must be strong enough to spread to high frequency on human timescales, but must also function within diverse and evolving social and regulatory frameworks (reviewed in [12,13]). ...
... High threshold self-sustaining gene drive mechanisms include various forms of engineered single-or multi-locus toxin-antidote systems [3,19,20,[26][27][28][29][30], and chromosome rearrangements such as translocations, inversions and compound chromosomes [4,31,32,33]. These drive using the phenomenon of frequency-dependent underdominance. ...
Article
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Gene drive elements promote the spread of linked traits, providing methods for changing the composition or fate of wild populations. Drive mechanisms that are self-limiting are attractive because they allow control over the duration and extent of trait spread in time and space, and are reversible through natural selection as drive wanes. Self-sustaining Cleave and Rescue (ClvR) elements include a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene, a tightly linked recoded version of the essential gene resistant to cleavage (the Rescue), and a Cargo. ClvR spreads by creating loss-of-function (LOF) conditions in which those without ClvR die because they lack functional copies of the essential gene. We use modeling to show that when the Rescue-Cargo and one or both components required for LOF allele creation (Cas9 and gRNA) reside at different locations (split ClvR), drive of Rescue-Cargo is self-limiting due to a progressive decrease in Cas9 frequency, and thus opportunities for creation of LOF alleles, as spread occurs. Importantly, drive strength and duration can be extended in a measured manner—which is still self-limiting—by moving the two components close enough to each other that they experience some degree of linkage. With linkage, Cas9 transiently experiences drive by hitchhiking with Rescue-Cargo until linkage disequilibrium between the two disappears, a function of recombination frequency and number of generations, creating a novel point of control. We implement split ClvR in Drosophila, with key elements on different chromosomes. Cargo/Rescue/gRNAs spreads to high frequency in a Cas9-dependent manner, while the frequency of Cas9 decreases. These observations show that measured, transient drive, coupled with a loss of future drive potential, can be achieved using the simple toolkit that make up ClvR elements—Cas9 and gRNAs and a Rescue/Cargo.
... Genetic control strategies are receiving an increased amount of attention as viable vector control approaches, and include sterile insect technique (SIT) [7][8][9][10][11][12], release of an insect carrying a dominant lethal (RIDL) [7,10,13], and potentially gene drive [14][15][16][17][18][19][20][21][22][23]. Gene drive involves the spread of a genetic element beyond Mendelian rates of inheritance [14,[23][24][25]. ...
... Synthetic gene drive mechanisms can take advantage of the CRISPR/Cas9 system, which allows targeting of the genome at a precise location to catalyze a double-stranded break with repair outcomes (non-homologous end joining or homology directed repair) determining whether the result is targeted disruption or copying of a cargo sequence. Population suppression approaches to vector control with genetic modifications seek to, in some way, prevent the female mosquito from being able to bloodfeed or mate, thus producing fewer or no offspring and leading to a population decline or collapse [15,[20][21][22]. Population replacement can couple a cargo, such as refractoriness to a pathogen, with a gene drive to potentially replace the native vector population with a new population less capable of transmitting the pathogen [16][17][18][19]. ...
Article
Full-text available
Aedes aegypti is a vector of dengue, chikungunya, and Zika viruses. Current vector control strategies such as community engagement, source reduction, and insecticides have not been sufficient to prevent viral outbreaks. Thus, interest in novel strategies involving genetic engineering is growing. Female mosquitoes rely on flight to mate with males and obtain a bloodmeal from a host. We hypothesized that knockout of genes specifically expressed in female mosquitoes associated with the indirect flight muscles would result in a flightless female mosquito. Using CRISPR-Cas9 we generated loss-of-function mutations in several genes hypothesized to control flight in mosquitoes, including actin (AeAct-4) and myosin (myo-fem) genes expressed specifically in the female flight muscle. Genetic knockout of these genes resulted in 100% flightless females, with homozygous males able to fly, mate, and produce offspring, albeit at a reduced rate when compared to wild type males. Interestingly, we found that while AeAct-4 was haplosufficient, with most heterozygous individuals capable of flight, this was not the case for myo-fem, where about half of individuals carrying only one intact copy could not fly. These findings lay the groundwork for developing novel mechanisms of controlling Ae. aegypti populations, and our results suggest that this mechanism could be applicable to other vector species of mosquito.
... The high efficiency of the CRISPR-Cas-mediated gene drive ensures that genetically modified organisms can spread readily, even when their insertions have fitness costs [50]. Alternatively, CRISPR-Cas-based population replacement strategies can lead to population suppression by imposing significant fitness costs (e.g., sterility) or gender biases (e.g., driving Y-chromosomes, X-chromosome shredders) [64][65][66]. Moreover, these systems can propagate recessive mutations causing lethality, infertility, or single-sex offspring [67]. ...
Article
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Population replacement refers to the process by which a wild-type population of insect pests is replaced by a population possessing modified traits or abilities. Effective population replacement necessitates a gene drive system capable of spreading desired genes within natural populations, operating under principles akin to super-Mendelian inheritance. Consequently, releasing a small number of genetically edited insects could potentially achieve population control objectives. Currently, several gene drive approaches are under exploration, including the newly adapted CRISPR-Cas genome editing system. Multiple studies are investigating methods to engineer pests that are incapable of causing crop damage or transmitting vector-borne diseases, with several notable successful examples documented. This review summarizes the recent advancements of the CRISPR-Cas system in the realm of population replacement and provides insights into research methodologies, testing protocols, and implementation strategies for gene drive techniques. The review also discusses emerging trends and prospects for establishing genetic tools in pest management.
... More than half of the kids will inherit the desired gene thanks to Gene Drive technology, which disturbs this process to locate the targeted gene's wild-type form and replace it with the desired/altered gene. By affixing Gene Drives to a desired/altered gene and delivering it into an organism's genome, gene-drive modification may be accomplished out (Akbari et al., 2013;Books, 2019). Therefore, the wild-type gene that would have been acquired from the wild-type parent would be recognized and molecularly deleted (cleaved) when an organism with the gene drives mates with the wild-type organism. ...
Chapter
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The global population is projected to reach almost 10 billion by 2050, demanding a 50% increase in agricultural production. In light of the increasing occurrence of pest and disease outbreaks, which jeopardize food security, it is crucial to adopt innovative approaches. Conventional approaches such as chemical pesticides have been found to be inadequate, prompting a shift towards biotechnology alternatives. Biotechnological interventions, such as gene transformation and genetic engineering, provide innovative approaches for controlling insect pests.The advancements in gene editing technologies, such as CRISPR-Cas9, offer possibilities for managing insect pests. RNA interference (RNAi) methods, including double-stranded RNA (dsRNA), have demonstrated potential in specifically eliminating pest species while leaving non-target species. The gene-drive approach modifies the inheritance of specific genes, providing a potent tool for managing insect pests. The book chapter explores the diverse applications of biotechnology in insect pest management, covering gene editing, RNAi, and gene-drive technologies.It highlights successful cases of gene editing in various insect species, such as fruit flies and the migratory locust, and discusses the potential for CRISPR-Cas9 to modify plants for insect resistance. In summary, the incorporation of biotechnology in agriculture provides inventive remedies to tackle the difficulties presented by rising insect prevalence, thereby promoting sustainable and robust food supply for the growing global population.
... 55 9.10 Gene drive systems with thresholds Finally, work is ongoing towards the creation of gene drive systems that will only spread into a population if they exceed a critical population frequency. [57][58][59] These systems have three desirable features for biosafety when the goal is local population replacement -accidentally released mosquitoes are unlikely to persist in the wild because they will inevitably be present at sub-threshold levels; mosquitoes released at super-threshold frequencies at an isolated release site are expected to spread transgenes locally while remaining at sub-threshold levels at nearby locations; and transgenes can be eliminated from the release site through a sustained release of wild mosquitoes diluting transgenes to sub-threshold levels. These desirable features are acknowledged in the first guidance document of the Sub-Working Group on LMMs, 16 although a proper ecological assessment will be required on a case-by-case basis when such a release is considered. ...
... One of the most attractive features of CRISPR/Cas9 gene drives is their potential to spread from very low initial release frequencies 6 , but this efficiency is also a cause for concern. The dangers of accidental release or issues around control in the field have promoted interest in less invasive, threshold-dependent gene drive systems that are more geographically confinable ("localized" [26][27][28][29][30][31] ). Split-drive systems, where one essential component of the drive does not itself benefit from biased inheritance, allow for safe and straightforward optimization and comparison of the different components of the drive, and provide many of the desirable effects of CRISPR/Cas9 homing gene drives with increased control [5][6][7][8] . ...
Article
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Aedes aegypti is the main vector of several major pathogens including dengue, Zika and chikungunya viruses. Classical mosquito control strategies utilizing insecticides are threatened by rising resistance. This has stimulated interest in new genetic systems such as gene drivesHere, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 and a separate multiplexing sgRNA-expressing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element drives through a cage trial population such that carrier frequency of the element increases from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system.
... As a result, the drive allele will be passed on to offspring at a super-Mendelian ratio, which generates an evolutionary force that allows the gene drive allele to increase exponentially in frequency in the population (Figure 1a). In addition to homing drives, there are several other possible drive mechanisms, such as sex-linked drives (Galizi et al., 2016;Prowse et al., 2019) and underdominance systems (Akbari et al., 2013;Champer, Kim, et al., 2020;Davis et al., 2001); for further details about the different types of gene drives and their molecular mechanisms, we refer readers to reviews by Champer et al. (2016) and Hay et al. (2021). While these different types of drives rely on distinct genetic mechanisms, the evolutionary dynamics they induce can often be quite similar. ...
Article
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Gene drive technology, in which fast‐spreading engineered drive alleles are introduced into wild populations, represents a promising new tool in the fight against vector‐borne diseases, agricultural pests and invasive species. Due to the risks involved, gene drives have so far only been tested in laboratory settings while their population‐level behaviour is mainly studied using mathematical and computational models. The spread of a gene drive is a rapid evolutionary process that occurs over timescales similar to many ecological processes. This can potentially generate strong eco‐evolutionary feedback that could profoundly affect the dynamics and outcome of a gene drive release. We, therefore, argue for the importance of incorporating ecological features into gene drive models. We describe the key ecological features that could affect gene drive behaviour, such as population structure, life‐history, environmental variation and mode of selection. We review previous gene drive modelling efforts and identify areas where further research is needed. As gene drive technology approaches the level of field experimentation, it is crucial to evaluate gene drive dynamics, potential outcomes, and risks realistically by including ecological processes.
... The desirability of these dispersal effects depends on the intended extent of gene drive spread; excess long-distance dispersal hamper drives intended for limited geographic spread, while spatially restricted dispersal slows progression of low-threshold drives intended for unrestricted spread. Models exploring spatial reproductive processes (e.g., premating dispersal) found those traits have a strong influence on drive speed and spread [44,62,63]. The interplay between life history, dispersal, and evolutionary trajectory varies among populations and has been difficult to predict. ...
Article
Engineered gene drives create potential for both widespread benefits and irreversible harms to ecosystems. CRISPR-based systems of allelic conversion have rapidly accelerated gene drive research across diverse taxa, putting field trials and their necessary risk assessments on the horizon. Dynamic process-based models provide flexible quantitative platforms to predict gene drive outcomes in the context of system-specific ecological and evolutionary features. Here, we synthesize gene drive dynamic modeling studies to highlight research trends, knowledge gaps, and emergent principles, organized around their genetic, demographic, spatial, environmental, and implementation features. We identify the phenomena that most significantly influence model predictions, discuss limitations of biological complexity and uncertainty, and provide insights to promote responsible development and model-assisted risk assessment of gene drives.
... These two aspects of migration, moving the gene drive out and moving other alleles in, are pivotal in establishing gene drive localisation. Gene drive localisation is an ideal goal for preliminary gene drive releases as it confines the effect of the gene drive spatially, which allows for long-term observation of the gene drive process (Akbari et al., 2013;Noble et al., 2019;Willis & Burt, 2021). ...
Preprint
Gene drives have the potential to address pressing ecological issues. Through the super-Mendelian inheritance of a gene drive, a trait can be spread through a population even in spite of a fitness cost. This ability to spread is both its greatest quality and detractor. We may not want a gene drive to spread universally. If a gene drive were designed to cause the collapse of a pest population, it may inadvertently cause the collapse of the entire species. Migration is the mechanism through which a gene drive can spread to distant populations. Understanding its effect on the progression of a gene drive is crucial to our ability to control a gene drive. While migration can spread the gene drive to other populations, equally it can bring in other alleles to the population that may disrupt the progression of the gene drive. Through our deterministic migration gene drive model we can assess the conditions in which a gene drive is likely to spread to unintended populations, and if a gene drive is likely to be displaced by incoming alleles.
... One of the most attractive features of CRISPR/Cas9 gene drives is their potential to spread from very low initial release frequencies (6), but this efficiency is also a cause for concern. The dangers of accidental release or issues around control in the field have promoted interest in less invasive threshold-dependent gene drive systems that are more geographically confinable (26)(27)(28)(29)(30)(31). Split-drive systems, where one essential component of the drive is not biased, allow for . ...
Preprint
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Aedes aegypti , the yellow fever mosquito, is the main vector of several major pathogens including yellow fever, dengue, Zika and chikungunya viruses. Classical mosquito control strategies, mainly utilizing insecticides, have had success in controlling other mosquito vectors in recent years, but are much less useful against Ae. aegypti , and even these methods are threatened by rising insecticide resistance. This has stimulated interest in new mosquito control mechanisms, notably genetic systems such as gene drives. However, the development of CRISPR/Cas9 gene drive systems has faced challenges such as low inheritance biasing rate, the emergence of resistance alleles, and the possibility of spreading beyond the intended population. Here, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm ( bgcn ) homolog to express Cas9 in the germline to find an expression timing more conducive to homing. We also created a separate multiplexing (targeting multiple different sites within the target gene) sgRNA-expressing homing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase ( kmo ) gene to limit the consequences of resistance alleles. This creates a ‘split’ gene drive such that one part does not drive, allowing control over geographic spread and temporal persistence. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element was driven through a cage trial population such that carrier frequency of the element increased from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system. Significance statement Mosquito-borne diseases affect millions of people worldwide, with the yellow fever mosquito ( Aedes aegypti ) being the principal vector of many viral diseases. Effective measures for controlling this mosquito are sorely needed. Gene drive systems have arisen as a potential tool for mosquito control due to their ability of biasing inheritance of a trait into a target population. Here, we assess a split gene drive, based on CRISPR/Cas9 endonuclease technology driving a target element into the mosquito population. Evaluated over successive generations in a replicated cage trial, the drive successfully biased its inheritance, increasing in frequency from 50% to up to 89%. Our results are encouraging for the potential use of this type of contained gene drive system for mosquito control in endemic areas.
... The maternal effect dominant embryonic arrest (Medea) drive uses an RNAi toxin and a zygotically expressed rescue element, but it has only been engineered in Drosophila [89,90]. Though the normal form only has an introduction threshold if it has a fitness costs, variants with multiple allele types possess an introduction threshold frequency even without fitness costs [91]. Efforts to bring Medea to mosquitoes have stalled due to highly specific component expression and target gene requirements. ...
Article
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Mosquitoes bring global health problems by transmitting parasites and viruses such as malaria and dengue. Unfortunately, current insecticide-based control strategies are only moderately effective because of high cost and resistance. Thus, scalable, sustainable, and cost-effective strategies are needed for mosquito-borne disease control. Symbiont-based and genome engineeringbased approaches provide new tools that show promise for meeting these criteria, enabling modification or suppression approaches. Symbiotic bacteria like Wolbachia are maternally inherited and manipulate mosquito host reproduction to enhance their vertical transmission. Genome engineering-based gene drive methods, in which mosquitoes are genetically altered to spread drive alleles throughout wild populations, are also proving to be a potentially powerful approach in the laboratory. Here, we review the latest developments in both symbionts and gene drive-based methods. We describe some notable similarities, as well as distinctions and obstacles, relating to these promising technologies.
... Along with providing an insight into the regulatory mechanism of genes, the CREs may have significant practical applications. These can be used to generate transgenic mosquitoes, refractory to disease transmission, for vector control strategies like population suppression or replacement (32)(33)(34). ...
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Significance Hematophagous Aedes aegypti mosquitoes spread devastating viral diseases. Upon blood feeding, a steroid hormone, 20-hydroxyecdysone (20E), initiates a reproductive program during which thousands of genes are differentially expressed. While 20E-mediated gene activation is well known, repressive action by this hormone remains poorly understood. Using bioinformatics and molecular biological approaches, we have identified the mechanisms of 20E-dependent direct and indirect transcriptional repression by the ecdysone receptor (EcR). While indirect repression involves E74, EcR binds to an ecdysone response element different from those utilized in 20E-mediated gene activation to exert direct repressive action. Moreover, liganded EcR recruits a corepressor Mi2, initiating chromatin compaction. This study advances our understanding of the 20E-EcR repression mechanism and could lead to improved vector control approaches.
... The precision-guided sterile insect technique (pgSIT) is newly developed technique that disrupts genes responsible for male fertility and female viability by using the accuracy and precision of CRISPR which ensures the emergence of only sterile adult males from the genetically modified eggs. A novel CRISPR-based system has been developed across species by Akbari et al. (2013) and initially employed in Drosophila and resulted in survival of only sterile males. In fact, the biallelic knockouts were generated simultaneously wherein recessive phenotype was converted to a dominant one. ...
... Numerous efforts have concentrated on defining an autosomal allele as genetically driven if more than 50% of progeny inherit the allele from a person bearing a single copy of the allele (Curtis 1968). Well-studied examples of such selfish elements or organisms include chromosomal rearrangements (Curtis 1968), transposons (Skipper et al. 2013), Medea elements (Chen et al. 2007;Ward et al. 2011;Akbari et al. 2014), homing endonuclease genes (HEGs) (Deredec et al. 2011;Windbichler et al. 2011;Alphey and Bonsall 2014), maternal effect lethal under dominant elements (Akbari et al. 2013), bacterial endosymbiont/parasite Wolbachia (Rasgon 2007), and the recently developed CRISPR/Cas9 system. CRISPR/Cas9 has been widely adopted and is now considered the de facto standard technique for genome editing using the GD feature. ...
Chapter
Advancements in genetic engineering have resulted in the development of mosquitoes with impaired vector competence, thereby limiting acquisition and transmission of pathogens. The main dengue (DENV) vector, Aedes aegypti, is an invasive species that have spread unwittingly across the world as a result of human trade and travel. The Ae. aegypti mosquito species has spread across tropical and subtropical regions, with higher presence in urban regions where rapid breeding patterns have shown in artificial containers. Identification of and treating an adequate number of mosquito breeding sites as a control measure have been done for the past couple of years, and yet improvement is far from the expectations, even with well-funded and well-organized initiatives. In order to stop the pathogen transmission, genetically modified mosquitoes (GMM) needs to be created and released. Despite many Aedes-related achievements, GMM creation has been challenging. The spread of particular genetic elements that impair vector competence, trigger deleterious recessive mutations, or skew a population's sex ratio can be used to prevent the spread of vector disease, or eradicate invasive organisms in a species-specific and eco-friendly manner. In recent years, genome editing strategies have evolved to make use of a variety of nucleases, ranging from sequence-specific zinc finger nucleases to modular TALENs (transcription activator-like effector nucleases) and most recently, RNA-guided nucleases adapted from bacterial adaptive immune systems, dubbed CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR associated systems). By combining these methods, a new era in gene editing had emerged. Generally, both of these gene editing technologies utilize sequence-specific nucleases to generate double-stranded DNA breaks (or nicks) in the target sequence, resulting in desired DNA modifications using endogenous DNA repair mechanisms. Since cells with DNA lesions are unable to divide further, the nuclease-generated strand breaks must be rapidly repaired by the cell to maintain the viability. CRISPR/Cas has been widely accepted for use in a variety of organisms, including insect species, with only minor optimization steps needed thus far. CRISPR/Cas9 technology transformed the process of engineering nucleases capable of cleaving complex genomic sequences. A complementary guide RNA (gRNA) directs the Cas9 endonuclease's operation to the specific DNA target site, enabling the editing of virtually any DNA sequence without complex protein engineering and selection procedures. Apart from genome editing, the specificity and flexibility of the CRISPR/Cas9 method enables unprecedented rapid development of genetically modified organisms with mutation systems for disease vector insect control. The stability and expression of the gene construct generated by CRISPR/Cas9 or any other method must be addressed before GMM are released, in order to make sure that pathogen transmission and formulation are interrupted robustly and completely. Spreading foreign antipathogen genes through gene drive strategies among wild mosquito populations strengthens the case for a more streamlined approach. Major fields that must be adequately assessed include risk evaluation and management, conducting studies to ensure human and environmental protection, developing effective control strategies built on comprehensive gene-driving systems, and adequately addressing the ethical, legal, and social consequences of GMM release. Although GMM is theoretically feasible as a disease control method, field releases should be made only when strong scientific evidence of human and environmental protection and effectiveness are presented, and public acceptance is addressed appropriately. This chapter discusses the diverse technological advances in generating Ae. aegypti mosquitoes which are resistant to dengue virus (DENV) and other diseases, as well as the biosafety and risk assessment of these procedures. Additionally, the chapter outlines a convincing path forward for developing successful genetic-based DENV control strategies based on CRISPR/Cas9, which could be expanded to control other arboviruses while maintaining biosafety.
... These risks are real, and could lead to political, economic or trade complications for countries that implement gene-drive technologies before the development of a global regulatory framework to govern their use. Technically, it might be possible to reduce these risks by designing gene drives that are temporally or spatially restricted in their activity (Marshall and Hay 2012;Akbari et al. 2013;Noble et al. 2016;Dhole et al. 2018). Nevertheless, the development of gene-drive technologies for biological control needs to be undertaken within both risk assessment (Webber et al. 2015;Breed et al. 2019) and risk mitigation frameworks (Dearden et al. 2018). ...
Article
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The ongoing use of 1080 toxin for the control of mammal pests in New Zealand remains highly contentious. Several reviews over the last 25 years identified information gaps and areas of concern, both social and scientific. In this paper these areas of concern are discussed and the extensive scientific and social research that has been undertaken to clarify and address them is reviewed. Although there has been a major national investment in research aimed at finding an alternative to 1080, that has not yet been fully achieved because of low or inconsistent efficacy and/or low cost-effectiveness of alternatives, regulatory difficulties in obtaining approval for aerial delivery of any alternative, and toxic residue concerns. Finding an alternative that has similar efficacy while satisfying the demands for species-selectivity, no residues, and humaneness is a continuing challenge. The most promising prospect appears to be through understanding the genome of the target animals and opportunities for genetic manipulation, either by developing species-specific designer lethal toxicants based on genome mining, or by gene editing to develop non-lethal technologies. Both will require considerable time and funding for research, and considerable effort and engagement to address social and regulatory hurdles.
... Each construct expresses its own toxin and an antidote to the toxin of the other locus. A proof-of-principle study was performed in D. melanogaster using an RNAi-based toxin system (Akbari et al., 2013). The team tested both single-and two-loci systems, all of which were able to invade a local population, though the two-locus system required a lower release threshold to invade. ...
Thesis
Insect pest control remains an important economic, environmental, and public health challenge. CRISPR/Cas9 gene drive (GD) is a novel genetic control strategy. GDs are genetic systems that can rapidly invade a population. This manuscript presents my efforts to develop gene drives in two important pest species, Anopheles gambiae, a major vector of malaria, and Drosophila suzukii, a global crop pest. The goals of this project were to develop a suppression gene drive in D. suzukii, to reduce population size, and a modification drive in An. gambiae, to reduce malaria transmission. While I was unable to produce a functional gene drive in D. suzukii, the efforts and protocols presented here can serve as a baseline for future work in this economically important crop pest. In An. gambiae, I successfully characterized two transgenic lines, one of which significantly blocks malaria transmission to a rodent model. Finally, I present my efforts to engineer a new modification gene drive strategy, indirect gene drive.
... Other such properties of interest are the speed of action, reversibility, and potential to be spatially confined to only target populations. The sensitivity of such fundamental properties of drive systems to drive parameters has been a topic of interest of numerous recent theoretical studies [19][20][21][22][23][24][25][26][27][28]. We have collated this material in the provided database. ...
Article
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Background Synthetic gene drive technologies aim to spread transgenic constructs into wild populations even when they impose organismal fitness disadvantages. The extraordinary diversity of plausible drive mechanisms and the range of selective parameters they may encounter makes it very difficult to convey their relative predicted properties, particularly where multiple approaches are combined. The sheer number of published manuscripts in this field, experimental and theoretical, the numerous techniques resulting in an explosion in the gene drive vocabulary hinder the regulators’ point of view. We address this concern by defining a simplified parameter based language of synthetic drives. Results Employing the classical population dynamics approach, we show that different drive construct (replacement) mechanisms can be condensed and evaluated on an equal footing even where they incorporate multiple replacement drives approaches. Using a common language, it is then possible to compare various model properties, a task desired by regulators and policymakers. The generalization allows us to extend the study of the invasion dynamics of replacement drives analytically and, in a spatial setting, the resilience of the released drive constructs. The derived framework is available as a standalone tool. Conclusion Besides comparing available drive constructs, our tool is also useful for educational purpose. Users can also explore the evolutionary dynamics of future hypothetical combination drive scenarios. Thus, our results appraise the properties and robustness of drives and provide an intuitive and objective way for risk assessment, informing policies, and enhancing public engagement with proposed and future gene drive approaches.
... Scientists are developing synthetic GDs, which are often mechanistically inspired by natural GDs (e.g., Medea, homing endonucleases) but developed from scratch, allowing them to be better understood and tailored for specific pathogens/vectors. There are several GD types with different characteristics including homing-based gene drives (HGDs) [25][26][27] and sexlinked meiotic drives 28,29 , which have been demonstrated in mosquitoes, other GD types include Medea and various under dominance systems [30][31][32] . In CRISPR HGDs 20,33 , CRISPRassociated protein 9 (Cas9) is guided by a programmable guide RNA (gRNA) to generate a double stranded break (DSB) in a precise location. ...
Article
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Mosquito-borne diseases, such as dengue and malaria, pose significant global health burdens. Unfortunately, current control methods based on insecticides and environmental maintenance have fallen short of eliminating the disease burden. Scalable, deployable, genetic-based solutions are sought to reduce the transmission risk of these diseases. Pathogen-blocking Wolbachia bacteria, or genome engineering-based mosquito control strategies including gene drives have been developed to address these problems, both requiring the release of modified mosquitoes into the environment. Here, we review the latest developments, notable similarities, and critical distinctions between these promising technologies and discuss their future applications for mosquito-borne disease control. Mosquito-borne diseases pose significant global health burdens. In this review, the authors explore Wolbachia and genome engineering approaches to mosquito-borne disease population control.
... UD elements consist of two constructs, one carrying a maternally expressed toxin and an embryonic antidote that rescues the lethal effect of the toxin located on the other construct, allowing only individuals carrying both constructs to be viable, and thus promoting the spread of the element. Synthetic UD have been proposed [73] and developed in D. melanogaster using miRNAs that target either myd88 (named maternal-effect lethal underdominance (UD MEL )) [74] or the haploinsufficient ribosomal protein-coding gene RpL14 [75]. Engineered underdominance has also been pursued through reciprocal chromosomal translocations [76,77] generating heterozygous individuals that are semi-sterile and less fit than homozygotes. ...
Article
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Recent advancements in genetic and genome editing research, augmented by the discovery of new molecular tools such as CRISPR, have revolutionised the field of genetic engineering by enabling precise site-specific genome modifications with unprecedented ease. These technologies have found a vast range of applications, including the development of novel methods for the control of vector and pest insects. According to their genetic makeup and engineering, these tools can be tuned to impose different grades of impact on the targeted populations. Here, we review some of the most recent genetic control innovations under development, describing their molecular mechanisms and performance, highlighting the sustainability potentials of such interventions.
... However, some genetic elements called meiotic drivers (MDs) are able to distort meiosis and become overrepresented among meiotic products (Sandler and Novitski 1957;Burt and Trivers 2006). Due to this ability to distort meiosis, MDs gain a selective advantage that allows them to increase in frequency in a population even when they impose fitness costs on their host organism (Hamilton 1967;Akbari et al. 2013;Pinzone and Dyer 2013;Kyrou et al. 2018). The ensuing genetic conflict between MDs and their hosts is known to affect several evolutionary processes (Rice 2013). ...
Article
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Meiotic drivers are selfish genetic elements that are able to become over‐represented among the products of meiosis. This transmission advantage makes it possible for them to spread in a population even when they impose fitness costs on their host organisms. Whether a meiotic driver can invade a population, and subsequently reach fixation or coexist in a stable polymorphism, depends on the one hand on the biology of the host organism, including its life‐cycle, mating system, and population structure, and on the other hand on the specific fitness effects of the driving allele on the host. Here, we present a population genetic model for spore killing, a type of drive specific to fungi. We show how ploidy level, rate of selfing, and efficiency of spore killing affect the invasion probability of a driving allele and the conditions for its stable coexistence with a non‐driving allele. Our model can be adapted to different fungal life‐cycles, and is applied here to two well‐studied genera of filamentous ascomycetes known to harbor spore killing elements, Neurospora and Podospora. We discuss our results in the light of recent empirical findings for these two systems. This article is protected by copyright. All rights reserved
... For example, Akbari & Matzen, then at the California Institute of Technology, designed and built a synthetic 'two-locus' gene drive system they termed 'maternal-effect lethal underdominance' (UD MEL ). Here the toxins are expressed maternally during egg-production, and it is the embryos that will die as a consequence unless they have the genes for the corresponding antidotes (Akbari et al. 2013). Males carrying the toxin genes will not express them. ...
... Various gene-drive concepts have been developed for proof-of-principle in Drosophila melanogaster (Diptera: Drosophilidae), Anopheles spp. (Diptera: Culicidae), and recently in Ae. aegypti (Akbari et al. 2013;Bier, 2015, 2016;Hammond et al. 2016;Kandul et al. 2019Kandul et al. , 2020Adolfi et al. 2020;Carballar-Lejarazú et al. 2020;Li et al. 2020). In this forum paper, we describe various antiviral effectors designed for Ae. ...
Article
Arthropod-borne viruses (arboviruses) such as dengue, Zika, and chikungunya viruses cause morbidity and mortality among human populations living in the tropical regions of the world. Conventional mosquito control efforts based on insecticide treatments and/or the use of bednets and window curtains are currently insufficient to reduce arbovirus prevalence in affected regions. Novel, genetic strategies that are being developed involve the genetic manipulation of mosquitoes for population reduction and population replacement purposes. Population replacement aims at replacing arbovirus-susceptible wild-type mosquitoes in a target region with those that carry a laboratory-engineered antiviral effector to interrupt arboviral transmission in the field. The strategy has been primarily developed for Aedes aegypti (L.), the most important urban arbovirus vector. Antiviral effectors based on long dsRNAs, miRNAs, or ribozymes destroy viral RNA genomes and need to be linked to a robust gene drive to ensure their fixation in the target population. Synthetic gene-drive concepts are based on toxin/antidote, genetic incompatibility, and selfish genetic element principles. The CRISPR/Cas9 gene editing system can be configurated as a homing endonuclease gene (HEG) and HEG-based drives became the preferred choice for mosquitoes. HEGs are highly allele and nucleotide sequence-specific and therefore sensitive to single-nucleotide polymorphisms/resistant allele formation. Current research efforts test new HEG-based gene-drive designs that promise to be less sensitive to resistant allele formation. Safety aspects in conjunction with gene drives are being addressed by developing procedures that would allow a recall or overwriting of gene-drive transgenes once they have been released.
... Among the principal areas of research are efforts to engineer control over the spatial spread and temporal persistence of the driving genetic element. Possible approaches include systems where the drive mechanism functions only when present above a threshold (e.g., Akbari et al. 2013;Oberhofer et al. 2019), which tends to limit the spatial spread of the genetic element, and generational limits on the persistence of the gene drive mechanism (e.g., Noble et al. 2019), after which natural selection will remove genes with fitness costs from the population. Another potential method for limiting spread is using a drive mechanism that operates on a specific genetic sequence that is prevalent only in a restricted subpopulation (e.g., Sudweeks et al. 2019). ...
Article
Gene drive approaches—those which bias inheritance of a genetic element in a population of sexually reproducing organisms—have the potential to provide important public benefits. The spread of selected genetic elements in wild populations of organisms may help address certain challenges, such as transmission of vector-borne human and animal diseases and biodiversity loss due to invasive animals. Adapting various naturally occurring gene drive mechanisms to these aims is a long-standing research area, and recent advances in genetics have made engineering gene drive systems significantly more technically feasible. Gene drive approaches would act through changes in natural environments, thus robust methods to evaluate potential research and use are important. Despite the fact that gene drive approaches build on existing paradigms, such as genetic modification of organisms and conventional biological control, there are material challenges to their evaluation. One challenge is the inherent complexity of ecosystems, which makes precise prediction of changes to the environment difficult. For gene drive approaches that are expected to spread spatially and/or persist temporally, responding to this difficulty with the typical stepwise increases in the scale of studies may not be straightforward after studies begin in the natural environment. A related challenge is that study or use of a gene drive approach may have implications for communities beyond the location of introduction, depending on the spatial spread and persistence of the approach and the population biology of the target organism. This poses a particular governance challenge when spread across national borders is plausible. Finally, community engagement is an important element of responsible research and governance, but effective community engagement for gene drive approaches requires addressing complexity and uncertainty and supporting representative participation in decision making. These challenges are not confronted in a void. Existing frameworks, processes, and institutions provide a basis for effective evaluation of gene drive approaches for public benefit. Although engineered gene drive approaches are relatively new, the necessities of making decisions despite uncertainty and governing actions with potential implications for shared environments are well established. There are methodologies to identify potential harms and assess risks when there is limited experience to draw upon, and these methodologies have been applied in similar contexts. There are also laws, policies, treaties, agreements, and institutions in place across many jurisdictions that support national and international decision making regarding genetically modified organisms and the potential applications of gene drive approaches, such as public health and biodiversity conservation. Community engagement is an established component of many decision-making processes, and related experience and conceptual frameworks can inform engagement by researchers. The existence of frameworks, processes, and institutions provides an important foundation for evaluating gene drive approaches, but it is not sufficient by itself. They must be rigorously applied, which requires resources for risk assessment, research, and community engagement and diligent implementation by governance institutions. The continued evolution of the frameworks, processes, and institutions is important to adapt to the growing understanding of gene drive approaches. With appropriate resources and diligence, it will be possible to responsibly evaluate and make decisions on gene drive approaches for public benefit.
... Sowing was done plot wise, 45 cm marker was used for rows marking and seeds were placed manually by keeping the plant to plant spacing at 15 cm. [1] reported that iron and zinc element in stress condition have an enhancing role on osmotic adjustment process (due to the increase of soluble carbohydrates). Under drought stress conditions the role of these elements can be seen as a contributor to osmotic regulation that with intervention in the synthesis of osmotic compounds for compatibility with stress and maintain turgor pressure performed their roles. ...
... In contrast, some forms of gene drive can be less invasive than a typical neutral transgene. 11 Although counterintuitive, these forms of gene drive only spread when they exceed a critical threshold; below the threshold they are driven in the opposite direction-out of the population. Thus, the mere presence or absence of a transgene or set of transgenes that can or cannot sustain gene drive is also not sufficient for the risk assessment process. ...
Article
Advances in recombinant DNA approaches have resulted in the development of transgene architectures that severely bias their own inheritance, a process commonly referred to as "gene drive."The rapid pace of development, combined with the complexity of many gene drive approaches, threatens to overwhelm those responsible for ensuring its safe use in the laboratory, as even identifying that a specific transgene is capable of gene drive may not be intuitive. Although currently gene drive experiments have been limited to just a few species (mosquitoes, flies, mice, and yeast), the range of organisms used in gene drive research is expected to increase substantially in the coming years. Here the defining features of different gene drive approaches are discussed. Although this will start with a focus on identifying when gene drive could or could not occur, the emphasis will also be on establishing risk profiles based on anticipated level of invasiveness and persistence of transgenes in the surrounding environment. Attention is also called to the fact that transgenes can be considered invasive without being considered gene drive (and vice versa). This further supports the notion that adequate risk assessment requires information regarding the specific circumstances a given transgene or set of transgenes is capable of invading a corresponding population. Finally, challenges in the review and evaluation of work involving gene drive organisms are discussed.
... For example: accidental releases are hard to contain; a phased pathway of local test trials is challenging for a technology so invasive; issues of governance arise where the intervention spreads outwith the original release area, or across new territories. It is beyond the scope of this technical article to address these in detail but these concerns have prompted interest in alternative gene drive systems that are less invasive or require a threshold of release frequency to be superseded in order for the drive to invade the population [20,37,38]. As an intermediate, 'split' gene drive systems, where the source of Cas9 is uncoupled from the element that is copied through homing, have also been proposed [39][40][41] and may allow useful optimization of the relevant components of a fully invasive gene drive prior to deployment. ...
Article
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Gene drives are selfish genetic elements that can be re-designed to invade a population and they hold tremendous potential for the control of mosquitoes that transmit disease. Much progress has been made recently in demonstrating proof of principle for gene drives able to suppress populations of malarial mosquitoes, or to make them refractory to the Plasmodium parasites they transmit. This has been achieved using CRISPR-based gene drives. In this article, I will discuss the relative merits of this type of gene drive, as well as barriers to its technical development and to its deployment in the field as malaria control. This article is part of the theme issue ‘Novel control strategies for mosquito-borne diseases'.
Article
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Background Threshold-dependent gene drives (TDGDs) could be used to spread desirable traits through a population, and are likely to be less invasive and easier to control than threshold-independent gene drives. Engineered Genetic Incompatibility (EGI) is an extreme underdominance system previously demonstrated in Drosophila melanogaster that can function as a TDGD when EGI agents of both sexes are released into a wild-type population. Results Here we use a single generation fitness assay to compare the fecundity, mating preferences, and temperature-dependent relative fitness to wild-type of two distinct genotypes of EGI agents. We find significant differences in the behavior/performance of these EGI agents that would not be predicted a priori based on their genetic design. We report a surprising temperature-dependent change in the predicted threshold for population replacement in an EGI agent that drives ectopic expression of the developmental morphogen pyramus. Conclusions The single-generation fitness assay presented here could reduce the amount of time required to estimate the threshold for TDGD strategies for which hybrid genotypes are inviable. Additionally, this work underscores the importance of empirical characterization of multiple engineered lines, as behavioral differences can arise in unique genotypes for unknown reasons.
Article
Genetic biocontrol aims to suppress or modify populations of species to protect public health, agriculture, and biodiversity. Advancements in genome engineering technologies have fueled a surge in research in this field, with one gene editing technology, CRISPR, leading the charge. This review focuses on the current state of CRISPR technologies for genetic biocontrol of pests and highlights the progress and ongoing challenges of using these approaches. Expected final online publication date for the Annual Review of Genetics, Volume 57 is November 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Article
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Genetic control strategies such as the sterile insect technique have successfully fought insect pests worldwide. The CRISPR (clustered regularly interspaced short palindromic repeats) technology, together with high-quality genomic resources obtained in more and more species, greatly facilitates the development of novel genetic control insect strains that can be used in area-wide and species-specific pest control programs. Here, we review the research progress towards state-of-art CRISPR-based genetic control strategies, including gene drive, sex ratio distortion, CRISPR-engineered genetic sexing strains, and precision-guided sterile insect technique. These strategies' working mechanisms, potential resistance development mechanisms, and regulations are illustrated and discussed. In addition, recent developments such as stacked and conditional systems are introduced. We envision that the advances in genetic technology will continue to be one of the driving forces for developing the next generation of pest control strategies.
Chapter
Development of genetic control strategies has stimulated interest due to environmental benefits of reduced pesticide use. The sterile insect technique can be seen as a precursor to newer technologies to achieve goals of pest suppression and pathogen interference. Genetic engineering techniques offer promise, but resistance mutations can hamper their success. These tactics generally involve release of male insects and require mating for efficacy in pest control. Therefore an understanding of mating behavior and population dynamics is critical.
Article
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Genetic-based technologies are emerging as promising tools to support vector population control. Vectors of human malaria and dengue have been the main focus of these development efforts, but in recent years these technologies have become more flexible and adaptable and may therefore have more wide-ranging applications. Culex quinquefasciatus , for example, is the primary vector of avian malaria in Hawaii and other tropical islands. Avian malaria has led to the extinction of numerous native bird species and many native bird species continue to be threatened as climate change is expanding the range of this mosquito. Genetic-based technologies would be ideal to support avian malaria control as they would offer alternatives to interventions that are difficult to implement in natural areas, such as larval source reduction, and limit the need for chemical insecticides, which can harm beneficial species in these natural areas. This mosquito is also an important vector of human diseases, such as West Nile and Saint Louis encephalitis viruses, so genetic-based control efforts for this species could also have a direct impact on human health. This commentary will discuss the current state of development and future needs for genetic-based technologies in lesser studied, but important disease vectors, such as C. quinquefasciatus , and make comparisons to technologies available in more studied vectors. While most current genetic control focuses on human disease, we will address the impact that these technologies could have on both disease and conservation focused vector control efforts and what is needed to prepare these technologies for evaluation in the field. The versatility of genetic-based technologies may result in the development of many important tools to control a variety of vectors that impact human, animal, and ecosystem health.
Article
Population suppression is an effective way for controlling insect pests and disease vectors, which cause significant damage to crop and spread contagious diseases to plants, animals and humans. Gene drive systems provide innovative opportunities for the insect pests population suppression by driving genes that impart fitness costs on populations of pests or disease vectors. Different gene-drive systems have been developed in insects and applied for their population suppression. Here, different categories of gene drives such as meiotic drive (MD), under-dominance (UD), homing endonuclease-based gene drive (HEGD) and especially the CRISPR/Cas9-based gene drive (CCGD) were reviewed, including the history, types, process and mechanisms. Furthermore, the advantages and limitations of applying different gene-drive systems to suppress the insect population were also summarized. This review provides a foundation for developing a specific gene-drive system for insect population suppression.
Chapter
The emergence of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (CRISPR/Cas) and its reengineering into a potent genome editing system has revolutionized life sciences. It has brought much excitement and hope in medical and agricultural research for unprecedented control over the redesigning of genomes. Based on CRISPR, many genome engineering tools have been developed and extensively used for the identification of new genes and therapeutic targets, functional genomics, gene therapies, and the development of transgenic animals and plants. The successful applications of CRISPR/Cas depend on the safe and efficient transportation of CRISPR/Cas reagents into the cell nucleus. In this chapter we discuss the merits and demerits of different cargo reagents used for genome editing through CRISPR/Cas. In addition, we detail several delivery methods reported for CRISPR/Cas, including physical, viral, and non-viral delivery methods. We also highlight different emerging delivery methods not currently reported for delivery of CRISPR/Cas reagents. Finally, we discuss available delivering methods of CRISPR/Cas components for plant genome editing.
Article
Gene drives are an emerging technology with tremendous potential to impact public health, agriculture, and conservation. While gene drives can be described simply as selfish genetic elements (natural or engineered) that are inherited at non-Mendelian rates, upon closer inspection, engineered gene drive technology is a complex class of biotechnology that uses a diverse number of genetic features to bias rates of inheritance. As a complex technology, gene drives can be difficult to comprehend, not only for the public and stakeholders, but also to risk assessors, risk managers, and decisionmakers not familiar with gene drive literature. To address this difficulty, we describe a gene drive classification system based on 5 functional characteristics. These characteristics include a gene drive's objective, mechanism, release threshold, range, and persistence. The aggregate of the gene drive's characteristics can be described as the gene drive's architecture. Establishing a classification system to define different gene drive technologies should make them more comprehensible to the public and provide a framework to guide regulatory evaluation and decisionmaking.
Article
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The avifauna endemic to islands is particularly susceptible to population declines and extinctions resulting from the introduction of non-native pathogens. Three pathogens of concern are the avian malaria parasites, the avian poxviruses, and West Nile virus—each of which can be transmitted by Culex quinquefasciatus, a highly adaptive and invasive mosquito. Culex quinquefasciatus has dramatically expanded its range in recent centuries and is now established throughout much of the tropics and sub-tropics, including on many islands that are remote from mainland landmasses and where this geographic separation historically protected island species from mosquito-borne diseases. The potential for ecological disruption by Cx. quinquefasciatus has been particularly striking in the Hawaiian Islands, where the introduction and transmission of avian malaria and avian poxvirus led to the extinction of several endemic bird species, with many more at risk. With Cx. quinquefasciatus now present in many insular communities and global trade and tourism increasing links between these areas, both to each other and to mainlands, there is growing concern that patterns of avian decline in Hawai‘i may be played out in other insular ecosystems. The implementation of traditional methods for Cx. quinquefasciatus control, including larval source management, is often impractical at large scale and when breeding sites are numerous and difficult to locate—typical issues associated with invasive species removal. One alternative approach would be the utilisation of genetic control methods, several of which have been successfully developed in other mosquitos such as Aedes aegypti and the malaria vector Anopheles gambiae. However, the development of similar tools for Cx. quinquefasciatus has been comparatively limited. Here we review the threat that Cx. quinquefasciatus poses as a vector of avian pathogens to island avifauna and discuss specific examples of at-risk bird populations on the islands of Hawai‘i, New Zealand and Galápagos. We also review the major options for the deployment of genetic control tools against Cx. quinquefasciatus, and discuss the current state of the field with a focus on radiation-based sterilisation, transgenic methods, and transinfections using the bacterial endosymbiont Wolbachia.
Chapter
Insects are responsible for considerable crop losses worldwide through their direct damage and the transmission of various diseases as well. Recently, novel techniques would replace the most frequently used chemical insecticides and help facilitate the sustainability in crop production in near future. Different strategies to overcome crop resistance against insect, especially Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated system (CRISPR/Cas) based genome editing and gene drives are becoming available for insecticide resistance management. Recent advances and applications of CRISPR/Cas9 both in plants and insects offer promising mechanism of deterrence to insect pests through improving resistance of Bt, knockout, or insertion of new genes; in depth understanding of plant response against insect pests provides routes to optimize plant defenses against insects. In addition, directed evolution may play an important role to combat insect resistance against Bt crops. Although, various genome editing techniques have been developed, however; CRISPR-based approaches for insect management in crops are growing rapidly so far. Therefore, recently CRISPR-mediated gene drives are being established as potential insect management approaches in agriculture.
Chapter
Gene drive approaches—those which bias inheritance of a genetic element in a population of sexually reproducing organisms—have the potential to provide important public benefits. The spread of selected genetic elements in wild populations of organisms may help address certain challenges, such as transmission of vector-borne human and animal diseases and biodiversity loss due to invasive animals. Adapting various naturally occurring gene drive mechanisms to these aims is a long-standing research area, and recent advances in genetics have made engineering gene drive systems significantly more technically feasible. Gene drive approaches would act through changes in natural environments, thus robust methods to evaluate potential research and use are important.
Article
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(1) Analytical models are described for a field population of adult Aedes aegypti mosquitoes, and used as foundations for the development of a multi-age-class simulation model. The models bring together published and unpublished data on the larval and adult ecology of A. aegypti in Wat Samphaya, Bangkok, Thailand. (2) The most appropriate analytical model is a generalization of a continuous time model used by Gurney, Blythe & Nisbet (1980) to describe Nicholson's blowfly populations. Despite uncertainty about egg-laying rate, local stability analysis firmly predicts that the population in Wat Samphaya is monotonically stable. Equilibrium analysis predicts that adult populations will be more sensitive to changes in death rate than to changes in either birth rate or number of larval breeding sites. Accurate prediction of equilibrium population size requires good estimates of parameters (beta s) describing density-dependent mortality. (3) Results of stability analysis with the simulation model accord with those of the analytical model: observed fluctuations in adult population size are unlikely to be driven cycles, but rather due to fluctuations in adult survivorship combined with strong density-dependent larval mortality. Equilibrium analysis reinforces the conclusion that the adult population is more sensitive to changes in adult survivorship than to changes in fecundity.
Book
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About this Book The sterile insect technique (SIT) is an environment-friendly method of pest control that integrates well into area-wide integrated pest management (AW-IPM) programmes. A first of its kind, this book takes a generic, thematic, comprehensive, and global approach in describing the principles and practice of the SIT. The strengths and weaknesses, and successes and failures, of the SIT are evaluated openly and fairly from a scientific perspective. The SIT is applicable to some major pests of plant, animal and human health importance, and criteria are provided to guide in the selection of pests appropriate for the SIT. This technology, using radiation to sterilize insects, was first developed in the USA, and is currently applied on six continents. For four decades it has been a major subject for research and development in the Joint FAO/IAEA Programme on Nuclear Techniques in Food and Agriculture, involving both research and the transfer of this technology to Member States so that they can benefit from improved plant, animal and human health, cleaner environments, increased production of plants and animals in agricultural systems, and accelerated economic development. A great variety of subjects are covered, from the history of the SIT to improved prospects for its future application. The major chapters discuss the principles, technical components, and application of sterile insects. The four main strategic options in using the SIT — suppression, containment, prevention, and eradication — with examples of each option, are described in detail. Other chapters deal with supportive technologies, economic, environmental, and management considerations, and the socio-economic impact of AW-IPM programmes that integrate the SIT. The 28 chapters were all peer reviewed before final editing.
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Gene drive systems are genetic elements capable of spreading into a population even if they confer a fitness cost to their host. We consider a class of drive systems consisting of a chromosomally located, linked cluster of genes, the presence of which renders specific classes of offspring arising from specific parental crosses unviable. Under permissive conditions, a number of these elements are capable of distorting the offspring ratio in their favor. We use a population genetic framework to derive conditions under which these elements spread to fixation in a population or induce a population crash. Many of these systems can be engineered using combinations of toxin and antidote genes, analogous to Medea, which consists of a maternal toxin and zygotic antidote. The majority of toxin-antidote drive systems require a critical frequency to be exceeded before they spread into a population. Of particular interest, a Z-linked Medea construct with a recessive antidote is expected to induce an all-male population crash for release frequencies above 50%. We suggest molecular tools that may be used to build these systems, and discuss their relevance to the control of a variety of insect pest species, including mosquito vectors of diseases such as malaria and dengue fever.
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The author has no financial interest in companies doing commercial GM exploitation. The author has no patents related to GM insects. The author is involved in the direction of academic research contracts through Imperial College London with WHO on international guidance for the deployment and implementation of GM mosquitoes for malaria and dengue control. Other partners in that WHO research contract do have commercial interests in GM insect exploitation. In 2012, the author will become involved in the direction of academic research on the design and implementation of risk analysis approaches relevant to GM mosquitoes as part of a research contract from the Gates Foundation to Imperial College London. The author is a member of a working group on GM mosquitoes for WHO and the FNIH. The author is a member of a working group on GM insects for the European Food Safety Authority. The working groups are unpaid, but cover travelling costs when needed for occasional meetings of the groups. The author is a College appointed director of a company called Agra-CEAS Consulting Ltd, which is part owned by Imperial College London, which has produced some reports on various aspects of GM technology in agriculture for various clients (such as the European Commission), but the author has not had any day to day involvement in any of those reports. The author acts as an academic reviewer for various national and international research funders evaluating research proposals that sometimes include GM technologies. No funding bodies or other organisations have influenced this manuscript. The author has not received any funding for this work.
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Author Summary Underdominance is a component of natural evolution: homozygotes – of either wildtypes or mutants – are advantageous. This can play a role in speciation and as a method to establish artificial genetic constructs in wild populations. The polymorphic state of wildtype and mutant alleles is unstable. However, in subdivided populations limited gene flow can counterbalance this effect. The maintenance of polymorphism sensitively depends on the amount of gene flow. In populations of finite size, the polymorphism is ultimately lost due to stochastic fluctuations, but there are long intermediate periods of polymorphism persistence. We analyze a simple population genetic model to characterize and explore the polymorphic phases depending on population size and genotypic fitness values. Even for large fluctuations (small population size), long periods of neither extinction nor fixation are possible. Since underdominance has been proposed as a genetic strategy in the pest management of disease vectors, it is important to understand the basic features of this system precisely, especially with a focus on gene flow between ecological patches. We assess different release strategies of potentially underdominant mutants, where one seeks to minimize the probability of fixation of the introduced allele but maximize the time to its extinction.
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One proposed strategy for controlling the transmission of insect-borne pathogens uses a drive mechanism to ensure the rapid spread of transgenes conferring disease refractoriness throughout wild populations. Here, we report the creation of maternal-effect selfish genetic elements in Drosophila that drive population replacement and are resistant to recombination-mediated dissociation of drive and disease refractoriness functions. These selfish elements use microRNA-mediated silencing of a maternally expressed gene essential for embryogenesis, which is coupled with early zygotic expression of a rescuing transgene.
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One strategy to control mosquito-borne diseases, such as malaria and dengue fever, on a regional scale is to use gene drive systems to spread disease-refractory genes into wild mosquito populations. The development of a synthetic Medea element that has been shown to drive population replacement in laboratory Drosophila populations has provided encouragement for this strategy but has also been greeted with caution over the concern that transgenes may spread into countries without their consent. Here, we propose a novel gene drive system, inverse Medea, which is strong enough to bring about local population replacement but is unable to establish itself beyond an isolated release site. The system consists of 2 genetic components--a zygotic toxin and maternal antidote--which render heterozygous offspring of wild-type mothers unviable. Through population genetic analysis, we show that inverse Medea will only spread when it represents a majority of the alleles in a population. The element is best located on an autosome and will spread to fixation provided any associated fitness costs are dominant and to very high frequency otherwise. We suggest molecular tools that could be used to build the inverse Medea system and discuss its utility for a confined release of transgenic mosquitoes.
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Two strategies to control mosquito-borne diseases, such as malaria and dengue fever, are reducing mosquito population sizes or replacing populations with disease-refractory varieties. We propose a genetic system, Semele, which may be used for both. Semele consists of two components: a toxin expressed in transgenic males that either kills or renders infertile wild-type female recipients and an antidote expressed in females that protects them from the effects of the toxin. An all-male release results in population suppression because wild-type females that mate with transgenic males produce no offspring. A release that includes transgenic females results in gene drive since females carrying the allele are favored at high population frequencies. We use simple population genetic models to explore the utility of the Semele system. We find that Semele can spread under a wide range of conditions, all of which require a high introduction frequency. This feature is desirable since transgenic insects released accidentally are unlikely to persist, transgenic insects released intentionally can be spatially confined, and the element can be removed from a population through sustained release of wild-type insects. We examine potential barriers to Semele gene drive and suggest molecular tools that could be used to build the Semele system.
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Genetically-modified (GM) mosquitoes have been proposed as part of an integrated vector control strategy for malaria control. Public acceptance is essential prior to field trials, particularly since mosquitoes are a vector of human disease and genetically modified organisms (GMOs) face strong scepticism in developed and developing nations. Despite this, in sub-Saharan Africa, where the GM mosquito effort is primarily directed, very little data is available on perspectives to GMOs. Here, results are presented of a qualitative survey of public attitudes to GM mosquitoes for malaria control in rural and urban areas of Mali, West Africa between the months of October 2008 and June 2009. The sample consisted of 80 individuals - 30 living in rural communities, 30 living in urban suburbs of Bamako, and 20 Western-trained and traditional health professionals working in Bamako and Bandiagara. Questions were asked about the cause of malaria, heredity and selective breeding. This led to questions about genetic alterations, and acceptable conditions for a release of pest-resistant GM corn and malaria-refractory GM mosquitoes. Finally, participants were asked about the decision-making process in their community. Interviews were transcribed and responses were categorized according to general themes. Most participants cited mosquitoes as one of several causes of malaria. The concept of the gene was not widely understood; however selective breeding was understood, allowing limited communication of the concept of genetic modification. Participants were open to a release of pest-resistant GM corn, often wanting to conduct a trial themselves. The concept of a trial was reapplied to GM mosquitoes, although less frequently. Participants wanted to see evidence that GM mosquitoes can reduce malaria prevalence without negative consequences for human health and the environment. For several participants, a mosquito control programme was preferred; however a transgenic release that satisfied certain requirements was usually acceptable. Although there were some dissenters, the majority of participants were pragmatic towards a release of GM mosquitoes. An array of social and cultural issues associated with malaria, mosquitoes and genetic engineering became apparent. If these can be successfully addressed, then social acceptance among the populations surveyed seems promising.
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We have isolated a new female sterile mutant from Drosophila melanogaster, which arrests the embryonic development during the transition from syncytial to cellular blastoderm. Cytological analysis of the mutant embryos indicates that pseudocleavage furrows in the syncytial blastoderm are abnormal but not completely disrupted. However, cleavage furrows during cellularization are totally disorganized, and no embryos can develop beyond this stage. Consistent with this observation, the expression of this gene peaks around the cellular blastoderm and not in any later developmental stages. Based on immunofluorescence experiments, the protein product of this gene is localized in both pseudocleavage furrows at the syncytial blastoderm and in the cleavage furrows during the cellularization stage. Sequence homology analysis demonstrates a modest, but statistically significant, similarity of this protein with the carboxyl-terminal domains of dystrophin and a family of proteins collectively known as apodystrophins. It is possible that this protein may play an essential role in organizing and maintaining a specialized cytoskeletal structure, a function also suggested for dystrophin and apodystrophins.
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The population structure of the Anopheles gambiae complex is unusual, with several sibling species often occupying a single area and, in one of these species, An. gambiae sensu stricto, as many as three "chromosomal forms" occurring together. The chromosomal forms are thought to be intermediate between populations and species, distinguishable by patterns of chromosome gene arrangements. The extent of reproductive isolation among these forms has been debated. To better characterize this structure we measured effective population size, N(e), and migration rates, m, or their product by both direct and indirect means. Gene flow among villages within each chromosomal form was found to be large (N(e)m > 40), was intermediate between chromosomal forms (N(e)m approximately 3-30), and was low between species (N(e)m approximately 0.17-1.3). A recently developed means for distinguishing among certain of the forms using PCR indicated rates of gene flow consistent with those observed using the other genetic markers.
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A novel form of underdominance is suggested as a mechanism that is able to drive desired genes into pest populations through the release of transgenic individuals over one or more generations. Such a mechanism is urgently needed by those working to reduce the impact of malaria by releasing strains of Anopheles, the vector of the disease, that are not susceptible to malaria parasites. We use simple population genetics models to quantify the benefits conferred when heterozygous genotypes, arising from matings between introduced and wild individuals, are not viable. In a randomly mating population, underdominant systems accelerate introgression of desired alleles and allow the release of individuals to be discontinued once the frequency of transgenic alleles attains a threshold. A set of two constructs, which together are selectively neutral but lethal when one is carried without the other, are found to produce dynamics that are characteristic of underdominant systems. When these constructs are carried on non-homologous chromosomes, then the ratio of released to natural born individuals need only be greater than 3:100 for introgression to occur. Furthermore, the threshold for the gene frequencies over which the introduced genes are expected to become fixed upon discontinuing the release of transgenic individuals is surprisingly low. The location of the threshold suggests that the introduced genes are expected to spread in space, at least locally. For the first time, the prospect of a practical drive mechanism for the genetic manipulation of pest populations is raised.
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MyD88 is an adapter protein in the signal transduction pathway mediated by interleukin-1 (IL-1) and Toll-like receptors. A Drosophila homologue of MyD88 (DmMyD88) was recently shown to be required for the Toll-mediated immune response. In Drosophila, the Toll pathway was originally characterized for its role in the dorsoventral patterning of the embryo. We found that, like Toll, DmMyD88 messenger RNA is maternally supplied to the embryo. Here we report the identification of a new mutant allele of DmMyD88, which generates a protein lacking the carboxy-terminal extension, normally located downstream of the Toll/IL-1 receptor domain. Homozygous mutant female flies lay dorsalized embryos that are rescued by expression of a transgenic DmMyD88 complementary DNA. The DmMyD88 mutation blocks the ventralizing activity of a gain-of-function Toll mutation. These results show that DmMyD88 encodes an essential component of the Toll pathway in dorsoventral pattern formation.
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The elegant mechanisms by which naturally occurring selfish genetic elements, such as transposable elements, meiotic drive genes, homing endonuclease genes and Wolbachia, spread at the expense of their hosts provide some of the most fascinating and remarkable subjects in evolutionary genetics. These elements also have enormous untapped potential to be used in the control of some of the world's most devastating diseases. Effective gene drive systems for spreading genes that can block the transmission of insect-borne pathogens are much needed. Here we explore the potential of natural gene drive systems and discuss the artificial constructs that could be envisaged for this purpose.
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Notch signalling, which is highly conserved from nematodes to mammals, plays crucial roles in many developmental processes. In the Drosophila embryo, deficiency in Notch signalling results in neural hyperplasia, commonly referred to as the neurogenic phenotype. We identify a novel maternal neurogenic gene, neurotic, and show that it is essential for Notch signalling. neurotic encodes a Drosophila homolog of mammalian GDP-fucose protein O-fucosyltransferase, which adds fucose sugar to epidermal growth factor-like repeats and is known to play a crucial role in Notch signalling. neurotic functions in a cell-autonomous manner, and genetic epistasis tests reveal that Neurotic is required for the activity of the full-length but not an activated form of Notch. Further, we show that neurotic is required for Fringe activity, which encodes a fucose-specific beta1, 3 N-acetylglucosaminyltransferase, previously shown to modulate Notch receptor activity. Finally, Neurotic is essential for the physical interaction of Notch with its ligand Delta, and for the ability of Fringe to modulate this interaction in Drosophila cultured cells. We present an unprecedented example of an absolute requirement of a protein glycosylation event for a ligand-receptor interaction. Our results suggest that O-fucosylation catalysed by Neurotic is also involved in the Fringe-independent activities of Notch and may provide a novel on-off mechanism that regulates ligand-receptor interactions.
Article
We have isolated a new female sterile mutant from Drosophila melanogaster, which arrests the embryonic development during the transition from syncytial to cellular blastoderm. Cytological analysis of the mutant embryos indicates that pseudocleavage furrows in the syncytial blastoderm are abnormal but not completely disrupted. However, cleavage furrows during cellularization are totally disorganized, and no embryos can develop beyond this stage. Consistent with this observation, the expression of this gene peaks around the cellular blastoderm and not in any later developmental stages. Based on immunofluorescence experiments, the protein product of this gene is localized in both pseudocleavage furrows at the syncytial blastoderm and in the cleavage furrows during the cellularization stage. Sequence homology analysis demonstrates a modest, but statistically significant, similarity of this protein with the carboxyl-terminal domains of dystrophin and a family of proteins collectively known as apodystrophins. It is possible that this protein may play an essential role in organizing and maintaining a specialized cytoskeletal structure, a function also suggested for dystrophin and apodystrophins.
Article
Permanent translocation heterozygotes are known from at least 57 species of flowering plants. They often share certain features of chromosome morphology, though none are universal or required. Many have apparently originated through hybridization of ancestors with different chromosome end arrangements. The population structure of organisms with this genetic system is similar to that of parthenogenetic organisms, ie there is relatively little variation within populations and greater amounts of differentiation between populations. Genetic heterozygosity is not necessarily associated with chromosomal heterozygosity. Heterosis may not be necessary to explain the apparent success of permanent translocation heterozygotes in the groups in which it occurs.-from Authors
Article
Matings of Triturus cristatus carnifex consistently produce two embryonic lethal phenotypes (fat-tailed and slim-tailed) and viable larvae in the 1:1:2 proportions expected for a balanced lethal system. A marker variant of chromosome 1 is used to show fat-tailed embryos are 1A/1A homozygotes, slim-tailed embryos are 1B/1B homozygotes and viable ones are 1A/1B heterozygotes. Another embryonic trait (dorsal blisters), which is expressed in the lethal phenotypes of some matings, is found to be incompletely linked to the recessive lethal factors.
Article
Insects act as vectors for diseases of plants, animals, and humans. Replacement of wild insect populations with genetically modified individuals unable to transmit disease provides a potentially self-perpetuating method of disease prevention. Population replacement requires a gene drive mechanism in order to spread linked genes mediating disease refractoriness through wild populations. We previously reported the creation of synthetic Medea selfish genetic elements able to drive population replacement in Drosophila. These elements use microRNA-mediated silencing of myd88, a maternally expressed gene required for embryonic dorso-ventral pattern formation, coupled with early zygotic expression of a rescuing transgene, to bring about gene drive. Medea elements that work through additional mechanisms are needed in order to be able to carry out cycles of population replacement and/or remove existing transgenes from the population, using second-generation elements that spread while driving first-generation elements out of the population. Here we report the synthesis and population genetic behavior of two new synthetic Medea elements that drive population replacement through manipulation of signaling pathways involved in cellular blastoderm formation or Notch signaling, demonstrating that in Drosophila Medea elements can be generated through manipulation of diverse signaling pathways. We also describe the mRNA and small RNA changes in ovaries and early embryos associated from Medea-bearing females. Finally, we use modeling to illustrate how Medea elements carrying genes that result in diapause-dependent female lethality could be used to bring about population suppression.
Article
Mosquito-borne diseases such as malaria and dengue fever pose a major health problem through much of the world. One approach to disease prevention involves the use of selfish genetic elements to drive disease-refractory genes into wild mosquito populations. Recently engineered synthetic drive systems have provided encouragement for this strategy; but at the same time have been greeted with caution over the concern that transgenes may spread into countries and communities without their consent. Consequently, there is also interest in gene drive systems that, while strong enough to bring about local population replacement, are unable to establish themselves beyond a partially isolated release site, at least during the testing phase. Here, we develop simple deterministic and stochastic models to compare the confinement properties of a variety of gene drive systems. Our results highlight several systems with desirable features for confinement-a high migration rate required to become established in neighboring populations, and low-frequency persistence in neighboring populations for moderate migration rates. Single-allele underdominance and single-locus engineered underdominance have the strongest confinement properties, but are difficult to engineer and require a high introduction frequency, respectively. Toxin-antidote systems such as Semele, Merea and two-locus engineered underdominance show promising confinement properties and require lower introduction frequencies. Killer-rescue is self-limiting in time, but is able to disperse to significant levels in neighboring populations. We discuss the significance of these results in the context of a phased release of transgenic mosquitoes, and the need for characterization of local ecology prior to a release.
Article
One strategy for controlling transmission of insect-borne disease involves replacing the native insect population with transgenic animals unable to transmit disease. Population replacement requires a drive mechanism to ensure the rapid spread of linked transgenes, the presence of which may result in a fitness cost to carriers. Medea selfish genetic elements have the feature that when present in a female, only offspring that inherit the element survive, a behavior that can lead to spread. Here, we derive equations that describe the conditions under which Medea elements with a fitness cost will spread, and the equilibrium allele frequencies are achieved. Of particular importance, we show that whenever Medea spreads, the non-Medea genotype is driven out of the population, and we estimate the number of generations required to achieve this goal for Medea elements with different fitness costs and male-only introduction frequencies. Finally, we characterize two contexts in which Medea elements with fitness costs drive the non-Medea allele from the population: an autosomal element in which not all Medea-bearing progeny of a Medea-bearing mother survive, and an X-linked element in species in which X/Y individuals are male. Our results suggest that Medea elements can drive population replacement under a wide range of conditions.
Article
Underdominance refers to natural selection against individuals with a heterozygous genotype. Here, we analyze a single-locus underdominant system of two large local populations that exchange individuals at a certain migration rate. The system can be characterized by fixed points in the joint allele frequency space. We address the conditions under which underdominance can be applied to transform a local population that is receiving wildtype immigrants from another population. In a single population, underdominance has the benefit of complete removal of genetically modified alleles (reversibility) and coexistence is not stable. The two population system that exchanges migrants can result in internal stable states, where coexistence is maintained, but with additional release of wildtype individuals the system can be reversed to a fully wildtype state. This property is critically controlled by the migration rate. We approximate the critical minimum frequency required to result in a stable population transformation. We also concentrate on the destabilizing effects of fitness and migration rate asymmetry. Practical implications of our results are discussed in the context of utilizing underdominance to genetically modify wild populations. This is of importance especially for genetic pest management strategies, where locally stable and potentially reversible transformations of populations of disease vector species are of interest.
Article
Advances in insect transgenesis and our knowledge of insect physiology and genomics are making it possible to create transgenic populations of beneficial or pest insects that express novel traits. There are contexts in which we may want the transgenes responsible for these traits to spread so that all individuals within a wild population carry them, a process known as population replacement. Transgenes of interest are unlikely to confer an overall fitness benefit on those who carry them. Therefore, an essential component of any population replacement strategy is the presence of a drive mechanism that will ensure the spread of linked transgenes. We discuss contexts in which population replacement might be desirable and the requirements a drive system must satisfy to be both effective and safe. We then describe the creation of synthetic Medea elements, the first selfish genetic elements synthesized de novo, with the capability of driving population replacement, in this case in Drosophila. The strategy used to create Drosophila Medea is applicable to a number of other insect species and the Medea system satisfies key requirements for scientific and social acceptance. Finally, we highlight several challenges to implementing population replacement in the wild.
Article
CHROMOSOME translocation heterozygotes (T/+) are usually semisterile, but translocation homozygotes (T/T) if viable are usually fully fertile. If such a viable translocation were produced in an insect pest, T/T insects could be reared in captivity and released into the wild, where matings with wild types (+/+) would produce T/+ progeny.
Article
Notch and its ligands are modified by a protein O-fucosyltransferase (OFUT1) that attaches fucose to a Serine or Threonine within EGF domains. By using RNAi to decrease Ofut1 expression in Drosophila, we demonstrate that O-linked fucose is positively required for Notch signaling, including both Fringe-dependent and Fringe-independent processes. The requirement for Ofut1 is cell autonomous, in the signal-receiving cell, and upstream of Notch activation. The transcription of Ofut1 is developmentally regulated, and surprisingly, overexpression of Ofut1 inhibits Notch signaling. Together, these results indicate that OFUT1 is a core component of the Notch pathway, which is required for the activation of Notch by its ligands, and whose regulation may contribute to the pattern of Notch activation during development.
Article
In Drosophila, the dorsoventral axis is set up by the action of the dorsal group of genes and cactus, which have been ordered genetically in a linear pathway. We have identified and characterised krapfen (kra) as a new member of the dorsal-group genes. kra encodes for the Drosophila homologue of MyD88, an adapter protein operating in the mammalian IL-1 pathway. Epistasis experiments reveal that kra acts between the receptor Toll and the cytoplasmic factor Tube. We show that there is a direct interaction between Kra and Tube presumably mediated by the death domains present in both proteins. Tube in turn interacts with its downstream effector Pelle through death domain association. We therefore suggest that upon Toll activation, Kra associates with Pelle and Tube, in an heterotrimeric complex.
Article
Genetic modification (GM) of mosquitoes (which renders them genetically modified organisms, GMOs) offers opportunities for controlling malaria. Transgenic strains of mosquitoes have been developed and evaluation of these to 1) replace or suppress wild vector populations and 2) reduce transmission and deliver public health gains are an imminent prospect. The transition of this approach from confined laboratory settings to open field trials in disease-endemic countries (DECs) is a staged process that aims to maximize the likelihood of epidemiologic benefits while minimizing potential pitfalls during implementation. Unlike conventional approaches to vector control, application of GM mosquitoes will face contrasting expectations of multiple stakeholders, the management of which will prove critical to safeguard support and avoid antagonism, so that potential public health benefits can be fully evaluated. Inclusion of key stakeholders in decision-making processes, transfer of problem-ownership to DECs, and increased support from the wider malaria research community are important prerequisites for this. It is argued that the many developments in this field require coordination by an international entity to serve as a guiding coalition to stimulate collaborative research and facilitate stakeholder involvement. Contemporary developments in the field of modern biotechnology, and in particular GM, requires competencies beyond the field of biology, and the future of transgenic mosquitoes will hinge on the ability to govern the process of their introduction in societies in which perceived risks may outweigh rational and responsible involvement.
The spread of genetic constructs in natural insect populations
  • H.R. Braig
  • G. Yan
  • H.R. Braig
  • G. Yan
The spread of genetic constructs in natural insect populations. In Genetically Engineered Organisms: Assessing Environmental and Human Health
  • H R Braig
  • G Yan
Braig, H.R., and Yan, G. (2001). The spread of genetic constructs in natural insect populations. In Genetically Engineered Organisms: Assessing Environmental and Human Health Effects, D.K. Letourneau and B.E. Burrows, eds. (Boca Raton, FL: CRC Press), pp. 251–314.
  • Z Kambris
  • H Bilak
  • R D 'alessandro
  • M Belvin
  • J L Imler
  • M Capovilla
Kambris, Z., Bilak, H., D'Alessandro, R., Belvin, M., Imler, J.L., and Capovilla, M. (2003). DmMyD88 controls dorsoventral patterning of the Drosophila embryo. EMBO Rep. 4, 64–69.
Gene drive systems for insect disease vectors
  • Sinkins
Sinkins, S.P., and Gould, F. (2006). Gene drive systems for insect disease vectors. Nat. Rev. Genet. 7, 427–435.